book of abstracts of symposium post-transcriptional gene regulation in plants

July 25-26 Providence, rhode island

Post-transcriPtional Gene reGulation in Plants

A satellite meeting of Plant Biology 2013

PLN013_satellite meeting_v2.indd 1 6/24/13 10:13 AM

Organizers: A.S.N. Reddy, Colorado State University, USA Andrea Barta, Medical University of Vienna, Austria John Brown, University of Dundee, UK Motoaki Seki, RIKEN, Japan Martin Crespi, CNRS, France Pam Green, University of Delaware. USA

Karen Browning, University of Texas, Austin

Sponsors:

College of Natural Sciences and Dept. of Biology

TABLE OF CONTENTS

Schedule 2-5

Thursday, July 25 2-3 Friday, July 26 4-5

Oral Presentation Abstracts Index

6

Oral Presentation Abstracts

7-26

Poster Abstracts

27-72

Participant List

73-81

Post-transciptional Gene Regulation in Plants 2

SCHEDULE Thursday, July 25 Time Event 7:00 am – 8:20 am

Registration

8:20 – 10:20 Session chair: Andrea Barta 8:20 – 08:30 Introduction / Opening remarks 8:30 – 9:00 Sarah Assmann Abstract No. 15: Hidden Codes in RNA: In Vivo Genome-wide Profiling of RNA Secondary Structure 9:00 – 09:20 Craig Simpson Abstract No. 97: Conservation of Alternative Exons in Plants 9:20 – 9:40 Marcelo Yanovsky Abstract No. 107: PICln, an SnRNP Assembly Related Protein, Plays a Role in the Regulation of Alternative Splicing, Developmental and Stress Responses in Arabidopsis 9:40 – 10:00 Vasiliki Zacharaki Abstract No. 119: FPA, a Regulator of Alternative Polyadenylation, is Closely Associated with Cleavage and Polyadenylation Factors in vivo 10:00 – 10:20 Ann Loraine Abstract No. 84: RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing

10:20 am – 10:40 am Coffee Break 10:40 am - 12:10 pm Session chair: Pam Green

10:40 – 11:10 Cecile Bousquet-Antonelli Abstract No. 28: XRN4 and LARP1 Are Required in a Heat-Mediated mRNA Decay Pathway Essential for Plant Acclimation to Heat Stress and Survival 11:10 – 11:30 Vinay Nagarajan Abstract No. 42: Transcriptomic Analysis Indicates Multiple Decay Pathways for XRN4-affected Transcripts in Arabidopsis 11:30 – 11:50 Dominique Gagliardi Abstract No. 60: Uridylation Protects Deadenylated mRNAs from 3' Trimming 11:50 – 12:10 Yukako Chiba Abstract No. 82: Involvement of Arabidopsis Deadenylases, AtCCR4a and AtCCR4b in Sugar Metabolism


12:10 am – 2:00 pm Lunch and Even number posters 2:00 pm – 3:50 pm Session Chair: Julia Bailey-Serres

2:00 – 2:30 M Crespi Abstract No. 120: Modulation of Alternative Splicing by Long Non-coding RNAs 2:30 – 2:50 Yuda Fang Abstract No. 17: Complementation of Hyponastic Leaves1 by Double-strand RNA Binding Domains of Dicer-like 1 in Nuclear Dicing Bodies 2:50 – 3:10 Srimathi Bogamuwa Abstract No. 23: P-body and Stress Granule Localized Tandem CCCH Zinc Finger Proteins are Involved in ABA and GA Mediated Plant Growth and Stress Responses 3:10 – 3:30 Peter Moffett Abstract No. 102: RNA Silencing and P-body Components in Compatible and Incompatible Interactions Between Plants and Viruses. 3:30 -3:50 Pablo Manavella Abstract No. 86: Exploring the miRNA Biogenesis Regulation by Luciferase-aided Fast-forward Genetics.

3:50 pm – 4:10 pm Coffee break

4:10 pm – 5:40 pm Session Chair: Martin Crespi 4:10 – 4:40 Motoaki Seki Abstract No. 51: Novel Antisense RNA Regulation Functions in Plant Abiotic Stress Responses 4:40 – 5:00 Jianhua Zhu Abstract No. 22: A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold-Responsive Gene Regulation, and Cold Tolerance in Arabidopsis thaliana 5:00 – 5:20 Mehdi Jabnoune Abstract No. 35: A Rice Cis-Natural Antisense RNA Enhances PHO1;2 Protein Level via Translational Control and Contributes to Phosphate Homeostasis and Plant Fitness 5:20 – 5:40 Nihal Dharmasiri Abstract No. 117: Involvement of Post-transcriptional Regulation of IBR5 in Plant Auxin Response.

6:00 pm – 10:30 pm Dinner Poster session 6:30 - 8:00 odd number posters 8:00- 9:30 even number posters


Friday, July 26 8:30 am -10:00 am Session Chair: Richard Jorgensen

8:30 – 9:00 Andrea Barta Abstract No. 133: Modulation of PPI-ase Activity by RNA Targets of AtCYP59 Implies a Novel Layer for Transcription Regulation 9:00 – 9:20 Chi Zhang Abstract No. 108: In vitro Identification of RNA Targets of Arabidopsis Puf RNA-binding Proteins 9:20 – 9:40 Li Tian Abstract No. 1: Identification and Characterization of a Novel RNA-binding Protein Involved in Glutelin mRNA Processing and Localization 9:40 – 10:00 Xiuren Zhang Abstract No. 40: Molecular Mechanism for Bi-directional Processing of Primary miRNAs with Branched Terminal Loops by Dicer-like 1 in Arabidopsis

10:00 am – 10:20 am Coffee break

10:20 am – 12:10 pm Session Chair: Karen Browning 10:20 – 10:50 Julia Bailey-Serres Abstract No. 29: High Resolution of Translational Dynamics Through Genome-wide Profiling of Ribosome Footprints in Arabidopsis 10:50 – 11:10 Shengben Li Abstract No. 123: microRNAs Inhibit the Translation of Target mRNAs on the Endoplasmic Reticulum in Arabidopsis 11:10 – 11:30 W. Allen Miller Abstract No. 89:Plant Viruses Reveal New Ways by which mRNAs Gain Access to the Translational Machinery 11:30 – 11:50 Joel Stafstrom Abstract No. 81: Genetic Analysis of the DRG-DFRP-SLH Pathway in Arabidopsis: Possible Involvement in Translational Initiation 11:50 – 12:10 Mar Castellano Abstract No. 21: Genome-Wide Translatome Analysis of Arabidopsis Seedlings Subjected to Heat Stress Identifies Regulatory Networks as Targets of Translational Control

12:10 pm – 2:00 pm Lunch and Poster session – odd numbers


2:00 pm – 3:30 pm Session Chair: Motoaki Seki 2:00 – 2:30 Anireddy Reddy Abstract No. 115: Global Analysis of Gene Expression and Alternative Splicing in a Splicing Regulator Mutant: Role of SR45 in Thermotolerance 2:30 – 2:50 Hou-Sung Jung Abstract No. 41: Correct RNA Processing is Critical for Plants to Respond to Excess Light 2:50 – 3:10 Lisa Hartmann Abstract No. 87: Changing Alternative Splicing Patterns during Early Photomorphogenesis in Arabidopsis thaliana 3:10 – 3:30 Shih-Long Tu Abstract No. 70: Genome-wide Analysis of Light-regulated Alternative Splicing in Plants

3:30 pm – 3:50 pm Coffee break

3:50 pm -5:20 pm Session Chair: Sarah Assmann 3:50 – 4:20 Richard Jorgensen Abstract No. 5: Conserved Peptide Upstream Open Reading Frames (CPuORFs) are Generally Associated with Regulatory Genes 4:20 – 4:40 Sergei Filichkin Abstract No. 132: Environmental Stress and Pathogen Challenge Regulate Unproductive Alternative Splicing of the Plant Circadian Clock Genes 4:40 – 5:00 Raquel Carvalho Abstract No. 72: Stress Responses Mediated by the Plant-specific SR45 and SCL30a Splicing Factors are ABA-dependent 5:00 – 5:20 Michael Hamilton Abstract No. 114: Detection of Putative Splicing Regulatory Elements from the Analysis of Discriminative Motifs Across Plants

5:20 pm – 5:30 pm Concluding Remarks .


ORAL PRESENTATION ABSTRACTS INDEX

SPEAKER ABSTRACT NO.

Assmann, Sarah 15

Bailey-Serres, Julia 29

Barta, Andrea 133

Bogamuwa, Srimathi 23

Bousquet-Antonelli, Cecile

28

Carvalho, Raquel 72

Castellano, Mar 21

Chiba, Yukako 82

Crespi, M 120

Dharmasiri, Nihal 117

Fang, Yuda 17

Filichkin, Sergei 132

Gagliardi, Dominique 60

Hamilton, Michael 114

Hartman, Lisa 87

Jabnoune, Mehdi 35

Jorgensen, Richard 5

SPEAKER ABSTRACT NO.

Jung, Hou-Sung 41

Li, Shengben 123

Loraine, Ann 84

Manvella, Pablo 86

Miller, W. Allen 89

Moffett, Peter 102

Nagarajan, Vinay 42

Reddy, Anireddy 115

Seki, Motoaki 51

Simpson, Craig 97

Stafstrom, Joel 81

Tian, Li 1

Tu, Shih-Long 70

Yanovsky, Marcelo 107

Zacharaki, Vasiliki 119

Zhang, Chi 108

Zhang, Xiuren 40

Zhu, Jianhua 22


ORAL PESENTER ABSTRACTS Abstract No. 1 Identification and Characterization of a Novel RNA-binding Protein Involved in Glutelin mRNA Processing and Localization Li Tian(1)*, Kelly A. Doroshenk(1), Toshihiro Kumamaru(2), Hikaru Satoh(2) and Thomas W. Okita(1) (1)Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA (2)Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan RNA-binding proteins (RBPs) play an important role in mRNA metabolism, including synthesis, maturation, transport, localization, and stability. In developing rice seeds, RNAs that code for the major rice storage proteins are transported to specific domains of the cortical endoplasmic reticulum (ER) by a regulated mechanism requiring RNA cis-localization elements or zipcodes. Putative trans-acting RBPs that recognized prolamine RNA zip codes, cis-sequences required for restricted localization to the protein body-ER, have been identified. Here, we identified a novel RBP, designated RBP-P, which specifically recognizes and binds to glutelin zipcode RNA, cis-sequences required for restricted localization of glutelin RNAs to cisternal-ER. RBP-P contains two RNA recognition motifs (RRMs) and is found in both the nucleus and cytosol. RBP-P exhibits the same expression pattern as glutelin synthesis during rice seed maturation. RNA-Immunoprecipitation (RNA-IP) and subsequent RT-PCR showed that RBP-P is associated with glutelin RNA in vivo. Likewise, in vitro RNA-protein UV-crosslinking assay indicated that recombinant RBP-P binds strongly to glutelin mRNA especially the 3?UTR and zipcode RNA. RBP-P also showed strong binding activity to a glutelin intron sequence, suggesting that RBP-P might participate in glutelin mRNA splicing. As 3?UTRs in plant mRNAs are generally U or AU-rich sequence and introns A or AU-rich sequence, we performed competition analysis against different ribopolymers and found that RBP-P binds efficiently to poly(U). A mutation in RBP-P induced abnormal accumulation of glutelin precursors and reduced amounts of acidic and basic subunits, a phenotype symptomatic of glutelin RNA mis-localization. Overall, these results support the role of RBP-P in glutelin mRNA metabolism including the splicing of the synthesized transcript to the mature form and the transport and localization to the cisternal-ER.

Abstract No. 5 Conserved Peptide Upstream Open Reading Frames (CPuORFs) are Generally Associated with Regulatory Genes Jorgensen, Richard, University of Arizona, Tucson, AZ and Ana E. Dorantes-Acosta, Universidad Veracruzana, Xalapa, Veracruz, Mexico Upstream open reading frames (uORFs) are common in eukaryotic transcripts, but those that encode conserved peptides occur in less than 1% of transcripts. The peptides encoded by three plant Conserved Peptide uORF (CPuORF) families are known to control translation of the downstream ORF in response to a small signal molecule (sucrose, polyamines, and phosphocholine). Hayden and Jorgensen (2007) found transcription factors to be statistically over-represented among genes that possess CPuORFs in angiosperms. By reassessing the functions of all known CPuORF gene families we now find that 22 of these 27 families play a variety of different regulatory roles, ranging from transcriptional control to protein turnover, and from small signal molecules to signal transduction kinases. Clearly, there is indeed a strong association of CPuORFs with regulatory genes. Importantly, 16 of these families play key roles in a variety of different biological processes: For instance, and most strikingly, the core sucrose response network includes three different CPuORF-possessing genes, creating the potential for sophisticated balancing of the network in response to three different molecular inputs at the level of translational control. We propose that the function of most CPuORFs is to modulate translation of a downstream major ORF (mORF) in response to a


signal molecule recognized by the conserved peptide and that because the mORFs of CPuORF genes generally encode regulatory proteins (many of which are centrally important in the biology of plants) CPuORFs play key roles in balancing (or 'fine-tuning') such regulatory networks.

Abstract No. 15 Hidden Codes in RNA: In Vivo Genome-wide Profiling of RNA Secondary Structure Ding, Yiliang, Penn State University, University Park, Pennsylvania, USA. Kwok, Chun Kit, Penn State University, University Park, Pennsylvania, USA. Tang, Yin, Penn State University, University Park, Pennsylvania, USA Bevilacqua, Philip C. Penn State University, University Park, Pennsylvania, USA. Assmann, Sarah M., Penn State University, University Park, Pennsylvania, USA. RNA structure plays critical roles in regulating numerous post-transcriptional events. Lack of genome-wide in vivo RNA structural data, however, has limited our understanding of how RNAs fold and regulate gene expression in living cells. We have developed a method that we term “RNA Structure-Seq”, which allows the high-throughput profiling of RNA secondary structures at single nucleotide resolution on a genome-wide scale in vivo. Application of this method to Arabidopsis thaliana seedlings yielded detailed single-nucleotide structural information across more than 10,000 transcripts. Analysis of this dataset reveals a three nucleotide periodic repeat in the structure of the coding regions that correlates with translation efficiency. We also find unique secondary structure patterns at sites of alternative polyadenylation and alternative splicing. Remarkably, a Gene Ontology analysis reveals that in vivo structures of mRNAs related to stress responses show much less similarity to in silico predicted structures than structures of mRNAs related to maintenance of cell function. These results suggest that stress-related RNAs adopt multiple structures while housekeeping-related RNAs have one structure that is strongly thermodynamically favored. Structure-Seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism. Abstract No. 17 Complementation of Hyponastic Leaves1 by Double-strand RNA Binding Domains of Dicer-like 1 in Nuclear Dicing Bodies Qi Liu, Qingqing Yan, Yin Liu, Fang Hong, Zenfei Sun, Leilei Shi, and Yuda Fang* National key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China MiRNAs are a class of small, regulatory RNAs that are found in almost all of the eukaryotes. Arabidopsis (Arabidopsis thaliana) miRNAs are processed from primary microRNAs (pri-miRNAs) mainly by the RNase III-like enzyme DICER-LIKE1 (DCL1) and its specific partner HYPONSTIC LEAVES1 (HYL1), a double-strand RNA-binding protein, both contain two double-strand RNA-binding domains (dsRBDs). These dsRBDs are essential for miRNA processing, but the functions of them are not clear. Here we report that the two dsRBDs (DCL1D12) of DCL1, and to some extent the second dsRBD (DCL1D2), complement hyl1 mutant, but not the first dsRBD of DCL1 (DCL1D1). DCL1D1 is diffusely distributed throughout the nucleoplasm, whereas DCL1D2 and DCL1D12 concentrate in nuclear dicing bodies (D-bodies) in which DCL1 and HYL1 colocalize. We show further that protein-protein interaction is mainly mediated by DCL1D2, while DCL1D1 plays a major role in binding of pri-miRNAs. These results suggest parallel roles between C-terminal dsRBDs of DCL1 and N-terminal dsRBDs of HYL1, and support a model in which Arabidopsis pri-microRNAs are


recruited to D-bodies through functionally divergent dsRBDs of microprocessor for accurate processing of plant pri-miRNAs.

Abstract No. 21 Genome-Wide Translatome Analysis of Arabidopsis Seedlings Subjected to Heat Stress Identifies Regulatory Networks as Targets of Translational Control Yanguez Emilio; Castro Sanz, Ana B., Fernandez-Bautista N. and Castellano M. Mar. Centre for Plant Genomics and Biotechnology. (INIA-UPM). Campus de Montegancedo. Pozuelo de Alarcón. 28223 Madrid. Spain. Heat stress is one of the most prominent and deleterious environmental threads affecting plant growth and development. Upon high temperatures, plants launch specialized gene expression programs that promote stress protection and survival. These programs involve global and specific changes at the transcriptional and translational levels; however the coordination of these processes and their specific role in the establishment of the heat stress response is not fully elucidated. We have carried out a genome wide analysis to monitor simultaneously the individual changes in the transcriptional and translational mRNA levels of Arabidopsis thaliana seedlings after the exposure to a heat shock stress. Our results demonstrated that, superimposed to transcription, translation exerts a wide but dual regulation of gene expression. For the majority of the mRNAs, translation is severely repressed causing a deep decrease in the association of the bulk of mRNAs to polysomes. However, some relevant mRNAs involved in different aspects of homeostasis maintenance follow a differential pattern of translation. Analysis of the sequence of the differentially translated mRNAs unraveled some special features that take part in the discrimination mechanisms for mRNA polysome loading. Among the identified differential translated genes stand out key regulators of the stress response highlighting the main role of translation in the early establishment of physiological response of plants to elevated temperatures. Abstract No. 22 A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold-Responsive Gene Regulation, and Cold Tolerance in Arabidopsis thaliana Guan Qingmei1, Wu Jianmin1, Zhang Yanyan1, Jiang Changhua1, Liu Renyi2, Chai Chenglin1 and Zhu Jianhua1,3 3To whom correspondence should be addressed: Jianhua Zhu ([email protected])(the presenter) 1Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742; 2Department of Botany and Plant Sciences, University of California, Riverside, CA 92521. Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis thaliana mutants for de-regulated expression of a firefly luciferase reporter gene under the control of the CBF2 (C-repeat binding factor 2) promoter (CBF2:LUC). An rcf1-1 (regulator of CBF gene expression 1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Ala-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOS4) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (Pseudo-response regulator 5 [PRR5], Shaggy-like serine/threonine kinase [SK12], MYB family transcription factor circadian 1 [CIR1], and SPFH/PHB domain-containing membrane-associated protein [SPFH]) that are mis-spliced in rcf1-1 revealed that these genes are cold-


inducible, positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants. Abstract No. 23 P-body and Stress Granule Localized Tandem CCCH Zinc Finger Proteins are Involved in ABA and GA Mediated Plant Growth and Stress Responses Srimathi P. Bogamuwa (1), Jie Qu (1), and Jyan-Chyun Jang (1, 2, 3) (1) Department of Horticulture and Crop Science, (2) Department of Molecular Genetics, and (3) Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA. Processing-Bodies (PBs) and Stress Granules (SGs) are oraganized cytoplasmic aggregations of messenger RiboNucleoProtein (mRNP) complexes, playing important roles in post-transcriptional regulation and epigenetic modulation of gene expression. While PBs are enriched with translation-repressed mRNAs and mRNA silencing and degradation machineries, SGs are aggregations of stalled pre-initiation complexes and translation regulators. The mammalian Tandem CCCH Zinc Finger (TZF) proteins play a central role in nucleating PBs and SGs. TZF can bind AU rich elements (AREs) at 3’UTR and recruit proteins to trigger mRNA degradation. Although PB/SG assembly and function has been extensively investigated in other systems, very little is known in plants. However, recent studies indicate that PBs and SGs are conserved, and PBs appear to play a role in mRNA stability in plants. The Arabidopsis TZF gene family consists of 11 differentially expressed members. TZF1 can shuttle between nucleus and cytoplasmic foci. It can bind AREs with specificity in vitro. Similar to TZF1, embryo specific TZF 4, 5 and 6 can co-localize with PB and SG markers in cytoplasmic foci. TZF1-associated cytoplasmic foci are dynamically assembled in response to various growth and stress cues. They are differentially assembled in stomata and stem cells, and can be induced by wounding and stress hormones in other cell types. Moreover, TZF6 can be assembled into PBs and SGs in embryos with the induction of stress hormone methyl jasmonate under the control of native TZF6 promoter. Reverse genetic analyses indicate that TZFs act as positive regulators of ABA response, and negative regulators of GA response, in part by differential regulation of ABA and GA responsive genes. Protein partner screen results indicate that TZFs may interact with ABA related stress responsive proteins. Together, we propose that plant TZF proteins play a pivotal role in hormone-mediated growth and stress responses through post-transcriptional regulation and protein-protein interaction.

Abstract No. 28 XRN4 and LARP1 are Required in a Heat-Mediated mRNA Decay Pathway Essential for Plant Acclimation to Heat Stress and Survival BOUSQUET-ANTONELLI Cécile(1) MERRET Rémy (1) DESCOMBIN Julie (1) JUAN Yu-ting (2) FAVORY Jean-Jacques (1) CARPENTIER Marie-Christine (1) CHAPARRO Cristian (3) CHARNG Yee-yung (2) DERAGON Jean-Marc (1) (1) LGDP_CNRS_University of Perpignan, 58 av Paul Alduy PERPIGNAN FRANCE (2) Academia Sinica, Agricultural Biotechnology Center, 128 Academia Road, Taipei, TAIWAN (3) Instituto Tecnologico Vale, Traverssa Boaventura da Silva, Belem, BRAZIL To survive adverse and ever changing environmental conditions, an organism must be able to adapt. It has long been established that the cellular reaction to stress includes the up-regulation of genes coding for specific stress-responsive factors. We demonstrate in the


present study that during the early steps of the heat stress response, 25% of Arabidopsis seedlings transcriptome is targeted for rapid degradation. Our findings demonstrate that this process is catalysed from 5' to 3' by the cytoplasmic exoribonuclease XRN4 which function is reprogrammed by the heat-sensing pathway. The bulk of mRNAs subject to heat-dependent degradation includes both the ribosome-released and polysome associated pools. The co-translational decay process is facilitated by LARP1, a heat specific cofactor of XRN4 required for its targeting to polysomes. Commensurate with their respective involvement at the molecular level, LARP1 and XRN4 are necessary for the thermotolerance of plants to long exposure to moderately high temperature with xrn4 null mutants being almost unable to survive. These findings provide for the first time mechanistic insights regarding a massive stress-induced post-transcriptional down-regulation and outline a potentially crucial pathway for plant survival and acclimation to heat stress. Abstract No. 29 High Resolutions of Translational Dynamics Through Genome-wide Profiling of Ribosome Footprints in Arabidopsis Juntawong, P.(1,2), Girke, T.(1), Bailey-Serres, J.(1) 1 Center for Plant Cell Biology and Department of Botany and Plant Sciences, Genomics Building, University of California, Riverside, CA 92521 2 Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand, 10900 mRNA translation is an energy demanding process that is tightly controlled by low oxygen stress in Arabidopsis thaliana. The translation state of an individual gene transcript can be quantitatively monitored by comparing the level of transcript associated one or more ribosomes (the translatome) to steady-state transcript abundance (the transcriptome). This can be accomplished through the isolation of ribosome-mRNA complexes by differential centrifugation or affinity purification of translating ribosomes (TRAP) using FLAG-tagged ribosomal protein L18 (RLP18) followed by high-through profiling analyses. Previous comparative transcriptome and translatome studies demonstrated that although most hypoxia-induced transcripts recruit ribosomes in proportion to their increase in abundance, over 65% of the cellular mRNAs are stable but poorly translated during hypoxia. These translationally repressed mRNAs are rapidly recruited back to polysomes upon reoxygenation. We hypothesize the initiation of translation is reduced during low oxygen stress as a means to curb energy consumption. Although translatome profiling is likely to provide information that more accurately reflects levels of synthesis of individual proteins, it does not provide quantitative information on the number or position of ribosomes along an mRNA. To obtain greater insight into translational regulation during hypoxia, ribosome footprint (RF) fragments were obtained by RNase I digestion of mRNA-ribosome complexes, sequenced (Ribo-seq) and mapped back to the genome. We compared total poly(A)+ RNA, polysomal poly(A)+ RNA and RFs of control and hypoxia stressed seedlings by use of RNA-sequencing. Ribosomes used for RF generation were obtained by centrifugation or TRAP. This analysis confirmed that hypoxia primarily regulates the initiation of translation, rather than elongation or termination. The results provided myriad new insights into dynamics in translation of uORF-containing and non-coding RNAs. This project was funded by National Science Foundation grants IOS-0750811 and MCB-1021969.

Abstract No. 35 A Rice Cis-Natural Antisense RNA Enhances PHO1;2 Protein Level via Translational Control and Contributes to Phosphate Homeostasis and Plant Fitness Jabnoune Mehdi(1)*, Secco David(1),Lecampion Cécile (2), Robaglia Christophe(2), Shu Qingyao(3) and Poirier Yves(1) (1) Department of Plant Molecular Biology, Biophore Building, University


of Lausanne, CH-1015, Lausanne, Switzerland; (2) Laboratoire de Génétique et Biophysique des Plantes, UMR 7265, CEA/CNRS, Aix Marseille Université, Faculté des Sciences de Luminy, 163 Avenue de Luminy, Marseille , France; (3) Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, China.. E-mail : [email protected] Cis-natural antisense transcripts (cis-NATs) are widespread in eukaryotes and most often associated with down-regulation of their associated sense genes. Unexpectedly, we found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice. OsPHO1;2, a gene involved in phosphate loading into the xylem, and its associated cis-NATPHO1;2 are both controlled from promoters active in the vascular cylinder. Under phosphate deficiency, expression of both cis-NATPHO1;2 and the OsPHO1;2 protein increased while OsPHO1;2 mRNA level remained stable. Down-regulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in OsPHO1;2 protein, impaired the transfer of phosphate from root to shoot and decreased seed yield. Constitutive over-expression of NATPHO1;2 in trans led to a strong increase of OsPHO1;2 protein, even under phosphate-sufficient conditions. Under all conditions tested, no changes occurred in OsPHO1;2 transcript level or splicing pattern, revealing that cis-NATPHO1;2 positively controls OsPHO1;2 protein accumulation from the sense transcript at the post-transcriptional level. Several experimental approaches were used to decipher the mechanism by which cis-NATPHO1;2 exert its positive effects on OsPHO1;2 protein synthesis. No polypeptide synthesis was detected from potential open reading frames present in cis-NATPHO1;2, indicating that it is most likely non-coding. Modulation of cis-NATPHO1;2 expression was not associated with changes in the nuclear export of OsPHO1.2 mRNA, nor was it associated with editing of the mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of OsPHO1;2 mRNA towards the heavier active polysomes. Together, these data indicate an unexpected role for a cis-NAT in promoting translation of its associated mRNA and provide a novel link between antisense RNA and Pi homeostasis.

Abstract No. 40 Molecular Mechanisms for Bi-directional Processing of Primary miRNAs with Branched Terminal Loops by Dicer-like 1 in Arabidopsis Zhu, Hongliang 1,2,4, Zhou, Yuyi1,2,5, Castillo, Claudia1,2, Lu, Amber1,2, Ge,Chunxiao2,3, Zhao,Ying-Tao6, Duan, Liusheng5, Li,Zhaohu5, Axtell,Michael J.7, Wang,Xiu-Jie6, and Zhang, Xiuren1,2,5* 1Department of Biochemistry and Biophysics 2Institute of Plant Genomics and Biotechnology 3Program of Molecular and Environmental Plant Sciences Texas A&M University, College Station, TX 77843, USA 4College of Food Science and Nutritional Engineering 5College of Agriculture and Life Science, China Agricultural University, Beijing, 100094, China 6State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, 10101, China 7Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, USA *Correspondence: [email protected]

miRNAs originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures. Accurate and efficient processing of pri-miRNAs ensures functional miRNAs. Here, using pri-miR165/166 family as a paradigm, we report the decisive role of pri-miRNA terminal loops in miRNA processing and accumulation. Specifically, we found that multi-branched terminal loops in pri-miR166s significantly suppressed miR166 expression in vivo. Unlike the canonical processing of pri-miRNAs, the terminal-branched pri-miRNAs were


processed by DCL1 complexes bi-directionally: from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In either case, DCL1 complexes canonically cut pri-miRNAs at a distance of 16~17 base pairs (bp) away from a reference single-stranded loop region. DCL1 could also adjust processing sites toward an internal loop through its helicase domain. Finally, genome-wide computational analysis and in vitro assays with a synthetic pri-miRNA revealed wide-spread presence of bi-directional processing of pri-miRNAs with branched terminal loops in Arabidopsis and rice. Thus, these results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complicated secondary structures. Abstract No. 41 Correct RNA Processing is Critical for Plants to Respond to Excess Light Hou-Sung Jung (1,2), Henry Priest (3,4), Todd C. Mockler (3,4) and Joanne Chory (2,5) (1) Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA; (2) Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA; (3) The Donald Danforth Plant Science Center, St. Louis, MO 63132, USA; 4) Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63110, USA; (5) Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA Excess light (EL) is too much light energy for plants to use for photosynthesis. EL decreases photosynthesis activity and prolonged exposure to EL causes destruction of plants? functional complexes such as PSII. Plants respond to EL in diverse ways including regulating nuclear gene expression to prevent and repair EL-driven damaging effects. The EARLY LIGHT-INDUCIBLE PROTEIN 2 (ELIP2) gene is a known EL-responsive nuclear gene; however its induction mechanism has not been reported. To understand the mechanism of EL-driven nuclear gene expression, we isolated tanorexia (tnr) mutants from EMS-mutagenized transgenic plants expressing the LUCIFERASE gene under the control of the ELIP2 gene promoter and terminator. In the tnr mutants, EL-driven ELIP2 induction levels drastically decreased. Positional cloning determined that TNR1 and TNR2 encode SPLICEOSOMAL TIMEKEEPER LOCUS1 (STIPL1), a predicted pre-mRNA splicing factor and LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 4 (LOS4), respectively. In tnr1/stipl1, the first intron of highly induced mRNAs under EL was retained. Intriguingly, in wild-type plants, we found that ASCORBATE PEROXIDASE 2 (APX2) mRNA, another highly induced gene by EL, was not fully spliced after exposure to EL, and that within two hoursAPX2 unspliced RNA disappeared. tnr2 is a newly isolated allele of los4, which is involved in cold stress response by regulating mRNA export from the nucleus to cytoplasm. After prolonged exposure to EL, the tnr1 tnr2 double mutant much more suffered from photo-oxidative stress than wild-type plants reflecting that mRNA processing is critical for photo-protection from photo-oxidative stress. Abstract No. 42 Transcriptomic Analyses Indicates Multiple Decay Pathways for XRN4-affected Transcripts in Arabidopsis Nagarajan, Vinay K., Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711 Green, Pamela J.*, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711 *Correspondence: [email protected] Arabidopsis exoribonuclease XRN4, a cytoplasmic homolog of yeast XRN1p, catalyzes mRNA degradation in the 5? ? 3? direction. Much of our understanding of XRN4 substrates in plants comes from polyA+ RNA. In yeast, deadenylation-dependent decapping is a major pathway of RNA decay, and XRN1p is involved in degrading uncapped RNA, including those with shortened polyA tails. However, the prominence of this pathway in plants is unknown. To


study the effects of XRN4 loss on the Arabidopsis transcriptome, the deep-sequencing technique, Parallel Analysis of RNA Ends or PARE was used to capture 5?-ends of decapped (5? monophosphorylated) polyA+ and polyA- RNA from the WT and xrn4 seedlings. Analysis of these libraries showed increased PARE sequence abundances corresponding to the 5?-ends of many mRNAs in xrn4, suggesting that these transcripts are decapped and likely degraded by XRN4. About 600 polyadenylated-decapped transcripts, including known NMD substrates and several with conserved upstream ORFs, showed >10-fold accumulation in xrn4. Yet, ~2,900 deadenylated-decapped transcripts showed >10-fold higher accumulation in xrn4 compared to the WT. RNASeq analysis indicates that, for transcripts showing >2-fold higher expression in xrn4 than WT, those that are deadaenylated outnumber those that are adenylated by more than 3:1, thus corroborating the PARE findings. Here, we demonstrate that xrn4 accumulates intermediates from multiple RNA decay pathways, and among them deadenylation-dependent decapping appears to be particularly prominent. Our results emphasize the role of XRN4 as a major player in 5? ? 3? mRNA turnover in Arabidopsis. This research was supported by the National Science Foundation under grant # MCB1021636

Abstract No. 51 Novel Antisense RNA Regulation Functions in Plant Abiotic Stress Responses Akihiro Matsui (1), Kei Iida (2), Maho Tanaka (1), Junko Ishida (1), Taeko Morosawa (1), Satoshi Takahashi (1,2), Tetsuro Toyoda (2), and Motoaki Seki (1) (1)RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, 230-0045; (2)RIKEN Advanced Center for Computing and Communication (ACCC), Wako, 351-0198. Plants respond and adapt to environmental stresses. We have analyzed RNA regulation mechanisms in plant stress responses. We have identified many stress-inducible, non-coding antisense RNAs by Arabidopsis tiling array analysis (Matsui et al. 2008 Plant Cell Physiol). The majority of these antisense RNAs showed co-expression with their sense mRNAs in response to stress. Accumulation of antisense RNA of a drought-inducible gene, RD29A, decreased in rdr1/2/6 mutants. RNA-seq revealed that RDR-mediated antisense RNA synthesis occurs in genome-wide in response to drought stress. Most of the antisense RNA loci was not linked to smRNA generation. Accumulation of RDR-generated antisense RNAs increased in xrn4, and decreased in dcp5, suggesting that the antisense RNAs are generated from degraded sense mRNAs. RNA decay rate analysis showed that RDR1/2/6 functions in the degradation of the sense mRNAs. We propose a novel RNA regulation mechanism where RDR-mediated synthesis of antisense RNAs functions in the degradation of sense mRNAs in plants under stress.

Abstract No. 60 Uridylation Protects Deadenylated mRNAs from 3' Trimming Sement, François (1), Ferrier, Emilie (1), Zuber, Hélène (1), Merret, Rémy (2), Deragon, Jean-Marc (2), Bousquet-Antonelli, Cécile (2), Lange, Heike (1) and Gagliardi*, Dominique (1) (1) Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, 12 rue du général Zimmer, 67000 Strasbourg, France. (2) Laboratoire Génome et Développement des Plantes, CNRS, Université de Perpignan Via Domitia, 66860 Perpignan Cedex, France.

Degradation of mRNAs is usually initiated by deadenylation, which promotes decapping and subsequent 5′ to 3′ degradation. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in


Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3' trimming of deadenylated mRNAs in Arabidopsis. We show that oligoadenylated mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3' ribonucleolytic attacks. This conclusion is further supported by an increase in 3' truncated transcripts detected in urt1 mutants. We propose that preventing 3' trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5' to 3' directionality of mRNA degradation. Importantly, uridylation prevents 3' shortening of mRNAs associated with polysomes suggesting that a key biological function of uridylation is to confer 5' to 3' polarity in case of co-translational mRNA decay. Abstract No. 70 Genome-wide Analysis of Light-regulated Alternative Splicing in Plants Wu, Hshin-Ping, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Su, Yi-shin, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Chen, Hsiu-Chen, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Chen, Yu-Rong, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Wu, Chia-Chen, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Lin, Wen-Dar, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Tu, Shih-Long, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Light is one of the most important factors influencing plant growth and development. Changes of light condition affect many developmental programs throughout the life cycle of plants. Light-sensing photoreceptors play major roles in regulating photomorphogenic changes via signal transduction and gene regulation. Although various levels of gene expression are modulated by light, regulation at the mRNA splicing step is less discussed. We performed mRNA sequencing to analyze transcriptome changes during de-etiolation in the single-cell-type stage of Physcomitrella patens and in the higher plant Arabidopsis thaliana. In addition to transcriptional regulation, light induces intensive alternative splicing in both organisms. Light-responsive intron retention preferentially occurred in transcripts for photosynthesis and translation in both Physcomitrella and Arabidopsis, which reveals that light-mediated splicing regulation is conserved among plants and has transcript specificity. Many light signaling gene transcripts were also alternatively spliced in responding to light changes. Moreover, intron retention was rapidly induced by light but misregulated in a moss mutant defective in red light sensing, suggesting the involvement of red light photoreceptors in splicing regulation. We also identified an exonic splicing element that potentially functions in the light-regulated intron retention regions. In summary, our results support that during photomorphogenesis, alternative splicing is rapidly fine-tuned by light to re-organize certain metabolic processes and modulate photomorphogenic gene regulation. Photoreceptors may be the determinants for regulating alternative splicing.

Abstract No. 72 Stress Responses Mediated by the Plant-specific SR45 and SCL30a Splicing Factors are ABA-dependent Carvalho, Raquel, Instituto Gulbenkian de Ciência, Oeiras, Portugal Carvalho, Sofia, Instituto Gulbenkian de Ciência, Oeiras, Portugal Barbosa, Inês, Instituto Gulbenkian de Ciência, Oeiras, Portugal Simpson, Craig,The James Hutton Institute, Dundee, Scotland, UK Brown, John, The James Hutton Institute, Dundee, Scotland, UK Baena-Gonzalez, Elena, Instituto Gulbenkian de Ciência, Oeiras, Portugal Duque, Paula, Instituto Gulbenkian de Ciência, Oeiras, Portugal


Stress responses mediated by the plant-specific SR45 and SCL30a splicing factors are ABA-dependent. As sessile organisms, plants evolved unique adaptive strategies to cope with environmental stress, with the basis of this capacity lying ultimately at the level of the genome. Alternative splicing provides a versatile means of regulating gene expression and generating proteome diversity likely to be crucial in plant responses to external cues. In agreement, several splicing factors and other RNA-binding proteins have been implicated in signal transduction of the stress phytohormone abscisic acid (ABA). Serine/arginine-rich (SR) proteins belong to one of the major classes of RNA-binding proteins active in alternative splicing. We have shown that the Arabidopsis SR-related splicing factor SR45 negatively regulates sugar signaling via downregulation of the ABA pathway. Indeed, a knockout mutant for the corresponding gene displays hypersensitivity to both glucose and ABA during early seedling development. Inhibition of ABA biosynthesis partially rescues the glucose phenotype, and the mutant displays hyperaccumulation of endogenous ABA in response to the sugar, which correlates with glucose overinduction of ABA biosynthesis and signaling genes. On the other hand, the plant-specific SR protein SCL30a negatively regulates ABA signaling to control salt and osmotic stress responses during seed germination in Arabidopsis. Loss of SCL30a function causes seed oversensitivity to high NaCl and mannitol concentrations as well as to the ABA hormone. Pharmacological and epistatic analyses combined with quantification of seed ABA levels indicate that the stress-related functions of this SR protein in germination depend on functional ABA signaling. Therefore, although responding to distinct external signals (high sugars and osmotic/salt stress), both the SR45 and SCL30a splicing factors converge on negatively regulating the ABA pathway during early plant development. This may allow seed germination and seedling growth under moderate stress, thus promoting plant tolerance to unfavorable environmental conditions. This work was funded by Grant PTDC/AGR-PRO/119058/2010 as well as Fellowships SFRH/BD/28519/2006 and SFRH/BPD/80073/2011 from Fundação para a Ciencia e a Tecnologia (FCT).

Abstract No. 81 Genetic Analysis of the DRG-DFRP-SLH Pathway in Arabidopsis: Possible Involvement in Translational Initiation Jennifer Kubic, Robert Srygler, Matthew Marcec and Joel Stafstrom*. Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115 USA. Email: [email protected] DRGs are highly conserved GTP binding proteins. Among plants, animals and fungi, amino acid identity within the DRG1 and DRG2 orthologous groups is 65-70%, and paralogs from one species are about 55% identical. This level of conservation suggests that DRGs may carry out an essential cell function in all organisms. Compared to wild type Arabidopsis plants, a drg1 drg2 double mutant is small and spindly, its leaves are yellowish and wrinkled, plants have reduced fertility, and seedling roots grow at about half the normal rate. In contrast, the equivalent genotype in yeast has no apparent phenotype. A bacterial two-hybrid system and other means were used to identify DFRP1 as a specific binding partner of DRG1, and DFRP2 as a specific partner of DRG2. In general, the presence of a particular DRG or DFRP protein is needed for the stability and normal accumulation of its binding partner. For example, a dfrp2 single mutant accumulates no detectable DRG2 protein. As predicted, a drg1 dfrp2 double mutant accumulated neither DRG1, DFRP2 nor DRG2 proteins, and it had a phenotype similar to that of the drg1 drg2 double mutant. We previously showed that DRG proteins co-purify with ribosomes, suggesting a role in regulating protein translation. Recent studies in yeast indicated that a drg1 drg2 slh1 triple mutant grows very slowly and is deficient in normal initiation of protein translation (SLH1 is


a DEAD box helicase). In Arabidopsis, the corresponding triple mutant, as well as a dfrp1 dfrp2 slh1 triple mutant, gives rise to embryo lethality. Consequently, we plan to study translation initiation in double mutants. Abstract No. 82 Involvement of Arabidopsis Deadenylases, AtCCR4a and AtCCR4b in Sugar Metabolism Suzuki, Yuya (1), Hirai, Masami Y., (2), Green, Pamela J., (3), Yamaguchi, Junji (1, 4), Chiba, Yukako (1, 4, 5) (1) Graduate School of Life Science, Hokkaido University, Sapporo, Japan; (2) Center for Sustainable Resource Science, RIKEN, Yokohama, Japan; (3) Delaware Biotechnology Institute, University of Delaware, Newark, USA; (4) Faculty of Science, Hokkaido University, Sapporo, Japan; (5) JST, Precursory Research for Embryonic Science and Technology, Kawaguchi, Japan. Sugars are important metabolites for plant growth and development serving as the energy source derived from photosynthesis as well as signaling molecules. Here, we present the involvement of mRNA turnover control in sugar metabolism. Removal of the poly(A) tail is the first and rate-limiting step of mRNA turnover in most of the transcripts in eukaryotes. Carbon Catabolite Repressor 4 (Ccr4) has been identified as the major cytoplasmic deadenylase in yeast. Arabidopsis has six homologs of this yeast Ccr4. Among them, AtCCR4a and AtCCR4b are most similar to yeast Ccr4. FLAG-tagged AtCCR4a or AtCCR4b exhibited poly(A)-specific RNA-degrading activity in vitro. Transient expression analysis using GFP fusions to AtCCR4a or AtCCR4b indicated that both are localized in specific granules, called P-bodies. To elucidate the functional significance of AtCCR4a and AtCCR4b in vivo, double mutants were constructed. Interestingly, the double mutants were insensitive to high levels of sucrose in media. The cellular concentration of sucrose was reduced and that of leaf starch was likely to be increased in the double mutants, while no difference was observed in glucose concentration. In addition, global gene expression profiling to compare double mutant and wild-type plants using microarray analysis revealed that one of the up-regulated genes in the double mutants is involved in starch biosynthesis. This suggests the possible involvement of AtCCR4s in regulating gene expression related to starch metabolism. Abstract No. 84 RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing Loraine, Ann (1) McCormick, Sheila (2) Estrada, April (1) Patel, Ketan (1) Peng, Qin (2) (1) Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, 600 Laureate Way, Kannapolis, NC 28081 (2) Plant Gene Expression Center, USDA-ARS/UC-Berkeley, Albany, CA, 94710 Pollen grains of Arabidopsis thaliana contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein coding genes expressed in pollen, including 289 assayed only by non-specific probe sets. Additional exons and


previously unannotated 5’ and 3’ UTRs for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 confirmed by PCR. Gapped read alignments revealed more than 1,900 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of on-going annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. Abstract No.86 Exploring the miRNA Biogenesis Regulation by Luciferase-aided Fast-forward Genetics Manavella, Pablo A.; Hagmann, Jörg; Weigel, Detlef. Max Planck Institute for Developmental Biology, Tübingen, Germany. The biogenesis of miRNAs in plants is a complex process that involves several steps. Between the transcription of a primary miRNA and the final incorporation of the mature miRNA into the RISC, several nuclear processing steps take place. A number of proteins that are involved in miRNA processing and function have been identified so far, some of which are essential and cause lethality in null-mutants, whereas others seem to play partially dispensable roles and only cause a range of morphological defects in mutant plants. Recently, a new high-throughput screen for the identification of unknown plant miRNA biogenesis cofactors was developed in our lab. The combination of a luciferase-based reporter system and state-of-the-art sequencing technologies enables the rapid identification of mutant genes reducing the drawbacks of re-discovery already characterized genes. Phenotypic analysis of the identified mutants showed a wide variety of morphological defects. At the same time, northern blots and qPCR reveled that the isolated mutants present abnormal functionality at different levels of the miRNA pathway. The SHORE mapping analysis revealed that the causal mutations lie on genes encoding an uncharacterized protein. Confirming the role of these proteins in the miRNA pathway, we detected alterations in the steady levels of pre-miRNAs, mature miRNAs and miRNA targets in the mutant backgrounds. Abstract No. 87 Changing Alternative Splicing Patterns during Early Photomorphogenesis in Arabidopsis thaliana Hartmann, Lisa, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen; Drewe, Philipp, Memorial Sloan-Kettering Cancer Center - Computational Biology Center, 1275 York Avenue, New York, NY 10065; Kahles, André, Memorial Sloan-Kettering Cancer Center - Computational Biology Center, 1275 York Avenue, New York, NY 10065; Rätsch, Gunnar, Memorial Sloan-Kettering Cancer Center - Computational Biology Center, 1275 York Avenue, New York, NY 10065; Wachter, Andreas, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen Most genes from higher eukaryotes contain introns which are removed from the precursor mRNA by splicing. Through the use of alternative splice sites, different mature mRNAs can be generated from the same gene. Alternative splicing (AS) is wide-spread in plants and affects, according to recent studies, approximately 60% of all multi-exon genes from Arabidopsis thaliana. With such high occurrence, AS could be an important mechanism to


coordinately adjust the transcriptome in adaptive and developmental processes. Focusing on photomorphogenesis, we compared the AS patterns of dark-grown seedlings exposed to light for different time periods. In an unbiased, transcriptome-wide approach, we used next-generation sequencing (RNA-seq) followed by computational analysis to identify AS events which are altered in the course of photomorphogenesis. We found the AS pattern of numerous genes to change light-dependently, among them several splicing regulators and light signaling components. Currently, we are characterizing the specific, biological functions of the alternative splice forms for some select candidate genes. To this end, we examine potential splicing regulatory function and consider regulation of expression through nonsense-mediated decay as well as the creation of altered proteins as possible outcomes of AS. Furthermore, we are investigating the role of these AS events in plant photomorphogenesis. In our future work, we would also like to identify the regulatory mechanisms underlying light-dependent AS control. Abstract No. 89 Plant Viruses Reveal New Ways by which mRNAs Gain Access to the Translational Machinery Kraft, Jelena, Plant Path and Micro Dept; Biochem, Biophys & Mol Biol Dept, Iowa State University, Ames, IA 50011 USA. Peterson, Mariko, Plant Path and Micro Dept; Biochem, Biophys & Mol Biol Dept, Iowa State University, Ames, IA 50011 USA. Treder, Krzysztof, Plant Path and Micro Dept; Biochem, Biophys & Mol Biol Dept, Iowa State University, Ames, IA 50011 USA. Hebrard, Eugenie, Institut de Recherche pour le Développement BP 64501, 34394 Montpellier, France. Brault, Véronique, INRA UMR SVQV, 28 rue de Herrlisheim BP 20507, 68021 Colmar, France. Ziegler-Graff, Véronique, Institut de Biologie Moléculaire des Plantes, 12 rue Général Zimmer, 67084 Strasbourg, France. Miller*, W. Allen, Plant Path and Micro Dept; Biochem, Biophys & Mol Biol Dept, Iowa State University, Ames, IA 50011 USA. and Institut de Biologie Moléculaire des Plantes, 12 rue Général Zimmer, 67084 Strasbourg, France. Plant viruses have evolved numerous strategies to gain a foothold in the host translation system. These include either cap-independent translation elements (CITE) in the 3 prime untranslated regions, or possibly the genome-linked proteins (VPg) at the 5 prime end of viral RNAs that lack a 3 prime CITE. Both the CITEs and the VPgs bind a subunit of translation initiation factor eIF4F. The efficient Barley yellow dwarf virus (BYDV)-like CITE (BTE) folds into a discrete structure in the absence of protein. It contains three to six helices (depending on the genus) protruding from a central hub. Footprinting studies reveal that a highly conserved helix, and some essential bases around the central hub, are bound by the eIF4G subunit of eIF4F. The N-terminal half of eIF4G, including the PABP and eIF4E binding sites, is not necessary for binding to the BTE or for BTE-mediated translation. An RNA-binding sequence in eIF4G, immediately upstream of the MIF4G domain is essential for binding and function. In contrast, the Panicum mosaic virus (PMV)-like CITE from various Tombusviridae requires the cap-binding eIF4E subunit of eIF4F, which it binds more tightly than any other uncapped RNA. We found that some PTEs function in mammalian translation systems, presumably because of the high structural similarity between mammalian and plant eIF4E. This is the first plant CITE known to function in animals. The polero- and sobemoviruses may initiate translation via VPg-eIF4F interactions that are required for infection. Poleroviruses appear to lack a CITE and we found that removal of the VPg from the RNA of the sobemovirus Rice yellow mottle virus drastically reduces translational efficiency in vitro. In summary, these viral mechanisms reveal some of the remarkable and diverse possibilities by which mRNAs can recruit the plant translational machinery.


Abstract No. 97 Conservation of Alternative Exons in Plants Kalyna et al., 2012. Nucleic Acid Res 40:2454-2469. Marquez et al., 2012 Genome Res. 22:1184-1195 Spensley, Mark 1,Simpson, Craig G 2, Brown, John WS 1,2 1.Division of Plant Sciences, College of Life Sciences, University of Dundee, James Hutton Institute, Dundee,DD2 5DA, Scotland. 2.Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, Scotland. Analysis of the link between alternative splicing (AS) and nonsense-mediated decay (NMD) using a high resolution RT-PCR system identified NMD sensitive transcripts that contain PTCs located within alternatively spliced exons (Kalyna et al., 2012). That some of these alternative exons were highly conserved among different plant species led to a genome-wide analysis of alternative exons in Arabidopsis. The analysis was based on annotated transcripts in TAIR (the Arabidopsis Information Resource) and assembled transcripts from our AS discovery RNA-seq analysis (Marquez et al., 2012). In addition, an unbiased computational screen for conserved regions within introns identified novel, conserved alternative exons. The alternative exons were either in frame, PTC-containing (PTC+) or induced frame shifts and were found in both coding sequence and UTRs. The in frame and PTC+ classes show enrichment in stress response and developmental genes and RNA-binding proteins respectively. We are assessing conservation of the alternative exons identified and have shown differential expression and AS of some of the genes under stress conditions. Abstract No. 102 RNA Silencing and P-body Components in Compatible and Incompatible Interactions Between Plants and Viruses Moffett, Peter Brosseau, Chantal Meteignier, Louis-Valentin El-Oirdi, Mohamed Ma, Xiaofang Département de Biologie, Université de Sherbrooke, 2500 Boulevard de l’université, Sherbrooke, Québec, Canada, J1K 2R1 To successfully infect their hosts, viruses must usurp the cell host machinery and overcome plant defense mechanisms. A major mechanism of constitutive antiviral immunity is ensured by RNA silencing, which relies on the recognition and degradation of viral double-stranded RNA into virus-derived small RNA (vsRNA) by DICER-like enzymes. Once incorporated into complexes containing members of the Argonaute (AGO) family of endonucleases, these vsRNA act as guides to target viral RNA for degradation or inhibition of translation. Induced resistance to viruses is afforded by the products of plant disease resistance (R) genes encoding NB-LRR proteins. Using several viruses, we have investigated the role of different AGO family members in both constitutive and induced anti-viral defenses. We find that specific AGO proteins are required for constitutive defenses to various plant viruses. Furthermore, we show that AGO2 variability between plants may play a role in determining host range. At the same time, R gene-induced anti-viral responses are dependent on a different set of AGO proteins, including AGO4, and these mechanisms appear to inhibit the translation of viral transcripts. In agreement with this, we find that induced defenses stimulate the formation of large numbers of RNA processing bodies (P-bodies). Although this translational regulation appears to target viral RNAs and not induce global translation shutdown, preliminary results suggest that certain cellular mRNAs are likewise regulated during a defense response. We also show that mutants in P-body components show enhanced susceptibility to plant viruses while at the same time showing enhanced R gene-mediated responses. Our results indicate a specialization in AGO function in different types of anti-viral defenses as well as link between RNA silencing, P-body function and anti-viral defenses.


Abstract No. 107 PICln, an SnRNP Assembly Related Protein, Plays a Role in the Regulation of Alternative Splicing, Developmental and Stress Responses in Arabidopsis Sanchez, Sabrina E.(1), Legris, Martina (1), Petrillo, Ezequiel (2) Seymour, Danelle K. (3) Mancini, Estefanía (1), Chernomoretz, Ariel (1), Weigel, Detlef (3), Brown, John (4), Simpson, Craig (4), Kornblihtt, Alberto R. (2), Yanovsky, Marcelo J. (1). (1) Fundación Instituto Leloir, Av. Patricias Argentinas 435, C1405BWE, Buenos Aires Argentina, (2) IFIBYNE, Pabellon II, Ciudad Universitaria, Buenos Aires, Argentina, (3) Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany, (4) Genetics Programme, SCRI, Invergowrie, Dundee DD2 5DA, UK Small nuclear ribonucleoproteins (snRNPs) are fundamental pieces of the spliceosome, the machinery that removes introns from pre-mRNAs in eukaryotes. pICln has been shown to act as a chaperone involved in snRNP assembly in mammalian cells. Despite the structural insights and mechanistic details described for pICln function, its biological function in vivo has not been elucidated yet due to the lack of pICln mutant organisms. Interestingly, we have found that pICln mutants are viable in Arabidopsis, allowing us to analyze its functional role during development and in response to different stress responses. Among other phenotypes, pICln mutants display flowering time defects and are hypersensitive to cold and salt stress. We show that the developmental and stress related phenotypes of Arabidopsis pICln mutants can be complemented by the human pICln gene, providing evidence the conservation of pICln function between humans and plants. Furthermore, high-throughput techniques allowed us to determine that only a small subset of splicing events, mainly ones that are alternatively spliced, is affected in picln mutants. Our results constitute the first proof for the involvement of pICln in the regulation of RNA processing in vivo and indicate that pICln function is to modulate alternative rather than constitutive splicing. Abstract No. 108 In vitro Identification of RNA Targets of Arabidopsis Puf RNA-binding Proteins Zhang, Chi, Department of Biological Sciences, University of Calgary, 2500 University Dr NW Calgary, AB T2N 1N4, Canada. Muench, Doug G., Department of Biological Sciences, University of Calgary, 2500 University Dr NW Calgary, AB T2N 1N4, Canada. Puf RNA-binding proteins control gene expression at the post-transcriptional level by functioning as translational repressors, promoting mRNA degradation, assisting in mRNA localization, and interfering with the maturation of RNAs. The Pumilio homology domain (PUM-HD) is a conserved region typically located at the C-terminus of Puf proteins, and this region binds to RNA in a sequence-specific manner. The Arabidopsis genome encodes up to 26 Puf proteins, each possessing eight or fewer Puf repeats in their PUM-HD. The binding surface of several Arabidopsis Puf proteins is highly conserved with non-plant Puf homologs, whereas others demonstrate significant variability in this region. This suggests that some plant Puf proteins have variable RNA target preferences. We have identifed the RNA target consensus sequences of several Arabidopsis Puf proteins using an iterative amplification-selection process (SELEX). The RNA consensus sequences of highly conserved Puf proteins were to their non-plant counterparts. In contrast, unique consensus sequences were identified for Arabidopsis Puf proteins that possess variable PUM-HDs. The PUM-HD RNA binding affinities and specificities were evaluated by mobility shift assay (EMSA). This work will assist in our efforts to decipher the binding code for the key amino acids in Puf repeats, and reveal potential plant genes that are regulated by plant Puf proteins.


Abstract No. 114 Detection of Putative Splicing Regulatory Elements from the Analysis of Discriminative Motifs Across Plants Hamilton, Michael, Computer Science Department, Colorado State University, Fort Collins, CO, USA Reddy, Anireddy SN, Department of Biology, Colorado State University, Fort Collins, CO, USA Ben-Hur, Asa, Computer Science Department, Colorado State University, Fort Collins, CO, USA Alternative splicing is a widespread phenomenon in plants, which is known to be regulated at several levels in response to a variety of conditions. Whereas recent studies have uncovered condition-specific splicing regulators in metazoans, the plant splicing code remains unexplored for the most part. In this work we present a first step towards this goal, which is the elucidation of putative splicing regulatory elements (SREs). Using gene annotations in 9 plant species (the dicots A. thaliana and G. max; the monocots O. sativa, Z. mays, B. distachyon, and S. bicolor; the moss P. patens; and the algae C. reinhardtii and V. carteri) we identify a collection of elements that are likely to function as splicing suppressors and splicing enhancers by comparing retained introns and their flanking exons with the corresponding regions in introns that are excised. By comparing the distribution of six-mers in the two populations of sequences, we identified elements that exhibit statistically significant over-representation. Specifically, we found multiple motifs over-represented in 3' exons of intron retention events across all species, suggesting the existence of conserved exonic splicing silencers (ESSs). In intronic sequences, conserved AU-rich motifs are present in sequences exhibiting intron excision, i.e., they function as intronic splicing enhancers (ISEs). Some of these ISEs are known to be associated with increased exon inclusion in metazoans. It is interesting to note that while we were able to identify putative ISEs and ESSs, we were unable to detect conserved ISSs and ESEs that function in intron retention. In conclusion, the motifs we detected are most likely functional considering their conservation across phylogenetically divergent photosynthetic eukaryotes and their similarity to known SREs in mammals. Abstract No. 115 Global Analysis of Gene Expression and Alternative Splicing in a Splicing Regulator Mutant: Role of SR45 in Thermotolerance Albaqami Mohammed(1,3), Hamilton Michael (2,3), Rogers Mark (2,3), Palusa Saiprasad G.(1), Xing D. (1), Ali Gul Shad (1), Ben-Hur Asa (2), and Reddy Anireddy S.N.(1) 1Department of Biology, Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA 2Computer Science Department, Colorado State University, Fort Collins, CO, USA. 3Contributed equally Serine/arginine-rich (SR) and SR-like RNA binding proteins are key regulators of pre-mRNA splicing. SR45, one of the SR-like proteins, regulates alternative splicing (AS) of pre-mRNAs of SR genes and multiple developmental processes. However, the role of SR45 in global regulation of AS and gene expression is not known. To address this and to gain insights into the mode of action of SR45 in controlling diverse processes we compared the transcriptomes of the sr45 mutant and wild type (WT) using high throughput sequencing. We identified over 1300 differentially expressed (DE) and 454 differentially spliced (DS) genes in the mutant. We detected 309 significant, differential intron retention (IR) events from 237 genes. Of these events, ~85% show higher rates of IR in the WT, suggesting a role for SR45 in promoting IR. In addition, we find evidence for 20% more 5' events and 33% more 3' events in WT, suggesting that SR45 plays a role in both 5' and 3' splice-site selection. The top over-represented Gene Ontology (GO) terms in DE and DS genes in the biological processes namespace are hormonal signaling and response to abiotic stresses including heat and in the molecular functions space is sequence-specific DNA binding


transcription factors (TF) activity, suggesting that AS of TFs likely mediates some functions of SR45. Based on GO enrichment, we analyzed heat stress responses in the mutant. The sr45 mutant was highly sensitive to high temperature (38oC and 45oC) at different developmental stages. Expression and splicing of several TFs and heat shock proteins implicated in heat tolerance are misregulated in the mutant. In addition, SR45 binds to an intron of a heat shock TF. These results suggest that SR45 is a positive regulator of heat stress response and that the expression and splicing of genes involved in heat signaling might mediate SR45 function in heat tolerance. Abstract No. 117 Involvement of Post-transcriptional Regulation of IBR5 in Plant Auxin Response Jayaweera, Thilanka - Department of Biology, Texas State University-San Marcos, San Marcos, TX 78666, USA. Dharmasiri, Sunethra - Department of Biology, Texas State University-San Marcos, San Marcos, TX 78666, USA. Quint, Marcel - Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA. Gray, William - Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA. Dharmasiri, Nihal - Department of Biology, Texas State University-San Marcos, San Marcos, TX 78666, USA. INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5) gene encodes a dual specificity phosphatase that regulates plant auxin responses. IBR5 is predicted to produce two transcripts, At2g04550.1 and At2g04550.3 through alternative splicing. Nevertheless the presence of second transcript, At2g04550.3, has not been proved experimentally. Previously characterized ibr5-1 null mutant shows many auxin related defects such as auxin insensitive primary root growth, defective vascular development, short stature, and defective lateral root development. Studies have shown that ectopic expression of At2g04550.1 transcript rescues some of the mutant phenotypes, but not others suggesting that At2g04550.3 transcript may be involved in regulating at least some developmental processes. Recently, we isolated two new auxin insensitive AtIBR5 alleles, ibr5-4 and ibr5-5. ibr5-4 is a catalytic domain mutant that was isolated through a genetic screening for picloram resistance, while ibr5-5 is a splice site mutant that was isolated as an enhancer of tir1. ibr5-5 produces an mRNA similar to the speculated IBR5 splice variant AT2G04550.3, probably encoding a truncated IBR5 with a functional catalytic domain. Both ibr5-1 and ibr5-4 produce less lateral roots while ibr5-5 does not show defect in lateral root development. Ectopic expression of At2g04550.3 rescues the lateral root defect in ibr5-1 and ibr5-4, but not the auxin resistant primary root growth. These results suggest that alternative splicing of IBR5 pre-mRNA to produce At2g04550.3 is necessary for the normal lateral root development. Additionally, we have shown that IBR5 enzyme activity is regulated by calcium/calmodulin pathway indicating that auxin responses are modulated through calcium signaling.

Abstract No. 119 FPA, a Regulator of Alternative Polyadenylation, is Closely Associated with Cleavage and Polyadenylation Factors in vivo Zacharaki, Vasiliki, (1), Rataj, Katarzyna, (1), Simpson, Gordon, (1), (2) (1)School of Life Sciences Research, University of Dundee, Dundee, UK (2)James Hutton Institute, Invergowrie, Dundee, UK Alternative cleavage and polyadenylation influences the coding and regulatory potential of mRNA and where transcription termination occurs. Although widespread, few regulators of this process are known. First identified because of its role in flower development, the Arabidopsis thaliana spen family protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. The characterization of FPA therefore provides an opportunity to reveal novel mechanisms by which poly(A) site selection can be controlled. Here we show that FPA


is closely associated with core components of the cleavage and polyadenylation apparatus in vivo. To understand how FPA might function, we developed a novel proteomics procedure based on the crosslinking of native protein interactions in living cells using formaldehyde. We then used statistical analysis of multiple biological replicates to compare proteins co-purified with FPA and the well-studied splicing factor U2B? as a control. We found that U2B? associates with known components of the U2snRNP and spliceosome as expected, but found little overlap between these proteins and those associated with FPA. Most proteins associated with FPA had domains connected to RNA processing and several had previously been identified as components of the same genetic pathway controlling flower development as FPA. Strikingly, almost all core components of the cleavage and polyadenylation machinery co-purified with FPA. We have substantiated these data with genetic analyses, revealing for example, that the function of FPA in promoting flower development requires a protein related to the cleavage, polyadenylation and termination factor Pcf11. Experiments designed to identify which of these co-purified proteins FPA interacts with directly are now in progress. These findings establish a simple, generally useful procedure for label-free in vivo proteomic analysis of proteins involved in RNA processing. In addition, they suggest that the mechanism by which FPA regulates poly(A) site choice involves close, and possibly direct, association with the cleavage and polyadenylation machinery. Alternative cleavage and polyadenylation influences the coding and regulatory potential of mRNA and where transcription termination occurs. Although widespread, few regulators of this process are known. First identified because of its role in flower development, the Arabidopsis thaliana spen family protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. The characterization of FPA therefore provides an opportunity to reveal novel mechanisms by which poly(A) site selection can be controlled. Here we show that FPA is closely associated with core components of the cleavage and polyadenylation apparatus in vivo. To understand how FPA might function, we developed a novel proteomics procedure based on the crosslinking of native protein interactions in living cells using formaldehyde. We then used statistical analysis of multiple biological replicates to compare proteins co-purified with FPA and the well-studied splicing factor U2B? as a control. We found that U2B? associates with known components of the U2snRNP and spliceosome as expected, but found little overlap between these proteins and those associated with FPA. Most proteins associated with FPA had domains connected to RNA processing and several had previously been identified as components of the same genetic pathway controlling flower development as FPA. Strikingly, almost all core components of the cleavage and polyadenylation machinery co-purified with FPA. We have substantiated these data with genetic analyses, revealing for example, that the function of FPA in promoting flower development requires a protein related to the cleavage, polyadenylation and termination factor Pcf11. Experiments designed to identify which of these co-purified proteins FPA interacts with directly are now in progress. These findings establish a simple, generally useful procedure for label-free in vivo proteomic analysis of proteins involved in RNA processing. In addition, they suggest that the mechanism by which FPA regulates poly(A) site choice involves close, and possibly direct, association with the cleavage and polyadenylation machinery. Abstract No. 120 Modulation of Alternative Splicing by Long Non-coding RNAs F. Bardou, F. Ariel, C. Sorin, A. Christ, C. Simpson, J. Brown, C. Lelandais, C. Hartmann and M. Crespi - CNRS Institut des Sciences du Végétal, SPS Saclay Plant Sciences, 91198 Gif-sur-Yvette, France ([email protected]) - Division of Plant Sciences, University of Dundee, the James Hutton Institute,Invergowrie, Dundee, UK


Non-protein coding RNAs (npcRNA) represent an emerging class of riboregulators, which act either directly in a long form or are processed to shorter miRNA and siRNAs. Plant and animals use small RNAs (microRNAs and siRNAs) as guide for post-transcriptional mRNA cleavage or translational inhibition and also epigenetic regulation in the nucleus. We have performed genome-wide bioinformatic analysis of transcriptomic databases and identified many Arabidopsis long npcRNAs. Eleven npcRNAs were antisense to protein-coding mRNAs whereas certain corresponded to miRNA or siRNA precursors. The expression pattern of a set of long npcRNAs in response to phytohormones and environmental stresses suggested a link with root growth and development. Interestingly certain 24nt siRNAs derived from these loci accumulate under the same conditions, suggesting an epigenetic link. Functional studies with one long npcRNA revealed unexpected relationships with alternative splicing (AS) during root development. Indeed, this npcRNA interacts with a novel class of AS regulators in Arabidopsis thaliana, the AtNSR proteins (for Nuclear Speckle RNA-binding protein). RNA immunoprecipitation assays demonstrate that AtNSRs interact with their mRNA targets and with the long npcRNA, nc351. The over-expression of nc351 in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Our results show that nuclear alternative splicing regulators can be hijacked by long npcRNA, to modulate alternative splicing patterns during development. Abstract No. 123 microRNAs Inhibit the Translation of Target mRNAs on the Endoplasmic Reticulum in Arabidopsis Li,Shengben,University of California, Riverside Liu,Lin,University of California, Riverside Zhuang,Xiaohong,Chinese University of Hong Kong Yu,Yu,University of California, Riverside Liu,Xigang,University of California, Riverside Ji,Lijuan,University of California, Riverside Raikhel,Natasha,University of California, Riverside Jiang,Liwen,Chinese University of Hong Kong Chen,Xuemei,University of California, Riverside MicroRNAs (miR) are key regulators of plant development and stress responses. They act by repressing their target genes expression at both the mRNA and protein level. In contrast to the molecular mechanism of miRNA-mediated mRNA cleavage, how miRNAs repress the translation of their target mRNAs, and where this regulation takes place is largely unknown. In our study, we found that ALTERED MERISTEM PROGRAM1 (AMP1) represses miRNA target protein synthesis without affecting miRNA accumulation and target mRNA cleavage. AMP1 is an integrated endoplasmic reticulum (ER) protein that interacts with AGONAUTE1 (AGO1), the major miRNA effector and a peripheral ER protein in Arabidopsis. Polysome analyses revealed that AMP1 largely affected the association of microRNA target transcripts with membrane-bound polysomes but not total polysomes. These results demonstrate that translation inhibition is an important function of plant microRNA, and the ER provides sub-cellular location for this regulation. Abstract No. 132 Environmental Stress and Pathogen Challenge Regulate Unproductive Alternative Splicing of the Plant Circadian Clock Genes Filichkin, Sergei, Oregon State University, Corvallis, Oregon, 97331, USA. Cumbie, Jason, Oregon State University, Corvallis, Oregon, 97331, USA. Chang, Jeff, Oregon State University, Corvallis, Oregon, 97331, USA. Jaiswal, Pankaj, Oregon State University, Corvallis, Oregon, 97331, USA. Dharmawadhana, Palitha, Oregon State University, Corvallis, Oregon, 97331, USA. Megraw, Molly, Oregon State University, Corvallis, Oregon, 97331, USA. Mockler, Todd, Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA. Several genes of the central plant circadian clock regulator undergo unproductive alternative splicing (AS), generating nonsense isoforms. We investigated alternations in


patterns of unproductive AS among the key components of the central oscillator that are driven by environmental stress and pathogen challenge. Under normal physiological conditions the fluctuations in the levels of the long intron retaining (IR) isoform of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) transcript were synchronized with oscillations of its constitutively spliced counterpart. In contrast, broad temperature fluctuations shifted relative ratios of the correctly spliced CCA1 mRNA towards IR nonsense transcript. Similar ratio shifts in alternatively spliced CCA1 isoforms were induced by different stress treatments such as thermo stress, high intensity light, etc. For example, poplar CCA1/LHY homolog responded to drought by increase in abundance of the nonsense transcript. A sharp increase of PTC+ CCA1 transcripts was also triggered by Pseudomonas syringae infection. Transcriptome analysis suggested that the expression and the AS patterns of several other key circadian genes were altered in the infected Arabidopsis leaves. During the course of infection the PTC+ mRNA of CCA1 accumulated in both the wild type plants and in the nonsense mediated mRNA decay (NMD)-impaired upf1-5 mutant. In contrast to other PTC+ circadian transcripts, the CCA1 mRNA did not trigger an NMD response. Conversely, inclusion of the early PTC into the REVEILLE2 (RVE2) transcript via a poison cassette exon event elicited a strong NMD response. The temperature modulation changed oscillating patterns of both CCA1 and RVE2 PTC+ isoforms. Our results suggest that both abiotic stress and pathogen infection could induce drastic shifts in production of nonsense isoforms. We propose a hypothesis that unproductive AS is a fine tuning mechanism regulating expression of several key circadian transcripts when rapid temporal adjustments in cyclical abundance of their protein coding mRNAs is required. Abstract No. 133 Modulation of PPI-ASE Activity By RNA Targets of ATCYP59 Implies a Novel Layer for Transcription Regulation Olga Bannikova1, Marek Zywicki2, Yamile Marquez1, Tatsiana Skrahina1, Maria Kalyna1 and Andrea Barta1 1. Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria 2. Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and RNomics, Innsbruck, Austria AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an RRM domain. Experiments in Arabidopsis as well as with the S. pombe ortholog have shown that these proteins bind to the C-terminal repeat domain (CTD) of RNA polymerase II and to influence transcription significantly. As the RRM domain is evolutionarily highly conserved and important for activity we employed a genomic SELEX method which is an unbiased approach to select potential RNA binding partners. Analysis of the selected RNAs revealed an G/C rich RNA binding motif with a preferential occurrence in exons .The binding was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Interestingly, genome-wide analysis showed that the consensus motif was present in at least 70 % of the annotated transcript and we show that this RNA motif is evolutionarily highly conserved. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Taken together, the available data suggest that this type of cyclophilins have an important function in transcription regulation.


POSTER ABSTRACTS

Abstract No. 2 Quantitative Analysis of the Complementarity Requirements of Plant MicroRNA-Target interactions Liu,Qikun*, Wang,Feng, Axtell,Michael J., Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 It is widely accepted that plant microRNAtarget interactions usually require a high degree of complementarity to trigger target cleavage. Since slicing is not necessarily the only path following microRNA target recognition, the complementarity requirements for target downregulation through the nonslicing processes remains largely unknown. On the other hand, in animals, microRNA function only requires limited complementarity and is usually followed by slicing independent downregulation. We are using dualluciferase reporters in Agro-infiltration based transient assays to systematically examine the complementarity requirements for microRNA function in plants. A group of base pairing patterns was designed specifically against plant microRNAs, such that effects of positioning ORF vs. 3UTR and complementarity on microRNA function were quantitatively monitored at both the mRNA and protein level. We found that base pairing patterns that are typical of the known configuration of animal microRNAtarget duplex did not trigger any detectable level of target downregulation. Switching the target site from the ORF to the 3UTR had no significant effect of target efficacy at either the mRNA or protein levels. Changes of the mRNA accumulation accounts for almost all observed regulatory effects. Comparison among those naturally occurring targets indicated that mismatches located near the miRNA 5ends were more disruptive to miRNA function than those occurred near miRNA 3ends. We also found that certain naturally occurring targets with unpaired bases only present at the miRNA 3ends had stronger strength than perfectly paired sites. Up to 3 mismatches could be tolerated at the 3end without affecting the target efficacy. However, extending the mismatched region to five at the 3end totally abolished target site function. Overall, our results indicate that the complementarity threshold required for miRNA function in plants is indeed more stringent than that in animals. This work was supported by NSF grant 1121438 to MJA. Abstract No. 3 Identification of Potential Effector for the Small Regulator Rab5 Nagamine, Ai, Institute of Biological Chemistry in Washington State University, Institute of Biological Chemistry Clark 299 PO box 646340 (for mailing), Pullman WA 99164-6340, United States; Kumamaru, Toshihiro, Faculty of Agriculture in Kyushu University, Fukuoka city, Fukuoka 812-8581, Japan; Satoh, Hikaru, Faculty of Agriculture in Kyushu University, Fukuoka city, Fukuoka 812-8581, Japan; Okita, Thomas W., Institute of Biological Chemistry in Washington State University, Institute of Biological Chemistry Clark 299 PO box 646340 (for mailing), Pullman WA 99164-6340, United States Rab5, the small regulatory GTPase, is widely distributed in eucaryotic organisms including yeast, mammals and plants. In addition to regulating early endocytosis, Rab5 plays a role in endoplasmic reticulum structure dynamics, chromosome congression, spindle stability, nuclear envelope breakdown and lamina disassembly in metazoans cells. In plants, Rab5 also controls the vacuolar transport of soluble cargo proteins from Golgi to vacuoles as shown in rice where a mutation in the Rab5 structural gene, GLUP4, results in the mis-sorting of seed storage proteins (glutelin and globulin). Instead of being transported to the storage vacuole, the storage proteins are secreted which are then subsequently packaged into large aborted endosome-like structures called paramural bodies. Rab5 mutation also


mis-targets glutelin RNAs to the protein body-ER and to the paramural body instead of its normal destination to the cisternal-ER. Rab5 is able to regulate many different processes as it interacts with specific effector proteins. To date, no effectors have been identified for the plant Rab5. To fill this notable gap in our knowledge on understanding the role of Rab5 in protein and RNA sorting, we screened for Rab5 interactive proteins, which might serve as effectors of this regulatory protein. Pulldown assays consisting of GTP-fixed GST-Rab5 (Q70L), GST, or GDP-fixed GST-Rab5 (S25N) were used to capture proteins in crude developing rice seed extracts. Proteins interacting specifically with the GTP-fixed GST-Rab5 (Q70L) were further analyzed by LC-MS. Several putative active-Rab5a interactive proteins from developing rice seeds were identified by mass spectrometry. They were a homolog for Wali7, an aluminum-induced wheat protein; Rab24, a key factor for tubule-ER direction and cell division as well as for vesicle transport in humans; and adenylate kinase, a key enzyme of energy homeostasis . Ongoing studies are being pursued to confirm the interaction of Rab5 by one of more of these potential effectors. Abstract No. 4 Finding a Novel Translationally Regulated microRNA Mutant Song, Charlotte, Charleston Southern University, 9200 University Blvd. Charleston, SC 29406 Aukerman, Milo, DuPont, Wilmington, DE Small RNAs have been found recently to play a major role in the regulation of gene expression in plants and animals. They are small double stranded RNAs that are only 21 – 24 nucleotides long. MicroRNAs (miRNAs) are a class of small RNAs that can act by cleavage of the target mRNA or by translational regulation. In animals, translational regulation is the predominant method used for regulation of the target, but in plants, it was believed to be mainly acting through cleavage. Recently, papers have shown that translational regulation is occurring in plants as well and may be widespread. We were interested in the translational regulation in plants, so an artificial microRNAs to target the chalcone synthase (CHS) transcript was made. Mismatches and loops were introduced into the artificial miRNA to shift regulation of the target from cleavage to translational regulation. We were successful in finding two conditions that were translationally regulated. EMS mutagenesis was done in the translationally regulated transgenic plants to get suppressors of the original phenotype. We were able to identify 969 mutants, but only 419 of these grew past the seedling stage. We have focused on genetically mapping 2 of these mutants and discovering their role in translational regulation. One of the mutants was mapped to the short arm of chromosome 2. Both mutants do not have known miRNA phenotypes, so it is very likely that they are novel mutants. We are continuing this work to characterize the mutants and find what genes are mutated. Abstract No. 6 Functional Characterization of an Abiotic Stress-inducible Transcription Factor AtERF53 in Arabidopsis thaliana En-Jung Hsieh and Tsan-Piao Lin Institute of Plant Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan Correspondence: Tsan-Piao Lin ([email protected]) Tel: 886 2 33662537 Fax: 886 2 23689564 AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) belongs to group 1 in the ERF family and is induced in the early hours of dehydration and salt treatment. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. Taking advantage of the RING domain ligase1/ RING domain ligase2 (rglg1rglg2) double mutant in which the AtERF53 can


express stably, we investigate the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and ABA treatment. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivition activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal apertutre of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expressions of many stress-related genes, such as DREB1A, COR15A, COR15B , PLC, P5CS1, cpHSC70s and proline and ABA metabolic-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, the genetic, molecular and biochemical result might explain how AtERF53 serving as a transcription factor contributes to abiotic stress tolerance in Arabidopsis. Abstract No. 7 The Importance Of Sample Preparation For Down Stream Applications: NGS, Sequencing, Microarray, RT-PCR, MdX, etc. Won-Sik Kim(1), Nezar Rghei(1) and Yousef Haj-Ahmad(1)(2) (1) Norgen Biotek Corp. 3430 Schmon Parkway, Thorold, Ontario, Canada, L2V 4Y6, (2) Brock University, 500 Glenridge Avenue, St. Catharines, ON, L2S 3A1 E-mail: [email protected], 1-866-667-4362 MicroRNAs are endogenous 20 to 24 nucleotide noncoding RNAs that play crucial posttranscriptional regulatory roles in plant and animals. Tremendous efforts are currently being undertaken to understand the profile of the entire miRNA population of a biological sample, which will provide useful information on miRNA activity. Many miRNA discovery tools, including micro arrays and Next-gen-based sequencing, have made it possible to comprehensively and accurately assess the entire miRNA repertoire. This presentation deals with the importance of sample preparation on downstream applications. A prerequisite for obtaining successful results from these approaches is an efficient method for total RNA purification without bias. The choice of the method of RNA purification is critical to the outcome of downstream analysis. This is made more significant in variations of the plant specimens and the high phenolics, starch and other inhibitors co-isolating with the RNA. The three most popular RNA purification methods (spin columns using Silicon Carbide, spin columns employing silica membrane and phenol/chloroform extraction) are compared in terms of quality, quantity and small RNA recovery from difficult and moderately challenging plant samples. Examples of microRNA study cases will be also discussed to highlight the importance of the RNA purification method used for different plant species. Abstract No. 8 Computational Prediction and qPCR Validation of miRNAs in the Fern Ceratopteris richardii Kharonov, Yevgeniy*, Tucker, Edward, Baruch College (CUNY) 17 Lexington Avenue, NY, NY 10010 MicroRNAs and pre-microRNAs were predicted from ESTs available for the fern Ceratopteris richardii. 11 unique pre-miRNAs were predicted from a total of 327,172 EST alignments. 89 unique mature miRNAs were predicted from a total of 76,940 EST alignments. Pre- miRNAs had an Expect value less than to e-12 while mature miRNAs had an Expect value less than e-6. Putative miRNAs were validated with qPCR. The role of several miRNAs in response to plasmolysis was investigated. 8 and 12-day-old C. richardii gametophytes were treated with 1.0 M mannitol for 30 minutes followed by rehydration in dH20. Mannitol/dH2O treatment induced plasmolysis that caused the breakdown of cell-cell adhesions leading to an aberrant


morphology termed the "secondary gametophyte." RNA isolation was performed pre-plasmolysis, 24, 48, and 72 hours post-plasmolysis. miR106b, miR181c, and miR914 and their respective precursors were detected. miR106b and pre-miR106b were up-regulated in both 8 and 12-day-old gametophytes. miR181c and pre-miR181c were up-regulated in both 8 and 12-day old gametophytes. miR914 and pre-miR914 were expressed at a near constant level in both 8 and 12-day-old gametophytes. miR914 is a miRNA conserved from blue-green algae with currently unknown targets. MiR106b-5p a regulator of cyclin-dependent kinase inhibitor 1A (p21/Cip1) was up-regulated post-plasymolysis and may be responsible for the release of cell cycle arrest typical in developing gametophytes. MiR181c was up-regulated and is thought to inhibit several key cell cycle proteins, including transportin1 a nuclear transporter, fidgetin a protein thought to sever microtubule and spindle formation, as well as DDX3X an ATP-dependent RNA helicase important for regulation of apoptotic pathway and G1/S transition of mitotic cell cycle. MiR181c may play a global role in gametophyte reprogramming post-plasmolysis. Cre-miR914 was detected and although it has no known targets, its detection contributes depth to the extent of which miRNA are conserved from alga to plant. Abstract No. 11 Dynamic Regulation of PvALF Mediated Expression of the Phas promoter and it Turning on the Seed Genes in Arabidopsis Sundaram Sabarinath*, Institute of Developmental and Molecular Biology and Department of Biology,Texas A&M University, College Station, TX 77843 3155, USA. Kertbundit Sunee Institute of Developmental and Molecular Biology and Department of Biology,Texas A&M University, College Station, TX 77843 3155, USA. Shakirov V Eugene Institute of Developmental and Molecular Biology and Department of Biology,Texas A&M University, College Station, TX 77843 3155, USA. Iyer m Lakshminarayan National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Building 38A, Room 5N50, Bethesda, MD 20894, USA. Jurícek Miloslav Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 16502 Prague 6, Czech Republic. Hall C Timothy Institute of Developmental and Molecular Biology and Department of Biology,Texas A&M University, College Station, TX 77843 3155, USA. Phaseolin is the most abundant protein stored in the seeds of the French bean, "" Phaseolus vulgaris "". Its expression is dependent on a seed-specific transcription factor, PvALF ("" Phaseolus Vulgaris "" ABI3 ortholog). In nature, phaseolin expression is spatially confined to embryogenesis, being stringently turned off during all other stages of plant development. Previous studies of ?-Phaseolin ("" phas "") illustrated that "" phas "" gene expression occurs in two discrete and sequential steps: potentiation, in which repressive chromatin architecture is remodeled through the activity of PvALF and transcriptional activation, initiated by abscisic acid (ABA). The tightly controlled inducible heterologous PvALF/phas-gus system was developed in Arabidopsis leaves to study the changes taking place during the transit from silent to active state. Using RNA-Seq, we explored the signaling and transcriptional landscape of the phas promoter in a heterologous system that recapitulates the two-step activation. Remarkably, expression of over 2000 genes from 11 functional categories coincided with changes in the transcriptional status of the phas promoter. Further it showed that PvALF is capable of activating native seed-specific genes when it was ectopically induced in vegetative tissues of transgenic plants. Downstream events mediated by PvALF included the expression of Cupin, the Arabidopsis homologue of Phaseolin and also genes that coordinate with PvALF to turn on seed development processes. Using gene network analysis of the chromatin and transcription factors induced in the system, in combination with loss-of-function genetic assays, we established for the first time that PvALF recruits novel transcription factors HBTFL1 and AIL5 to activate phas expression. The


comprehensive data obtained from RNA-Seq survey of genome-wide expression, coupled with the functional analysis of candidate genes, can provide a new insight to the functional pathway of epigenetic regulation of gene expression and its interactions with the seed specific B3 TFs utilize transcriptional networks to induce chromatin remodeling and, hence, tissue-specific gene expression. Abstract No. 12 Computational Methods for Prediction of Tertiary Structures of microRNAs and Ago-MiRNA Ribonucleoprotein Formation Author: Maxat Askarbekov, Yevgeniy Kharonov, and Edward B. Tucker Baruch College (CUNY), Natural Science Department, 17 Lexington Ave, New York City, NY, 10010. MicroRNAs are small regulatory RNAs (22nt) that are derived from non-protein-coding genes and function as posttranscriptional regulators of gene expression. MiRNA genes are processed into mature miRNA that are incorporated into the miRNA-induced silencing comlex (RISC). MiRNA guide RISC toward the mRNA that is targeted for repression. The design of computational methods for RISC formation and activity are preliminary and challenging. Typically, two-dimensional microRNA secondary structures are predicted and their formation energies are analyzed. These studies are limited in scope and application. Three-dimensional (3D) tertiary structures have the advantage of providing a more accurate representation of RNA-protein complexes. However, the accurate modeling of 3D structures is still a difficult process. In this study we used complex computational methods, including 2D and 3D folding prediction (Mfold, UNAFold, Chimera, Glide), nondeterministic polynomial complex problems (HHpred, RAPTOR), as well as kinetic and thermodynamic property analysis in a step-wise process to model miRNAs that were predicted to exist in the organism Ceratopteris richardii. We were able to successfully model miRNA in association with the protein Argonaute, a member of RISC. We were able to predict structures that are very close to known crystal structures by using the energy values of RNA recognition motifs within the ribonucleoprotein complex as well as electrostatic and H-bond interactions that were characterized. This study is essential for further accurate research of RISC and their mechanism of action. Abstract No. 13 Silencing Vital Genes of Bemesia Tabaci Using RNA Interference Approach and the Potential use of Technology in Development of Transgenic Crops Verma Praveen Chandra*, Thakur Nidhi, Upadhyay Santosh Kumar, Chandrashekar Krishnappa, Singh Pradhyumna Kumar Plant Molecular Biology & Genetic Engineering Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001 (India) Tel & Fax: +91-0522-2205698 (Direct); Tel: +91-0522-2297944; Fax: +91-0522-2205836, 2205839 E. mail: [email protected] * Presenting Author Eukaryotic organisms, including insects, possess common machinery for sequence specific gene silencing which is triggered by the presence of double stranded RNA. The silencing caused by small RNAs is called RNA interference in animals and post transcriptional gene silencing in plants. Ds RNA mediated silencing of the essential genes can induce cessation of feeding and ultimately morbidity of insects. However, this requires efficient uptake of dsRNA either by injection or oral delivery. Therefore expression of dsRNA directed against appropriate target genes in transgenic plants can be used for development of insect resistant crops. Artificially synthesised dsRNAs and siRNAs of five important genes - actin ortholog, ADP/ATP translocase, ?-tubulin, ribosomal protein L9 (RPL9) and V-ATPase A subunit were able to cause 29-97% mortality after 6 days of feeding. The most effective amongst these genes was V-ATPase A subunit. Here we report enhanced insect resistance in


transgenic tobacco expressing dsRNA construct- pBI101-dsvatpse A in comparison to wild type tobacco plants. The T2 transgenic tobacco lines showed relatively high expression level of desired transcripts as examined by DOT-BLOT assay and qRT-PCR. These plants showed considerable protection against whiteflies as compared to pBI101-dsASAL control plants. Due to heavy infestation, the control plant drooped while the transgenic plants did not show any considerable damage. Meanwhile, the bioassays conducted with total small RNAs isolated from transgenic and wild type tobacco leaves showed up to 26-45.8% mortality as compared to wild type. This study confirmed that the longer dsRNA were being diced into smaller siRNAs by the endogenous dicer machinery of the plants and supports the earlier evidence of mortality of whiteflies upon feeding on artificially synthesized dsRNA targeting insect-associated genes when mixed with artificial diet. As a further study we are generating and analyzing whitefly resistant v-ATPase-dsRNA-expressing cotton (Gossypium hirsutum) plants for its application in agriculture. Abstract No. 14 SMG1 Functions in the Nonsense-Mediated mRNA Decay Pathway of Plants and is Important for Normal Growth *Lloyd, James, Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom Lang, Daniel, Faculty of Biology, University of Freiburg, Freiburg, Germany Zimmer, Andreas, Faculty of Biology, University of Freiburg, Freiburg, Germany Reski, Ralf, Faculty of Biology, University of Freiburg, Freiburg, Germany Davies, Brendan, Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom The regulation of gene expression is not simply confined to the activity of a promoter but can occur at many stages, including mRNA degradation. Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA decay pathway. It was first characterised as a pathway degrading transcripts with premature stop codons arising from mutations or alternative splicing. However, NMD also directly targets many non-aberrant transcripts and is important for normal growth and development. For example, NMD is needed for a normal response to pathogens in Arabidopsis thaliana and NMD is regulated during mammalian brain development. In animals, it is well known that the kinase SMG1 activates the NMD pathway when a premature stop codon is recognized but no NMD-associated kinase has been characterized outside the animal kingdom. Here we report that SMG1, whilst missing from fungi and A. thaliana, is ubiquitous across the rest of the plant kingdom, functions in the NMD pathway of moss and is needed for normal moss development. An RNA-seq analysis of transcripts regulated by SMG1 in moss revealed that NMD is important for regulating the unfolded protein response and is also involved in the DNA repair pathway. Taken together, we have shown that SMG1 is an ancient kinase, which functions in the NMD pathway in moss. Furthermore, NMD is important for normal moss development. Abstract No. 18 Stress-induced miR-106b Cell Cycle Re-programming in Ceratopteris richardii Gametophyte Cells Edward B. Tucker and Yelena Kozachkova, Baruch College (CUNY), 17 Lexington Ave, New York City, NY, 10010. Email: [email protected] Plasmolysis of fern gametophyte with 1M mannitol for 30 min followed by rehydration resulted in reprogramming the cells such that each individual rehydrated cell developed into a new gametophyte. Each differentiated gametophyte cell became an undifferentiated stem cell. While the pattern of division in the preplasmolyzed developing gametophyte is controlled via cell-to-cell communication through the plasmodesmata, after plasmolysis, plasmodesmata are disrupted and these cells are reprogrammed into spore-like cells. The plasmolyzed/rehydrated gametophyte develops into a plant that looks like a head of leaf


lettuce, with each leaf being a complete gametophyte derived from one of the cells of the original prothallus. We isolated total RNA from fern gametophytes: (a) before plasmolysis and (b) 24, 48 and 72 hrs after plasmolysis/rehydration and had System Biosciences complete qPCR profiling, using OncoMir array analysis. There were many homologies between fern and human miRNAs, and of the 100 miRNAs analyzed, the expression of 23 trended down and 2 trended up. miR-106b decreased from 123.2 to 0.79 and miR-108c from 879.2 to 20.9 (standard U6 snRNA = 1) in the 48 hr rehydrated gametophyte. Supported by PSC-CUNY Abstract No. 19 Long-term N Fertilization Alters Overall Bacterial Community Structure and Abundance of N Cycling Bacterial Gene Copies at Harvard Forest, MA, USA Turlapati Swathi 1,Minocha Rakesh 2, Bhiravarasa Premsai1, Sweeney Karen 2, Tisa Louis 3, Thomas William 3,4, Minocha Subhash 1 1Department of Biological Sciences, University of New Hampshire, Durham, NH 03824 2USDA Forest Service, Northern Research Station, Durham, NH 03824 3Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH 03824 4Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH 03824, USA Environmental N deposition from fossil fuel emissions is considered a threat to forested ecosystems worldwide. Soil microbes act as indices of soil fertility and plant growth. The impact of twenty years of annual application of N (ammonium nitrate) on the forest soil bacteria was studied using pyrosequencing of 16S rRNA genes. The abundance of N cycling bacterial genes (amoA, nirS and nosZ) was also investigated using qPCR. DNA was extracted from 30 soil samples (three treatments x two horizons x five subplots) collected from untreated (control), low N-amended (LN; 50 kg ha-1 yr-1) and high N-amended (HN; 150 kg ha-1 yr-1) plots. A total of 1.3 million sequences were obtained through pyrosequencing and processed using QIIME program. Principal coordinate analysis (PCoA) confirmed that bacterial communities differed among soil horizons and between treatments. While 28 to 35% of the total taxa (6936) were common to all three treatments, the rest were specific to one treatment or common to two. Data revealed that the abundance of nitrifying bacteria (amoA gene) was higher only in HN mineral soil as compared to control. Among denitrifying bacteria, while no differences were observed for nirS gene, nosZ gene copies were higher in LN organic soil compared to control. Soil C, N, cation exchange capacity, N03-, NH4+, acidity and soil pH were found to influence the amoA, nirS and nosZ gene abundance in mineral soil. Majority of the amoA clones generated from HN soils corresponded to the genus Nitrosospira. The changes observed in the bacterial community structure may be a cumulative outcome of N-driven soil base cation changes, net changes in aboveground plant productivity, as reported earlier by our and other groups. Future studies must examine the linkages between belowground phylogenetic and functional gene diversity with plant productivity in forested ecosystems. Abstract No. 20 DDM1 Promotes Telomere Maintenance in Arabidopsis thaliana Xie, Xiaoyuan & Shippen, Dorothy, Dept of Biochemistry and Biophysics, Texas A&M Univ, 2128 TAMU, College Station, TX 77843-2128 Deficient in DNA Methylation1 (DDM1) encodes a nucleosome remodeling protein that is essential for maintaining DNA methylation in Arabidopsis thaliana. Hypomethylation in ddm1 mutants leads to increasing transposon activity in the sixth generation of the mutant and ultimately to unresolved DNA double-strand breaks (DSBs). Previous studies also show that DDM1 participates in subtelomeric heterochromatin formation. Here, we examine how


mutation of DDM1 affects telomere length homeostasis and telomerase activity. We found that bulk telomere length remains within the wild type range (2-5kb) until the sixth generation, when it declines precipitously to 1.5-2kb. Telomere shortening was not associated with a sudden drop in telomerase activity. We also found no evidence for chromosome end-to-end fusions in the sixth generation mutants. However, the mutant displayed a significant increase in extrachromosomal telomeric circles. This observation indicates that telomere shortening in late-generation mutants might be caused by deletional recombination, an outcome associated with compromised chromosome end protection. Altogether, these data argue that DDM1 promotes telomere maintenance by restricting telomere recombination. We are currently investigating whether epigenetic modification of subtelomeric DNA by DDM1 contributes to this mode of regulation. Abstract No. 25 Alternative Splicing of Rgh3 Transcripts Regulates Protein Abundance in the Spliceosome Martin Federico 1, Fouquet Romain 1, Fajardo Diego 1, Gault Christine 1, Settles A Mark 1 1 Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL Alternative RNA splicing produces multiple mRNA species from individual genes increasing protein diversity and regulating gene expression. Genome sequencing projects have shown that about 42% to 45% of intron-containing genes in plants are alternatively spliced, but little is known about how alternative splicing is controlled. The rough endosperm3 (rgh3) mutant causes developmental defects that are either seed or seedling lethal. Rgh3 encodes a U2AF35 related protein (URP), which is a predicted RNA splicing factor. U2AF35 proteins identify splice acceptor sites during RNA processing and function through protein-protein interactions by creating complexes with U2AF65 and other Serine/Arginine rich-proteins. Semi-quantitative RT-PCR analyses of alternatively spliced genes showed that rgh3 affects splicing in a subset of genes supporting a role for RGH3 in alternative splicing. Rgh3 is alternatively spliced, producing at least 19 different spliced variants. Interestingly, only one variant is predicted to encode a full-length URP ortholog containing an N-terminal acidic domain followed by two zinc fingers flanking a UHM domain and a C-terminal RS-like domain. Several Rgh3 splice variants produce truncated proteins missing one to several domains. GFP fused to full-length RGH3 localized to the nucleolus and nuclear speckles. Moreover, GFP fused to the endogenous truncated protein variants showed that while the acidic domain contains a nuclear localization signal, the RS-like domain enhances nuclear localization and is also important for protein recruitment to nuclear speckles. The UHM domain is a modified RRM domain that allows protein-protein interaction and in RGH3 it enables co-localization with U2AF65. Bi-molecular fluorescence complementation assays proved that U2AF65 interacts with U2AF35 as well as with RGH3. These results indicate that RGH3 participates in the U2-type spliceosome and suggest that its function is regulated by alternative splicing by creating truncated protein variants that are excluded from the spliceosome. Abstract No. 26 A Phosphorylation on T218 is Required for the Distinct Function of SR45.1 in the Regulation of Flower Petal Development Zhang, Xiao-Ning, Saint Bonaventure University, Saint Bonaventure, NY 14778, USA. Mo, Cecilia, Saint Bonaventure University, Saint Bonaventure, NY 14778, USA. Mount, Stephen, University of Maryland, College Park, MD 20742, USA. Cooper, Bret, USDA-ARS, Beltsville, MD 20705, USA.


RNA splicing is a crucial step in the production of meaningful mature messenger RNAs (mRNA) in eukaryotic cells. We have previously described isoform-specific phenotypes associated with SR45, which encodes a protein with roles in spliceosome assembly. SR45 is also orthologous to RNPS1, a peripheral component of the exon-exon junction complex (EJC) which functions in multiple ways to assure the quality and availability of mRNAs in the cell. Thus, SR45 is in a unique position to couple mRNA splicing events with the EJC. SR45 produces two alternatively spliced isoforms, SR45.1 and SR45.2, which differ by only 21 nucleotides, making SR45.1 seven amino acid residues longer than SR45.2. Although these isoforms are expressed similarly, they seem to play distinct roles in a tissue-specific manner. Overexpression of SR45.1 and SR45.2 separately in sr45-1 suggests that SR45.1 restores normal flower petal development while SR45.2 restores normal root growth. Here, we further investigate the difference between these two isoforms. We hypothesize a phosphorylation modification on either threonine-218 (T218) or serine-219 (S219) within the seven amino acids unique to SR45.1. Immunoprecipitation of SR45.1:GFP protein from transgenic plants and analysis of its phosphorylation pattern by mass spectrometry confirms that there is only one phosphorylation event at either T218 or S219. Genetic experiments silencing or mimicking the phosphorylation event for these two residues, shows a correlation between a phosphorylation on threonine-218 and normal petal development. A kinase, AFC2, has been shown to interact with SR45 in vitro. The inability of SR45.1 to affect floral development in an afc2 mutant suggests that AFC2 may be required for phosphorylation of T218. Abstract No. 27 Visualizing the Sub-cellular Location of mRNAs Xu, Muyun, Barrero, Jose, White, Rosemary, Gubler, Frank, Helliwell, Chris, CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia After transcription of an mRNA there are many post-transcriptional processes that can act to affect the expression of the protein it encodes. The transport and storage of mRNAs are two of these processes. mRNAs can be assembled into granules by association with a wide range of RNA binding proteins. These granules can be transported to specific cytoplasmic destinations or be associated with processing or storage of mRNAs and hence regulate the activity of the mRNA. To visualize the sub-cellular locations of mRNAs in plant cells we are using the MS2 and ?N22 aptamer-tagging systems which were developed for animal systems but have been modified for use in plants (Schönberger et al., 2012). In these systems fluorescently-tagged RNA binding protein binds to an mRNA to which an RNA aptamer has been added. We have been testing the efficacy of these systems in planta using control RNAs and a PROFILIN mRNA (previously shown to be localized at the tip of root hairs in maize). We have also tagged a number of seed-expressed Arabidopsis RNA binding proteins (mainly from the PUMILIO-like family) with GFP to determine their sub-cellular locations, and will use these for RNA immunoprecipitation to identify the associated RNAs. We are now using the system to characterize mRNAs expressed at various stages of seed development to understand how post-transcriptional regulation contributes to the expression of these genes. Reference: Schönberger J, Hammes UZ and Dresselhaus T. (2012) In vivo visualization of RNA in plants cells using the ?N?? system and a GATEWAY-compatible vector series for candidate RNAs. Plant J, 71, 173-81.


Abstract No. 35 A Rice Cis-Natural Antisense RNA Enhances PHO1;2 Protein Level via Translational Control and Contributes to Phosphate Homeostasis and Plant Fitness Jabnoune Mehdi(1)*, Secco David(1),Lecampion Cécile (2), Robaglia Christophe(2), Shu Qingyao(3) and Poirier Yves(1) (1) Department of Plant Molecular Biology, Biophore Building, University of Lausanne, CH-1015, Lausanne, Switzerland; (2) Laboratoire de Génétique et Biophysique des Plantes, UMR 7265, CEA/CNRS, Aix Marseille Université, Faculté des Sciences de Luminy, 163 Avenue de Luminy, Marseille , France; (3) Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, China.. E-mail : [email protected] Cis-natural antisense transcripts (cis-NATs) are widespread in eukaryotes and most often associated with down-regulation of their associated sense genes. Unexpectedly, we found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice. OsPHO1;2, a gene involved in phosphate loading into the xylem, and its associated cis-NATPHO1;2 are both controlled from promoters active in the vascular cylinder. Under phosphate deficiency, expression of both cis-NATPHO1;2 and the OsPHO1;2 protein increased while OsPHO1;2 mRNA level remained stable. Down-regulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in OsPHO1;2 protein, impaired the transfer of phosphate from root to shoot and decreased seed yield. Constitutive over-expression of NATPHO1;2 in trans led to a strong increase of OsPHO1;2 protein, even under phosphate-sufficient conditions. Under all conditions tested, no changes occurred in OsPHO1;2 transcript level or splicing pattern, revealing that cis-NATPHO1;2 positively controls OsPHO1;2 protein accumulation from the sense transcript at the post-transcriptional level. Several experimental approaches were used to decipher the mechanism by which cis-NATPHO1;2 exert its positive effects on OsPHO1;2 protein synthesis. No polypeptide synthesis was detected from potential open reading frames present in cis-NATPHO1;2, indicating that it is most likely non-coding. Modulation of cis-NATPHO1;2 expression was not associated with changes in the nuclear export of OsPHO1.2 mRNA, nor was it associated with editing of the mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of OsPHO1;2 mRNA towards the heavier active polysomes. Together, these data indicate an unexpected role for a cis-NAT in promoting translation of its associated mRNA and provide a novel link between antisense RNA and Pi homeostasis. Abstract No. 36 Characterization of GCN2 in Maize: Analysis of GCN2 Phosphorylation of Eukaryotic Translation Initiation Factor 2a and Identification of Targets of GCN2 Regulation Jia, Mo, Eason, Bethany, Gibbon, Bryan, Department of Biology, Baylor University, One Bear Place #97388, Waco, TX, 76798 General control non-derepressible-2 (GCN2), first discovered in yeast as a regulatory protein kinase, plays an important role in cellular responses to amino acid availability. It phosphorylates the α subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), which in turn decreases the general rate of protein synthesis in response to amino acid starvation. The phosphorylation of eIF2 α enhances the translation of the transcription factor GCN4 by overcoming the inhibitory effect of the GCN4 upstream open reading frames (uORFs), resulting in increased expression of over 500 amino acid synthesis genes. The GCN2-like kinases are highly conserved among eukaryotes, but little is known about GCN2 regulation in plants, especially in maize. Six Mutator insertion mutations of gcn2 were identified, of which gcn2-1 was intensively studied and confirmed as a null mutant by western blot analysis. 5' rapid amplification of cDNA ends (RACE) was used to detect aberrant transcripts in the gcn2-1 mutant. Phosphorylation of eIF2α was detected in


response to herbicide treatment only in wild type maize seedlings, indicating that GCN2 kinase activity was abolished in gcn2-1 mutant. Preliminary sequence analysis indicated Opaque2 (O2) to be the maize homologue of GCN4. O2 protein accumulation was increased after sectioned ears were incubated on media containing 20 ppb herbicide, suggesting that O2 could be under GCN2 regulation. Further sequence analysis that was based on genes predicted to function as transcription factors, expressed in seedling tissues and share the similar multiple uORF structures with GCN4 and Opaque2 indicated other possible GCN2 targets . Abstract No. 37 Analysis of the Arabidopsis AtTCTP2 gene Toscano-Morales,Roberto. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN); Av. Instituto Politécnico Nacional 2508 Col. San Pedro Zacatenco PC 07360 Xoconostle-Cázares,Beatriz. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN); Av. Instituto Politécnico Nacional 2508 Col. San Pedro Zacatenco PC 07360 Ruiz-Medrano,Roberto. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN); Av. Instituto Politécnico Nacional 2508 Col. San Pedro Zacatenco PC 07360 The Translationally Controlled Tumor Proteins (TCTP) are eukaryotic proteins involved in the regulation of proliferation, cell growth, and many other important processes. Arabidopsis harbors two TCTP genes. One of them (AtTCTP1) has been studied in detail, while the other (AtTCTP2) has been suggested to be a pseudogene. We have previously shown that AtTCTP2 is expressed in different organs, albeit at lower levels than AtTCTP1. Furthermore, an AtTCTP2 insertional mutant shows severe defects in growth, indicating that this is a functional gene. The AtTCTP1 transcript levels in this mutant are higher than in wild type plants and thus cannot compensate for the loss of function of AtTCTP2. We had also shown AtTCTP2 expression/localization patterns and quantify its expression levels. We also analyzed the growth kinetics of tobacco calli transformed with AtTCTP2. Results of these experiments will be presented. Abstract No. 38 Vitis Vinifera L. microRNAs and Their Role in Phenotypic Plasticity Lopes Paim Pinto, Daniela, Scuola Superiore Sant'Anna, Inst of Life Sciences, Pisa, Italy. Belli Kullan, Jayakumar,Scuola Superiore Sant'Anna, Inst of Life Sciences, Pisa, Italy. Stephen Horner, David, Univ of Milan, Dept of Life Sciences,Milano, Italy. Meyers, Blake, Univ of Delaware, Dept of Plant and Soil Sciences, Newark, United States. Enrico Pè, Mario,Scuola Superiore Sant'Anna, Inst of Life Sciences, Pisa, Italy. Mica, Erica, Scuola Superiore Sant'Anna, Inst of Life Sciences, Pisa, Italy. Plant species can adjust to different environments and/or climate changes through phenotypic plasticity, a re-programming of the genome in response to the environment.Vitis vinifera L. is one of the most plastic plants known, a single genotype being able to produce berries with different quality, thus different wine characteristics, depending on the microenvironment where it is cultivated. Understanding the molecular mechanisms involved in the interaction between genetic composition and the environment is crucial for modern viticulture, given that wine quality depends on the combination of terroir and cultivar. Focusing on both conserved and lineage-specific miRNAs expressed during berry development and maturation, here we describe results obtained by applying an experimental design in which the 2 varieties Cabernet Sauvignon and Sangiovese, growing in adjacent vineyards, were analyzed in three different environments (Bolgheri, Montalcino and Riccione). A total of 48 small RNA libraries (4 developmental stages, 2 varieties, 3


environments and 2 biological replicates) obtained from small RNA fractions of berries were produced and sequenced. All data were analyzed using a custom made bioinformatics pipeline, coupled with statistical tests to identify differentially expressed miRNAs. We expect that our bioinformatics analyses will allow the identification of both conserved and lineage-specific MIR genes, greatly improving our knowledge on miRNAs involved in berry development and maturation and/or in response to environmental stimuli. Furthermore, our experimental design should facilitate the identification of specific miRNAs and their targets putatively involved in genotype x environment interactions. Abstract No. 39 Methods to Quantify Mature miRNA Levels and Target Cleavage by qPCR Damodaran, Suresh, Department of Plant Science, South Dakota State University, Brookings, SD 57007 Adhikari, Sajag, Department of Plant Science, South Dakota State University, Brookings, SD 57007 Turner, Marie, Department of Plant Science, South Dakota State University, Brookings, SD 57007 Subramanian, Senthil, Department of Plant Science, Department of Biology & Microbiology, South Dakota State University, Brookings, SD 57007 Reliable and accurate quantification of active miRNA levels and cleavage products resulting from their activity is crucial for functional studies. We compared existing methods for miRNA qPCR and developed a novel method to quantify target cleavage. “polyA tailing” and “Hairpin priming” or “stem-loop RT” are two most commonly used methods to generate cDNAs for miRNA qPCR. We reasoned that inhibition of polyadenylation by 2’-O-methylation in 3’ ends of plant miRNAs might render the polyA tailing method less-suited to generate cDNA for plant miRNA qPCR assays. We compared the suitability of these methods to assay miRNAs and show that hairpin priming is better suited than polyA tailing to generate cDNA for plant miRNA qPCR. Secondly, there are no easy methods available to quantify the extent of miRNA cleavage other than the qualitative “modified 5’-RACE” and the global “degradome/PARE” analyses. We developed a novel qPCR method, quantitative amplification of cleaved ends (qACE), to quantify the level of cleaved target molecules resulting from miRNA activity. First, we ligated adapters to cleaved 5’ends of polyA RNA molecules. We then performed qPCR using a forward primer designed from the adapter-cleavage site junction and a gene-specific reverse primer. We used the method to assay cleavage products in miRNA over-expression lines and miRNA deficient lines. We show that the method specifically detects cleavage products and that by calculating the ratio of cleaved vs full-length transcripts for each miRNA target, one can estimate the level of miRNA activity. In summary, we present appropriate qPCR methods for reliable and accurate quantification of active miRNA molecules and their cleavage products. Abstract No. 44 Isolation and Molecular Characterization of a miR169 from Brassica species Kumari G.1, Kusumanjali K. Srivastava PS and Das S.3 1Deapartment of Biotechnology, Jamia Hamdard, New Delhi-110062, 2Deapartment of Botany, University of Delhi, New Delhi110007, 3Department of Genetics, University of Delhi, South Campus, New Delhi – 110021, India E-mail address of [email protected] In plants, miRNAs play various developmental and physiological roles by cleaving its target mRNAs. Drought and high salinity are the most severe environmental abiotic stresses and cause crop losses all over the world. Thus, improving crop water-use efficiency is possible by manipulating miRNAs, such as miR169, to regulate genes responsible for drought stress responses. MiR169 widespread in the plant kingdom and plays important roles in development as well as responses to environmental cues. Brassicas are characterized by an extensive range of morphological diversity in various aspects of plant character and also the


ability to adapt to a wide variety of habitats and environment. We performed in-silico analysis to identify BAC clones harboring miR169 orthologs from Brassica genome. Our analysis of miR169, the regulator of NFYA5 from Brassica genome revealed the existence of paralogous sequences, arisen as a result of duplication events. The mature 21 nt miRNAs encoded by the paralogous segments however remains unchanged. We have isolated, cloned and sequence characterized miR169 members from B. juncea, and B. rapa, B and revealed the sequence polymorphism between the species. Molecular characterization of miR169a in Brassica will allow us to understand their role in modulating stress responses, growth and development processes and also in evolution of diversity. Abstract No. 45 Small RNAs Show an Association to Heterosis for Grain Yield in a Maize Breeding Population Seifert, Felix, University of Hamburg, Biocenter Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststrasse 18, 22609 Hamburg, Germany; Thiemann, Alexander, University of Hamburg, Biocenter Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststrasse 18, 22609 Hamburg, Germany; Frisch, Matthias, Justus-Liebig-University Giessen, Biometry and Population Genetics, Heinrich-Buff-Ring 26. 35392 Giessen, Germany; Melchinger, Albrecht E., University of Hohenheim, Plant Breeding, Fruwirthstrasse 21, 70599 Stuttgart, Germany; Scholten, Stefan, University of Hamburg, Biocenter Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststrasse 18, 22609 Hamburg, Germany Heterosis also known as hybrid vigour describes the superior performance of hybrids over their distantly related parents regarding various traits. Although this phenomenon is widely used in animal and plant breeding, and potential genetic mechanisms were defined, its molecular basis is not unveiled. Small RNAs (sRNAs) are hypothesized to contribute to the formation of heterosis by maintaining genome integrity and gene regulation by various mechanisms. Such mechanisms are silencing of repetitive regions, regulation of transcription by guiding DNA methylation, and post-transcriptional gene regulation by degradation or translational inhibition Previously published analyses studied the change of small RNA expression in hybrids of two parental inbred lines. These studies show changes in the expression levels of sRNAs in hybrids and motivated us to analyze the association of small RNAs to the formation of heterotic traits in a large population. We are currently performing a study analyzing the sRNA expression of a maize breeding factorial comprising 7x14 inbred crosses and three selected hybrids with different levels of heterosis by sRNA-sequencing. This sequencing data is integrated with existing field data for all hybrids in the factorial as well as transcriptome data of the inbred parents. Initial results show a significant and high correlation of differentially expressed sRNAs between parents with mid parental heterosis of grain yield. The identified sRNAs mainly belong to the class of 22 and 24 nt long sRNAs in agreement with previous studies of other maize genotypes and plant species. Although 24 nt sRNAs are known to mainly arise from highly repetitive regions, the heterosis-associated sRNAs predominantly belong to non-conserved or low copy regions of the maize genome. Abstract No. 47 HESO1 Binds AGO1 to Uridylate miRNAs and 5’ RNA Fragments Generated by AGO1 Cleavage in Arabidopsis Bin Yu, Guodong Ren Meng Xie and Carissa Vinovskis Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588–0660 3’ untemplated uridine addition (uridylation) is a common modification that regulates miRNA stability and activity in animals and plants. How terminal uridyl transferases recognize


miRNAs is poorly understood. Uridylation also occurs at the 3’ termini of the 5’ RNA fragments (5’ fragments) generated by miRNA-mediated target RNA cleavage. The enzyme uridylating 5’ fragment has not been identified. Consequently, the biological significance of 5’ fragment uridylation is unknown. Here, we show that HESO1, a miRNA uridyl transferase in Arabidopsis, uridylates 5’ fragments to trigger their degradation through a mechanism that is likely different from 3’-to-5’ trimming activity. Furthermore, we find that HESO1 interacts with AGO1, a major effector protein for miRNA-mediated gene silencing, in an RNA-independent manner, and is able to uridylate AGO1-bound miRNAs in vitro. Based on these results, we propose that HESO1 binds AGO1 to uridylate both miRNAs and 5’ fragments. Abstract No. 48 Systems Biology Based Analyses of Water Stress Responses in Soybean (Glycine max) Prateek Tripathi, South Dakota State University, Department of Biology & Microbiology, SDSU Brookings South Dakota 57007 Roel Rabara, South Dakota State University, Department of Biology & Microbiology, SDSU Brookings South Dakota 57007 Neil Reese, South Dakota State University, Department of Biology & Microbiology, SDSU Brookings South Dakota 57007 Qingxi Shen, Department of Biological Sciences, School of Life Sciences, University of Nevada, Las Vegas, NV 89119 Paul Rushton,South Dakota State University, Department of Biology & Microbiology, SDSU Brookings South Dakota 57007 We have used systems level approaches to understand the water stress responses in soybean and role of WRKY transcription factors. The number of genes in the WRKY transcription family was determined to generate a comprehensive data set from soybean for further analysis. Soybean plants were grown under hydroponic conditions, removed and subjected to dehydration. Plants underwent dehydration stress as confirmed by measuring total water content, osmotic potential, stomatal conductance and phytohormone level. We found that drought intensity ranged from severe to extreme, as confirmed by rehydration experiments. Transcriptome analyses using a custom made NimbleGen oligoarray containing all gene models from the Glyma1.0v of the soybean genome showed that over 3000 genes and 25% of soybean WRKY genes are up regulated at least 8-fold in either root or leaf tissue. Promoters of up-regulated genes were enriched with ABRE-like motifs and two novel motifs. Metabolite analyses revealed a flow of nitrogen into amino acid metabolism and an elevation of amino acids due to de novo biosynthesis. This was confirmed by proteomics studies performed using Shotgun Mass-spectrometry (MuDPIT) and it constitutes a potential dehydration tolerance strategy. Strikingly, coumestrol an isoflavonoid was a prominent metabolite found to accumulate over 100-fold in roots during dehydration. This led to our hypothesis that increases in coumestrol level might promote dehydration-induced reactive oxygen species scavenging. Promoter activity of GmWRKYA and GmWRKYB in soybean hairy roots and GmWRKYC in tobacco BY-2 cells suggests that WRKY gene activation during water stress is largely ABA-dependent. Presence of MeJA-like elements in the promoter region of GmWRKYD as revealed by PLACE database signifies its potential as a suitable candidate for understanding cross talk. These results provide many novel insights into the roles of WRKY transcription factors during water stress in soybean and provide valuable inputs towards a systems-wide understanding of water-stress signaling. Abstract No. 50 A Novel Type of Dicer-Like Gene Affects Heterochromatic siRNA Accumulation in Physcomitrella Patens Coruh, Ceyda, Penn State University Cho, Sung Hyun, Penn State University Shahid, Saima, Penn State University Axtell, Michael, Penn State University


In eukaryotes, microRNAs (miRNAs) and short-interfering RNAs (siRNAs) exert RNA-mediated gene silencing, a phenomenon involved in viral resistance, gene regulation, and genome maintenance. In flowering plants, most endogenous small RNAs are heterochromatic siRNAs that emanate from repeats and intergenic regions. The Arabidopsis heterochromatic siRNA pathway is well understood, and includes a Dicer family member (DCL3), three Argonautes (AGO4, AGO6, AGO9), an RNA-dependent RNA polymerase (RDR2), and two plant-specific DNA-dependent RNA polymerases, Pol IV and Pol V. The existence of heterochromatic siRNAs outside of flowering plants is unclear, as gymnosperms and the lycophyte Selaginella have been reported to lack heterochromatic siRNAs, but the moss Physcomitrella expresses DCL3-dependent small RNAs with features similar to known heterochromatic siRNAs. Through extensive small RNA sequencing efforts, we have curated a comprehensive set of reference annotations for Physcomitrella small RNA producing genes. We find tens of thousands of highly repetitive, intergenic loci that produce mostly 23-24 nt siRNAs. In addition to the previously reported dcl3 mutants, have also obtained and characterized Physcomitrella rdr2, polIV (largest sub-unit), and polV (largest sub-unit) mutants. Small RNA sequencing and subsequent differential expression analysis confirms that the Physcomitrella homologs have similar roles as those known in Arabidopsis: rdr2 and polIV mutants lose nearly all heterochromatic siRNA accumulation, while dcl3 and polV mutants have much milder effects. We also identified a Physcomitrella "minimal Dicer" (mDCL), which lacks the N-terminal helicase and dsRNA-binding domains typical of most eukaryotic Dicers. Interestingly, mDCL is responsible for 23 nt siRNA production at RDR2/PolIV-dependent heterochromatic siRNA loci, and in the absence of mDCL, 24 nt siRNA production dramatically increases at these loci. Overall, our data demonstrate the presence of a canonical heterochromatic siRNA pathway in Physcomitrella, and provide the first functional characterization of a novel type of Dicer-Like gene in plants. Abstract No. 52 HEN1 Coordinates the Expression of Both Positive and Negative Regulators in Arabidopsis Photomorphogenesis Tsai, Huang-Lung, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Li, Yi-Hang, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Hsieh, Wen-Ping, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Lin, Meng-Chun, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Ahn, Ji Hoon, Creative Research Initiatives, Division of Life Sciences, Korea University, Anam dong 5 ga, Seongbuk-Gu, Seoul 136-701, Korea. Wu, Shu-Hsing, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Light regulates many growth and developmental processes in plants. Plants use multiple photoreceptors to perceive and interpret the light signal inputs. The regulation of plant growth and development by light is achieved by global transcriptome adjustment, translational control and multi-layered post-translational modification of proteins. The transcriptional activation and repression of many light-responsive genes has been well documented, but the impact of post-transcriptional regulation on conveying light signals has been less addressed. Here we show that optimal photomorphogenesis in Arabidopsis requires proper biogenesis of small regulatory RNAs, which play pivotal roles in the post-transcriptional regulation of gene expression. Arabidopsis carrying a mutation in HUA ENHANCER 1 (HEN1), which encodes a methyltransferase required for stabilizing multiple classes of small regulatory RNAs, showed a light-hypersensitive phenotype. This finding indicated that HEN1 is a negative regulator of Arabidopsis photomorphogenesis. We provide both molecular and genetic data to show that light-activated HEN1 expression depends on the functional photoreceptors phyA, phyB, cry1 and a key transcriptional regulator, HY5. We


also demonstrate the involvement of small regulatory RNAs, miR157d and miR319, in moderating the expression of a positive regulator, HY5, and negative regulators, TCPs, respectively, for Arabidopsis to achieve optimal photomorphogenic development. Abstract No. 53 Role Small Regulatory RNAs in Increase of Rape Plants Tolerance to Parasitic Nematode Heterodera schachtii Victoria Tsygankova,Instituteof Bioorganic Chemistry & Petrochemistry, Natl. Acad. of Sci. of Ukraine, Kyiv, Murmanskaya str., 1, 02260, phone (044)558-53-88; E-mail: [email protected] Alla Yemets, Institute of Food Biotechnology and Genomics, Natl. Acad. of Sci. of Ukraine, Kyiv, Osypovskogo str., 2A, phone (044)434-37-77 e-mail: [email protected], Yaroslav Blume, Institute of Food Biotechnology and Genomics, Natl. Acad. of Sci. of Ukraine, Kyiv, Osypovskogo str., 2A, phone (044)434-37-77 e-mail: [email protected] Role Small Regulatory RNAs in Increase of Rape Plants Tolerance to Parasitic Nematode Heterodera schachtii Plant endoparasitic root cyst nematode Heterodera schachtii Schmidt damages sugar beet and oilseed rape plants in world’s developed countries. The annual and cumulative damage to world agricultural production due to nematode infestations are estimated at over 125 billion USD. The modern RNAi-Technology is new and perspective strategy in nematode disease management. The discovery of small regulatory RNAs (si/miRNAs) that play a key role in Posttranscriptional Gene Silencing has allowed to realize gene expression control and to increase plant resistance to these pathogenic organisms. In genetic-engineering experiments we have constructed a vector with constitutive expression of antinematodic dsRNA antisense to the conservative region 8H07 gene Heterodera schachtii and have conducted A. tumefaciens-mediated genetic transformation of rape plants. The expression of antinematodic dsRNA in transgenic rape plants have been confirmed and a high level of their silencing activity (decrease expression of 8H07 gene) have been obtained both in nematodes and in infected plants. In laboratory studies the considerable increase of transgenic rape plant resistance to root parasitic nematode Heterodera schachtii was shown. Abstract No. 54 Changes in Small RNAs Transcripts Levels During Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce Yakovlev Igor A., Norwegian Forest and Landscape Institute, PO box 115, 1431, Ås, Norway Vivian-Smith, Adam, Norwegian Forest and Landscape Institute, PO box 115, 1431, Ås, Norway Fossdal, Carl Gunnar, Norwegian Forest and Landscape Institute, PO box 115, 1431, Ås, Norway Epigenetic memory in Norway spruce permanently affect the timing of bud burst and bud set, vitally important adaptive traits, in this long-lived forest species. Epigenetic memory marks are established in response to the temperature conditions prevailing during early seed formation. Somatic embryogenesis closely mimics the natural processes of epigenetic memory formation in seeds and result in epigenetically different plants in a predictable temperature dependent manner with respect to altered phenology. The main task of the current study is to continue identification and characterization of sRNAs components in the small non-coding part of Norway spruce transcriptomes during formation of epigenetic memory in somatic embryos. To expand our knowledge of these plant gene regulators, we applied high-throughput sequencing methods to Norway spruce. Extensive characterization of small RNAs in Norway spruce during different stages of embryogenesis under different temperature treatments allowed identifying around 3000 ta-siRs, originated from 50 TAS. We showed that Norway spruce poses a variety of ta-siRs with distinct temperature dependent expression patterns, and these ta-siRs target large amount of spruce genes with


a wide range of functions, including also genes involved in epigenetic regulation. We report the identification of 438 novel putative miRNAs in Norway spruce transcribed from158 miR genes and confirming 413 genes as targets for the 99 spruce novel and conserved miRNAs and 120 conserved miRNAs from other tree species and Arabidopsis. Based on identified miRNAs we studied their expression patterns during different stages of SE in dependence on the epitype inducing temperature prevailing during SE growth and leading to establishing of epigenetic marks. We showed that Norway spruce sRNAs originating from the same loci varying considerably by quantity and length. 24-nt ta-siRs were prevailing among smRNAs, followed by 21-nt ta-siRs. Unknown mechanisms provide fine-tuning of sRNAs pool content participating in the epigenetic memory. Deep characterization of sRNAome changes is undergoing. Abstract No. 58 Identification and Analysis of Ice Plant ((Mesembryanthemum crystallinum ) microRNA by High-Throughput Sequencing and Their Association with Stress Response Chih-Pin Chiang, Hung-Cheng Hsieh, and Hungchen E. Yen Department of Life Sciences, National Chung Hsing University, 250 Kuo Kwang Rd., Taichung 40227, Taiwan. E-mail:[email protected] Salinity is one of the major abiotic stresses that affect plant growth and development worldwide. Ice plant (Mesembryanthemum crystallinum L.), this halophyte has unique features for tolerating high saline environments. The expressions of several salt-induced genes of ice plant have been identified and the corresponding proteins have been characterized their roles in the mechanism of salt tolerance. However, the detailed transcriptional and post-transcriptional gene regulation of ice plant was still lacking. MicroRNAs (miRNAs) are endogenous 20 to 24-nucleotide small RNAs (sRNA) that modulate pivotal events in plant development and stress responses by transcriptional or post-transcriptional gene silencing. High-throughput sequencing provides the means to characterize and profile quantitatively small RNAs in diverse plant species, including Arabidopsis, rice, maize, barely, peanut, olive, and others. Here, we used high-throughput sequencing and computational analyses in an attempt to identify the miRNA population in ice plant. Two sRNA libraries of control and salt-treated ice plant seedlings were sequenced and totally 270 conserved miRNAs belonging to 38 families were detected. In addition, the stem-loop precursor structures of 19 conserved miRNAs and 24 novel miRNAs were identified from the ice plant transcriptome. Most of them paired with the star sequences, with two exceptions. Furthermore, we predicted potential targets of these putative miRNAs based on a sequence homology and subjected to GO analysis. Our results suggested that expression of miRNAs and their predicted targets would be useful in exploring the significance of the post-transcriptional gene regulation of ice plant under salt stress. Abstract No. 59 Exploring Messenger RNA Ribonucleoprotein Complexes and Their Functions in Plants Hummel, M.1, Sorenson, R.1, Juntawong, P.1, Oki, J. 1, and Bailey-Serres, J1. 1. University of California Riverside, Botany and Plant Sciences, Center for Plant Cell Biology (CEPCEB), Riverside, CA, 92521 Post-transcriptional regulation of mRNA stability and translation is important for plant development and stress survival. RNA binding proteins (RBPs) play key roles in these processes, which often take place in heterogenous mRNA-ribonucleoprotein complexes (mRNPs). The aim of the NSF Arabidopsis 2010 ?mRNPome Project? is to assign functions to poorly characterized RBPs of Arabidopsis. Representative members of several families of RBPs have been targeted to evaluate cellular localization, mRNA targets, and biological


function. Poly(A) binding proteins (PABPs) bind to the 3? polyadenylated tail of mRNAs to provide stability and enhance translation. PABPs however also bind an A-rich region within their own 5? untranslated region, which may regulate their translation. Arabidopsis encodes eight PAB genes. A survey of PAB T-DNA insertion mutants identified pab2-1 with reduced and pab2-2 with increased PAB2 mRNA abundance, respectively. pab2-2 seedlings are sensitive to germination on sugar, abscisic acid and paclobutrazol, a gibberellin biosynthesis inhibitor. PAB8 mRNA also accumulates in pab2-2 and PAB2 overexpression seedlings, suggesting positive regulation by PAB2 of other PABs. Based on comparison of total and polysomal mRNA, the elevated PAB2 and PAB8 mRNAs are actively translated. Western blot analysis confirmed that pab2-2 accumulates high levels of a PAB that has altered electrophoretic mobility. There was no change in the proportion of PAB2 mRNA in total and polysomal mRNA populations in the pab2-2 as compared to Col-0, indicating that high levels of a modified PAB did not affect PAB2 mRNA translation. Polysome analysis in combination with a PAB immunopurification procedure will be used determine if PAB2 levels affect mRNA translation in Col-0, pab2-2 and 35S:PAB2-mRFP lines. To date, our results indicate that differential accumulation of PAB2 impacts accumulation of PAB and other mRNAs. This project is funded by National Science Foundation, MCB-1021969 and MCB-1022435, ?Collaborative Research: Arabidopsis 2010: Deciphering mRNP Networks?. Abstract No. 61 Investigating the Role of Histone H2A.Z Abundance in Modulating Responses to Phosphorus- and/or Iron-deficiency Zahraeifard, Sara, RamanaRao, Mangu V. , DiTusa, Sandra F., Joshi, Rohit, Bedre, Renesh, Baisakh, Niranjan, and Smith, Aaron P. Department of Biological Sciences, Louisiana State University The scarcity of phosphorus (P) and iron (Fe) in soil renders them as important limiting factors in productivity of crops. Modulation of plant response to fluctuation of P and Fe levels is achieved by means of global transcriptional networks comprised of transcription factors and signaling components that regulate downstream genes involved in nutrient uptake, assimilation, and utilization. In spite of the significant role of chromatin structure in controlling gene expression in eukaryotes, the relevant mechanisms involved in regulating P and Fe transcriptional networks have not been investigated so far. We are interested in identifying the chromatin-level mechanisms involved in regulating the uptake, assimilation, and utilization of P and Fe homeostasis in rice. Recently, it was shown in the case of Arabidopsis that the replacement of the H2A.Z histone variant for the canonical H2A histone is necessary for proper expression of many P deficiency response genes. It was shown that mutation of Arabidopsis ARP6 disrupts H2A.Z deposition at a number of P homeostasis genes, resulting in their de-repression as well as mis-regulation of a number of Fe homeostasis genes. Also, chromatin immunoprecipitation experiments showed that Arabidopsis H2A.Z deposition at some target genes is affected by phosphorus deficiency. We want to investigate the role of H2A.Z deposition in P- and/or Fe-deficiency in rice. To that end, a polyclonal antibody against rice H2A.Z has been made and rice ARP6RNAi transgenic lines have been constructed. As a next step, genome-wide analyses including H2A.Z ChIP-seq of DNA isolated from P- and/or Fe-deficient wild type and ARP6RNAi root tissues, as well as microarrays of P- and/or Fe-deficient RNA samples will be carried out to determine the role of H2A.Z deposition in controlling responses to P and/or Fe deficiency. Accordingly, thorough understanding of the chromatin-level mechanisms that regulate nutrient homeostasis serves to provide new opportunities for developing crop plants with improved nutrient uptake, nutrient use-efficiency, and an enhanced ability to adapt to low-nutrient conditions.


Abstract No. 62 Analysis of Alternatively Spliced mRNA Transcripts in Plant Genes Using a Highly Sensitive Surface Enhanced Raman Spectroscopy Kadam, Ulhas, Horticulture & Landscape Architecture and Agricultural & Biological Engineering, Purdue University, West Lafayette, IN 47907 Schulz, Burkhard, Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN 47907 Irudayaraj, Joseph, Agricultural & Biological Engineering, Purdue University, West Lafayette, IN 47907 We demonstrate a surface enhanced Raman spectroscopy (SERS) technique, which combines a highly sensitive analytical spectroscopy platform with DNA hybridization to identify and quantify direct targets of alternatively spliced genes in plants. Our understanding of the molecular control of many genetic regulatory networks requires the specific identification and quantification of mRNA transcripts in plants. We develop a unique SERS array format to detect splice variants of specific mRNA from oligonucleotides extracted from plant leaf tissues using a sandwich DNA hybridization format of specific sequences related to target splice junction by SERS. Alternative splicing (AS) is a process, which increases the size of the transcriptome and proteome to enhance the physiological capacity of cells. AS of two plant genes, AtDCL2 and AtPTB2 was investigated using non-fluorescent Raman probes under Raman spectroscopy based ‘sandwich assay’. In this approach, using Raman probes conjugated to gold nanoparticles, we showed its capability to recognize nucleic acid sequences specific to DCL2 and PTB2 splice junction variants. This report is the first application of SERS-based approach for detection of alternative splice variants in plants leaf tissues. Further, we show that the detection sensitivity of up to 0.1 fM using non-fluorescent Raman tags is possible. Moreover, we were able to identify and quantify the splice junctions of specific gene targets. This approach could be applicable to study highly sensitive and specific changes in gene expression and alternative isoforms of genes of high significance in plan breeding and agriculture. Abstract No. 63 Characterization of Lactoylglutathione lyase and FHA1 Homolog Genes for Aluminum Stress Sasikiran reddy Sangireddy1, Sarabjit Bhatti1, Ikenna Okekeogbu1, Suping Zhou1 and Roger J. Sauvé1. 1. Department of Agricultural and Environmental Sciences, College of Agriculture, Human and Natural Sciences. Tennessee State University, 3500 John A Merritt Blvd, Nashville, TN 37209 Aluminum (Al) ion is highly cytotoxic to plants at pH levels below 5.0. Plants are continuously faced with Al stresses, which limit their growth and production. Through proteomics experiments, Lactoylglutathione lyase and FHA1 homologs have been identified as being susceptible to Al stress (Unpublished data). Characterization of these genes was achieved by overexpression strategy. The open-reading frame (ORF) region of each gene was amplified from the cDNA clone, and BamH1 and XhoI restriction sites were added at the 5, and 3, termini. PCR products were cloned onto pSAT RNAi vector. After digestion with BamHI and XhoI, the gene fragment was cloned onto a pSAT RNAi vector which was digested with the same pair of enzymes. The ligation reaction was done using the protocol outlined by Promega (Madison, WI). The ligation product was transfected into E.coli competent cells (Genhunter, Nashville, TN), and positive clones were selected on LB plate containing ampicillin (100mg/l) (Sigma-aldrich, StLouis, MO). Plasmid DNA from positive clones was isolated by using a QIAprep® Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced to confirm the insert in the plasmid. The expression cassette (promoter-cDNA insert-terminator) on the pSAT-RNAi vector was isolated with (PI-PSPI) enzyme and sub cloned onto the pRCS2-ocs-bar binary vector. The construct has been transferred into agrobacterium strain, LBA4404. Ten lines of transgenic plants (Money maker) have been


produced through agrobacterium mediated transformation and are being evaluated for Al stress. Keywords: Aluminum Stress, Lactoylglutathione lyase, FHA1 homolog and pSAT RNAi Abstract No. 64 Arabidopsis thaliana VOZ Transcription Factors are Required for Proper Expression of microRNAs Implicated in Development and Stress Responses Abdel-Ghany, Salah E. (1); Celesnik Helena (1); Dolata Jakub (2); Bielewicz Dawid (2); Szweykowska-Kulinska Zofia (2); Reddy Anireddy S.N.(1) (1) Department of Biology. Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA (2) Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland. VOZs (Vascular plant One-Zinc finger proteins) are highly conserved plant-specific transcription factors (TFs). In Arabidopsis, there are two VOZs (AtVOZ1 and AtVOZ2). Recent genetic studies have revealed that they are functionally redundant. Plants lacking both VOZs (voz1 voz2) exhibited several developmental phenotypes including late flowering, delay in transition from juvenile to adult phase, early senescence, and small siliques (Yasui et al., 2012; Celesnik et al., 2013). In addition, the voz double mutant showed increased tolerance to abiotic stresses and enhanced susceptibility to biotic stresses, indicating that VOZs function as both negative regulators of abiotic stresses and positive regulators of biotic stresses (Nakai et al., 2013). microRNAs (miRNAs), a class of short RNAs, are important modulators of gene expression. They have emerged as key regulators of growth, development and stress responses. However, little is known about the TFs that regulate miRNAs expression. As the double mutant of VOZ TFs showed developmental and stress response phenotypes, we analyzed pri-miRNA and miRNA profiles using qPCR and miRNA microarray, respectively, in wild type and voz double mutant plants. The expression of 66% (43 out of 65) and 55% (104 out of 190) of the tested miRNAs and pri-miRNAs, respectively, is significantly changed, mostly downregulated, in the double mutant. Among the upregulated miRNAs, primary as well as mature miR395 is highly upregulated in the double mutant. Both miR395 and VOZs are predominantly expressed in vascular tissues, consistent with a possible direct regulation of miR395 expression by VOZs. A systematic analysis of all the affected miRNAs has revealed that all of them are known to function in developmental phase transition, flowering time, flower development, hormonal signaling and stress responses, suggesting a regulatory link between the VOZs and these miRNAs, and hinting that some of the observed phenotypes in the double mutant may be due to altered levels of miRNAs. Abstract No. 65 Differential Recruitment of Splice Variants Encoding SR Proteins to Polyribosomes During Development and in Response to Stresses Saiprasad G. Palusa1, Julia Bailey-Serres2 and Anireddy S.N. Reddy1 1Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA 2Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California 92521 Precursor-mRNAs (pre-mRNAs) of serine/arginine-rich (SR) proteins in plants are extensively alternatively spliced. We have previously shown that in Arabidopsis about 100 distinct splice variants are generated from 14 SR genes. Furthermore, the splicing pattern of SRs is changed in different tissues and in response to abiotic stresses. About half of the splice variants are potential targets of nonsense-mediated decay (NMD) and encode truncated proteins. Among these, 25 splice forms were confirmed to be real NMD targets.


However, it is not known i) if all splice variants are recruited to polyribosomes (polysomes), ii) if there is a preferential recruitment of specific splice isoforms, and iii) if there is a differential recruitment of splice variants during development and in response to stresses. To address these we analyzed the association of splice variants of all SR genes with polysomes in seedlings, different tissues and in seedlings exposed to heat and cold stress. We monitored spliced SR variants in total RNA and polysomal RNA isolated from immunoaffinity-purified polysomes. In two week-old seedlings, many splice variants (about 40%) are not recruited to polysomes. Among the ones associated with polysomes are all the functional isoforms that code for full-length proteins as well as some candidates of putative and confirmed NMD targets. Fourteen of the 25 confirmed NMD targets are recruited to polysomes. Also, among the recruited splice forms there is preferential recruitment of some over others. Analysis of polysomal-associated splice forms in different tissues also revealed predominant recruitment of functional isoforms along with a few NMD candidates. Furthermore, we observed differential recruitment of isoforms in different tissues. Heat and cold stress dramatically enhanced or reduced recruitment of specific splice variants. Together, our studies reveal differential recruitment of splice variants to polysomes during development and in response to stresses. Abstract No. 66 Deciphering the Transcriptional Network for the Phloem-Mobile RNA StBEL5 Sharma Pooja1 , Lin Tian1, and Hannapel, David J1 1Plant Biology Major, Iowa State University, Ames, Iowa 50011. E-mail:[email protected] StBEL5 is a BEL1-like transcription factor that is expressed in potato and functions as a mobile RNA. BEL1-like transcription factors are members of the TALE superclass that interact with Knotted1-like partners to regulate expression of target genes involved in numerous aspects of plant development. The heterodimer of StBEL5 and POTH1, a KNOTTED1-type transcription factor, binds to a tandem TTGAC-TTGAC motif that is essential for regulating transcription. In potato, StBEL5 and its Knox protein partner regulate tuberization by targeting genes that control growth. RNA movement assays demonstrated that under short days, StBEL5 transcripts originating in the leaf move through the phloem to stolon tips, the site of tuber induction. Recent results also demonstrate movement of StBEL5 into roots correlated with increased root growth. Because of the important role the StBEL5/KNOX complex plays in regulating development, we have focused on putative target genes involved in hormone synthesis and signaling. Using the potato genome as a resource, a search of upstream sequences for the tandem TTGAC motif revealed the presence of the motif in several genes involved in gibberellin, auxin, and cytokinin metabolism. By using mobile StBEL5 in an induction system, distinct expression profiles for StBEL5 target genes in both roots and stolons were compiled. Gel-shift assays are being used to confirm the interaction of the BEL5/POTH1 complex to the motifs present in these target gene promoters. Binding of tandem motifs occurs independent of the linker spacing between core motifs and with all four DNA strand orientations for the two core motifs: head-to-head, tail-to-tail, head-to-tail (same strand), and tail-to-head (same strand). These results suggest that StBEL5 and its partners are involved in a complex developmental network that regulates expression in select organs through the long-distance transport of its mRNA. Abstract No. 68 Genome-wide Analysis of microRNAs Associated with Flower Development in Rice Dong-Hoon Jeong, Sunhee Park, Jixian Zhai, Blake C. Meyers, and Pamela J. Green Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA ([email protected])


MicroRNAs (miRNAs) are non-coding small RNAs that regulate target gene expression at the posttranscriptional level. Plant miRNAs have been identified that contribute to a variety of biological process, including flower development. Functional studies examining Arabidopsis miRNAs have identified several miRNAs that play crucial roles in flower development by repressing key regulatory genes in floral organ identity. Although these miRNAs are well conserved in rice, their role in rice flower development had not been well characterized. To better understand the role of rice miRNAs in flower development, small RNA libraries were constructed from rice floral organs including glume, palea/lemma, lodicule, anther, and pistil as well as panicle tissues from six different stages. We also constructed PARE (Parallel Analysis of RNA Ends) degradome libraries from anther and pistil. Both small RNA and PARE libraries were deeply sequenced by Illumina technology and analyzed to identify differentially regulated miRNAs and to investigate their target cleavage functions. From the small RNA libraries, we identified most miRNAs that were previously reported as well as tens of new miRNAs using a computational pipeline. Among these miRNAs, about 20% were preferentially expressed in a specific floral organ. One of these, miR172, was mainly expressed in anther where the expression of its target gene, APETALA2, is low. In contrast, a new miRNA, which targets the mRNA of a MADS box gene involved in anther identity, was preferentially expressed in pistil. Analysis of PARE data revealed precise cleavage of these target RNAs in the particular floral organ where the corresponding miRNA is expressed. We have identified more than 170 miRNAs that were differentially expressed during panicle development. Of these, some miRNA variants showed differential expression patterns in different stages. Additional studies are underway to investigate the association of floral-organ-preferential miRNAs with the expression of their target genes. Funded by USDA. Abstract No. 69 In vivo Interaction Studies of Viral Proteins Using Negative-stranded Tospoviruses as a Model System Tripathi, Diwaker,Washington State University, Pullman, WA 99164 Dietzgen, Ralf, Queensland Agricultural Biotechnology Center, The University of Queensland, Australia 4072 Goodin, Michael, University of Kentucky, KY 40546 Pappu, Hanu, Washington State University, Pullman, WA 99164 Interaction studies of viral proteins provide important information about their replication in the host plants. These studies further help understand the infection process and develop a molecular strategy to control viral diseases. Using advanced cloning and imaging techniques, we are developing an interaction map of tospoviral proteins. Tospoviruses are economically important viruses as they are involved in billion dollar global crop production. Molecular interactions between cognate proteins of tospovirus species may be of crucial signi?cance for the understanding of pathogenesis, evolution, and development of efficient and durable control strategies for these viruses, which frequently exist as mixed infections in the same host plant. Earlier studies in our lab have suggested that during a mixed infection, Tomato spotted wilt virus (TSWV) turns a restrictive host into a permissive one for another distinct tospovirus, Iris yellow spot virus(IYSV). The mechanism(s) behind this observation are not clear. Here we describe the findings of interaction studies of IYSV and TSWV proteins. Viral genes of both viruses were separately cloned into binary pSITE-BiFC vectors using the Gateway system. Expression clones were agroinfiltrated into a nuclear marker line of N. benthamiana expressing cyan fluorescent protein fused to Histone 2b. Confocal microscopy was used to visualize the fluorescence of the expressed fusion proteins containing two complementary halves of yellow fluorescent protein. Our results suggest that IYSV proteins interact with each other and with their counterparts coded by TSWV in a doubly infected plant. Findings of this research will facilitate a better understanding of function of tospoviral proteins in infected host plants. Developing a common molecular strategy to control mixed pathogen infections in the fields will provide a better alternative to


less efficient chemical control of pathogen damage Abstract No. 71 Diurnal Changes of Polysome Loading Track Sucrose Content in the Rosette of Wildtype Arabidopsis and the Starchless pgm Mutant Liput, Magdalena; Kumar Pal, Sunil; Piques, Maria; Ishihara, Hirofumi; Stitt, Mark; Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1 14476 potsdam In plants, the newly fixed carbon (C) in the light drives growth during the day; while, throughout the night, growth depends on reserves accumulated in the light, such as starch. Unless plants coordinate their growth with diurnal changes in the C supply, they will experience acute C starvation during the night. Protein synthesis is a major component of cellular growth and is an energy-intensive process. We analysed polysome loading in rosettes of Arabidopsis thaliana Col-0 wild-type (WT) and the pgm starchless mutant rosettes during the diurnal cycle, and also in an extended night, and under low CO2 conditions. Polysome loading was high during the light period (60-70%), decreased to 40-45% during the night and to <20% in the extended night. In the pgm mutant, it fell below 25% early in the night. The overall polysome pattern closely tracks changes in sucrose levels and the polysome levels correlates positively with sucrose content. Quantification of cytosolic, plastidial and mitochondrial rRNA species using qRT-PCR, showed that polysome loading was strongly light dependent in plastids, remained high for much of the night in the cytosol and was always high in mitochondria. Total ribosome abundance did not show significant diurnal changes. However, the pgm mutant contained fewer plastidial ribosomes but more mitochondrial ribosomes than WT plants. Modelling the rate of protein synthesis, along with associated energy costs, throughout the diurnal cycle revealed the necessity of having lower polysome levels at night to balance protein synthesis with the availability of carbon from starch breakdown. Our results show: (i) how dynamically polysome loading and, by implication, the rates of protein synthesis change during the diurnal cycle in non-stressed plants; (ii) that polysome loading changes independently in the cytosol, plastids and mitochondria; and (iii) a close coordination of protein synthesis with the momentary C supply. Abstract No. 73 Characterizing the Putative Oligomerization of Plant High-affinity Phosphate Transporters Batista, Elena J., Louisiana State University, 202 Life Sciences Bldg. Baton Rouge, Louisiana 70803 Olivier, Danielle, M., Louisiana State University, 202 Life Sciences Bldg. Baton Rouge, Louisiana 70803 Kato, Naohiro, Louisiana State University, 202 Life Sciences Bldg. Baton Rouge, Louisiana 70803 Smith, Aaron P., Louisiana State University, 202 Life Sciences Bldg. Baton Rouge, Louisiana 70803 Phosphorus is an essential macronutrient taken up by plants in the form of phosphate, a form that is not readily accessible to plants. The sharp concentration gradient existing between the plant and soil exemplifies the importance of genes induced to cope with phosphate starvation, including high-affinity phosphate transporters (Phts). In plants, there is evidence that oligomerization of transporter isoforms is an important aspect of transport function and can confer several structural and functional advantages to monomers, including modifications in allosteric control, affinity, catalysis and stability. In vivo split assays from our lab suggest homomeric and heteromeric interactions occur among Arabidopsis root high-affinity phosphate transporters Pht1;1 and Pht1;4. An area of probable contacts between the monomers has been detected using UCSF Chimera software. Interestingly, amino acid sequences of the putative protein interface of Pht1;1 and Pht1;4 compared with remaining Arabidopsis Pht1 members, yeast high affinity transporters, and


other plant high-affinity phosphate transporters contain a highly conserved region. Preliminary split-luciferase complementation studies suggest this area may participate in monomer-monomer interaction. Results of key residue(s) involved in Pht1;1 and Pht1;4 oligomerization are currently being studied and will be presented. The putative oligomerization of these Pht isoforms may lead to a better understanding of the specificities for phosphate, as well as arsenate, in Phts. In turn, this knowledge could aid in crop development and phytoremediation strategies. Abstract No. 74 Towards a Map of the Landscape of Translational Regulation in Arabidopsis Albrecht G von Arnim, Bayu S Tiruneh, Ju Guan, Anamika Missra, Qidong Jia, Byung-Hoon Kim Department of Biochemistry Cell and Molecular Biology, The University of Tennessee Knoxville, TN 37996-0840, USA We are employing sucrose gradient fractionation and microarray analysis to examine the translation states of Arabidopsis mRNAs. High resolution profiles of ribosome density across the Arabidopsis transcriptome show that most mRNAs have one broad distribution of ribosomes and a minority of mRNAs has distinct populations of untranslated and highly translated molecules. We performed the first global analysis of polysome loading defects in a ribosomal mutant and discovered partial overlap with defects in an initiation factor. We are exploring new experimental conditions to obtain a global view of changes in translation states, for example over the diurnal and circadian light dark cycles. A large fraction of the Arabidopsis transcriptome is subject to diurnal translational control. Meta-analyses of translation regulation are beginning to sort mRNAs into regulons of translational control. For example, a subset of mRNAs for ribosomal proteins routinely stands out as a distinct translational regulon. Moreover, the response to herbicide, which induces amino acid starvation, resembles the response to several other environmental stresses. These analyses are beginning to shed light on the landscape of translational regulation. Abstract No. 75 Post-transcriptional Regulation of Nitrogen Assimilation in Marine Diatoms - the Role of RNA-binding Proteins (1) Perera, Minoli, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 (2) Sohini Ghoshroy, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 (3) Jessica Alexander, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 (4) Deborah Robertson, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 Post-transcriptional regulation of gene expression in eukaryotes is a complex and dynamic process that involves an extensive repertoire of RNA-binding proteins (RBPs). RBPs form dynamic associations with mRNAs, mediating maturation events in the nucleus and regulatory events in the cytoplasm, including mRNA localization, stability, decay, and translation. Diverse RBPs have been shown to interact with functionally related collections of mRNAs, indicating a multilayered and extensive regulatory system functioning at the RNA regulon level. The regulation of nitrogen (N) assimilation in marine diatoms is under tight control. While regulation occurs at many levels, we propose that coordinated post-transcriptional regulation of genes encoding N transporters and assimilatory enzymes allows for rapid metabolic response to perturbations in nutrient source and supply and are mediated by changes in mRNA stability. We hypothesize that changes in transcript stability are brought about by RBPs that combinatorially regulate these functionally related mRNAs, consistent with the RNA operon and RNA regulon hypotheses. We identified several well conserved RBP families in the genomes of three diatoms. We predict that RBPs will selectively bind to the 3’-untranslated regions (UTRs) of N assimilating mRNAs and that


different RBPs will be present under nitrate-replete vs. nitrogen-starved conditions. We cloned and sequenced the 3’UTRs of transcripts for five N assimilating enzymes from Thalassiosira pseudonana, synthesized biotinylated mRNAs, and used these in binding assays with protein extracts obtained from T. pseudonana cells grown under different culture conditions, in order to pull down specifically bound proteins and examine the RBP profiles. We detected RBPs for all five transcripts. Interestingly, the binding profiles differed between transcripts as well as culture conditions. Future experiments are aimed at identifying the RBPs through protein sequencing and characterizing their functions. Abstract No. 76 Proteome Expression of Maturing Tomato Seeds under Aluminum Stress Okekeogbu, Ikenna*; Sangireddy, Sasikiran; and Zhou, Suping. Department of Agricultural Sciences, College of Agriculture, Human and Natural Sciences, Tennessee State University, Nashville, TN 37209 The primary effect of soil acidity is aluminum (Al) toxicity, which occurs on about 75% of total acid soil area. Under acidic condition (pH<5), Al is solubilized from its nonphytotoxic form into toxic forms such as the trivalent cation, Al3+ which inhibits plant growth and development. In this study, we used maturing tomato fruits to provide a natural acidic condition (4.3-4.5) in order to localize Al and determine how the toxic ions affect protein expression in the maturing seeds. Tomato (Solanum lycopersicum cv. Micro-Tom) plants were grown in hydroponic tanks filled with Magnavaca’s solution, pH 4.5 - 4.6. For Al treatment, the hydroponic solution was supplemented with 50 µM AlK (SO4)2. 12H2O, and the control were refreshed with only the Magnavaca’s solution. Seed tissues isolated from matured green fruits were stained with morin (2’, 3, 4’, 5, 7-pentahydroxyflavone) to detect Al in situ. Differentially expressed proteins between the Al-enriched seeds and controls were identified using two-dimensional gel electrophoresis (DIGE) analysis. 54 protein spots showed significant difference (?1.3 fold, p< 0.05) between Al treated and control seed proteomes, with 28 protein spots being induced, and 26 spots suppressed. The Proteome analysis resulted in the identification of 20 significant proteins, out of which 10 were up regulated and 10 down regulated. The identified proteins are involved in metabolic pathways, gene expression and cell division, chaperones and protectants, and phytohormone-biosynthesis. The activities of 7 different antioxidant enzymes were also assessed. They include glutathione reductase (GR), glutathione S-transferase (GST), peroxidase (POX), catalase (CAT), ascorbate peroxidase (AsPOX), carbonate anhydrase, and hydroxypyruvate reductase (HPR). Based on these results, a molecular model of ion toxicity from endogenous Al during seed maturation and germination is being evaluated. Further works to identify the genes that are involved in these mechanisms are ongoing. Abstract No. 77 Long-term N Fertilization Alters Overall Bacterial Community Structure and Abundance of N Cycling Bacterial Gene Copies at Harvard Forest, MA, USA Turlapati Swathi 1,Minocha Rakesh 2,Bhiravarasa Premsai 1, Sweeney Karen 2,Tisa Louis 3,Thomas William 3,4,Minocha Subhash 1 1Department of Biological Sciences, University of New Hampshire, Durham, NH 03824 2USDA Forest Service, Northern Research Station, Durham, NH 03824 3Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH 03824 4Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH 03824, USA Environmental Nitrogen deposition from fossil fuel emissions is considered a threat to forested ecosystems worldwide. Soil microbes act as indices of soil fertility and plant growth. The impact of twenty years of annual application of N (ammonium nitrate) on the


forest soil bacteria was studied using pyrosequencing of 16S rRNA genes. The abundance of N cycling bacterial genes (amoA, nirS and nosZ) was also investigated using qPCR. DNA was extracted from 30 soil samples (three treatments x two horizons x five subplots) collected from untreated (control), low N-amended (LN; 50 kg ha-1 yr-1) and high N-amended (HN; 150 kg ha-1 yr-1) plots. A total of 1.3 million sequences were obtained through pyrosequencing and processed using QIIME program. Principal coordinate analysis (PCoA) confirmed that bacterial communities differed among soil horizons and between treatments. While 28 to 35% of the total taxa (6936) were common to all three treatments, the rest were specific to one treatment or common to two. Data revealed that the abundance of nitrifying bacteria (amoA gene) was higher only in HN mineral soil as compared to control. Among denitrifying bacteria, while no differences were observed for nirS gene, nosZ gene copies were higher in LN organic soil compared to control. Soil C, N, cation exchange capacity, N03-, NH4+, acidity and soil pH were found to influence the amoA, nirS and nosZ gene abundance in mineral soil. Majority of the amoA clones generated from HN soils corresponded to the genus Nitrosospira. The changes observed in the bacterial community structure may be a cumulative outcome of N-driven soil base cation changes, net changes in aboveground plant productivity, as reported earlier by our and other groups. Future studies must examine the linkages between belowground phylogenetic and functional gene diversity with plant productivity in forested ecosystems. Abstract No. 78 Water Deficit Responses Regulated by microRNAs in Phaseolus Vulgaris Reyes, Jose, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor. México. Sosa-Valencia, Guadalupe, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor. México. De la Rosa, Carlos, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor. México. Perez-Morales, Beatriz, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor. México. Garcia, Alma, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor. México. Arteaga-Vazquez, Mario,Instituto de Biotecnología y Ecología Aplicada, Universidad Veracruzana, 91090 Xalapa, Veracruz, México. Sunkar, Ramanjulu,Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA. Covarrubias, Alejandra, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor. México. In Mexico, as in other developing countries, common bean (Phaseolus vulgaris) is an important crop legume, corresponding to one of the main sources of protein uptake for human consumption. However, water deficit is a major constraint limiting crop production. In order to contend with different environmental adversities, such as drought, plants have developed a series of mechanisms at the physiological, cellular and molecular level. To obtain novel insights into the mechanisms involved in plant responses to water deficit, we have focused on the analysis of microRNAs as regulators of this response at the post-transcriptional level. Within a multiprotein complex called RISC (for RNA-Induced Silencing Complex), the microRNA (a small RNA of 20-24 nts) is bound to the effector protein ARGONAUTE 1 (Ago1), and recognizes a target mRNA by RNA:RNA base-pairing. RISC subsequently directs mRNA down-regulation by Ago1-mediated mRNA cleavage or by translational inhibition. We have identified common bean microRNAs that are expressed under water deficit conditions, including several legume-specific microRNAs. Their characterization along with new data from high-throughput sequencing of small RNA populations will be presented. Still, mRNA target identification has been hindered by lack of genomic data. Thus, we have combined different strategies to identify miRNA targets: bioinformatical prediction of targeted transcripts, biochemical analysis of the AGO1 protein and of its interacting RNAs,


and a high-throughput sequencing analysis of cleaved mRNAs to identify miRNA targets under water deficit conditions. Comparison of small RNAs obtained by high-throughput sequencing and transcript sequences, along with the recent public release of the P. vulgaris complete genome sequence, is leading us to identify elements of gene regulation by microRNAs. Our results will help to better understand strategies used by common bean and other legumes to cope with adverse environmental conditions. Abstract No. 79 Arabidopsis SC35 and Two SCL Subfamily Members of Serine/Arginine-rich Proteins Function as Redundant Positive Regulators of Heat Stress Response Kelsey C. Peters*, Jenna Ermisch*, Saiprasad G. Palusa, Julie Thomas and Anireddy S.N. Reddy Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA *Undergraduate students, contributed equally. Serine/arginine-rich (SR) proteins, a family of non-snRNP proteins, play critical roles in both constitutive and alternative splicing. In Arabidopsis, there are 18 SR genes and fourteen of them undergo alternative splicing. Furthermore, the splicing pattern of the SRs is significantly altered in response to diverse stresses. In the present study, we investigated the roles of SC35 and two members (SCL30a, SCL33) of the plant-specific SC35-like (SCL) subfamily of SR proteins in hormonal and stress (heat and salt) responses using three single (scl30a, scl33, sc35), three double (scl30a scl33, scl30a sc35, scl33 sc35) and one triple (scl30a scl33 sc35) mutants. Five of the seven mutants, all except sc35 single and scl33 sc35 double mutant, were found to be sensitive to glucose. However, no effect was observed on any of the mutants in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. We subjected all seven mutants to heat stress by exposing the seeds or seedlings to 380C or 450C for 4 hours. At 380C no difference in germination or seedling growth was observed in any of the mutants as compared to wild type. However, at 450C the triple mutant showed reduced seed germination and bleaching of seedlings whereas none of the single or double mutants showed these phenotypes. Quantification of total chlorophyll and carotenoid content in control and heat-treated (450C) wild type and the triple mutant seedlings showed a significant reduction of both in the mutant seedlings. In the presence of salt (NaCl), germination and seedling growth of all mutants was similar to wild type. Our results suggest a role for these SRs in glucose sensing and a functional redundancy of tested SR proteins in heat stress response. Abstract No. 80 The Arabidopsis Splicing Regulator, SR45, Confers Salt Tolerance in an Isoform-Dependent Manner Albaqami Mohammed(1), Laluk K(1), Abdel-Ghany Salah E.(1), Zhang Xiao-Ning(2) and Mount Stephen M.(3) and Reddy Anireddy S.N.(1) (1)Department of Biology and Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA (2)Saint Bonaventure University, Saint Bonaventure, NY 14778, USA (3)Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20740, USA A number of stresses have been shown to regulate alternative splicing of precursor mRNAs in plants. Serine/arginine-rich (SR) and SR-like proteins that regulate splicing also undergo extensive alternative splicing in response to various stresses. Hence, reprogramming of the transcriptome through changes in pre-mRNA splicing is thought to be important for stress tolerance. We have previously shown that SR45, an SR-like protein, interacts with several spliceosomal proteins (U170K, SCL33 and U2AF35) and plays an important role in plant


growth and development. Its two alternatively spliced isoforms (long and short) that differ in eight amino acids have distinct biological functions during development. Here, we investigated the role of SR45 and its splice variants in salt stress using sr45 mutant and transgenic lines complemented with either the long or short form. The sr45 mutant, at different developmental stages, was highly sensitive to NaCl and other salts as compared to wild type. Interestingly, this salt sensitive phenotype was fully rescued by the long isoform but not the short one, suggesting that only the long isoform functions in salt tolerance, likely by regulating the expression and/or the splicing pattern of genes involved in salt stress. Further molecular analyses have revealed that the relative expression and the splicing pattern of some genes involved in salt overly sensitive (SOS), ABA and other stress signaling pathways were affected in sr45 mutant and the long isoform is needed for normal splicing and expression of these genes. In addition to the relative changes in the expression of some splicing isoforms of salt stress responsive genes, new splicing isoforms were identified, suggesting the importance of the SR45 long isoform in fine-tuning gene expression under salt stress. These results suggest an important role for the long splice form of SR45 as a positive regulator of salt tolerance. Abstract No. 83 Visualizing RNA-Seq Data with Integrated Genome Browser 7.0 Loraine, Ann Vora, Hiral Gulledge, Alyssa Wang, Fuquan Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, 600 Laureate Way, Kannapolis, NC 28081 Ever-improving sequencing technologies are enriching our view of plant transcriptomes. The cost of sequencing is decreasing, making RNA-Seq projects of increasing sophistication more accessible. However, working with these vast data sets can be daunting. To help scientists manage, analyze, visualize, and share their data, we developed Integrated Genome Browser 7.0, a fast, flexible, and free genome visualization tool. IGB’s highly configurable navigation and interactive data analysis tools are unique among currently available genome browsers. Using IGB, one can move easily and rapidly through a genome to investigate regulated splicing of genes and confirm results from statistical analysis. Another distinguishing feature is IGB’s support for interactive analyses, such as creating exon-exon junction features from read alignments in RNA-Seq data. IGB also supports data sharing and the publication process. Using IGB’s simple Web-based IGBQuickLoad system, one can easily share data with collaborators, reviewers, and the public. This presentation will demonstrate how we used IGB to investigate and share RNA-Seq data sets produced in-house or harvested from the Short Read Archive. Abstract No. 85 NEW Tools to Monitor PBs and SGS Dynamics in Arabidopsis FAVORY Jean-Jacques (1) DESCOMBIN Julie (1) DERAGON Jean-Marc (1) BOUSQUET-ANTONELLI Cecile (1) 1: LGDP-UMR5096 University of Perpignan CNRS 58 av. Paul Alduy 66860 PERPIGNAN FRANCE For each living organism it is of prime importance to be able to adapt to changes of environmental conditions. For plants, as sessile organisms, the fine tuning of the gene expression in an ever changing environment is even more vital. Post-transcriptional regulations play a crucial role in gene expression control. In animals two types of cytosolic ribonucleoprotein (RNP) structures have been described in response to translation repression: stress granules (SGs) and processing bodies (PBs). SGs are dynamic aggregates of untranslated mRNAs in association with translation initiation factors and PBs are RNA protein aggregates containing untranslated mRNAs associated with the mRNA decapping machinery. Recent studies showed that some key factors involved in the formation of the PBs and SGs are common to plants and animals. These aggregates are visible as


cytoplasmic foci which can be followed using fluorescent microscopy approaches. To study the dynamics of these RNP in Arabidopsis, stable transgenic lines expressing GFP and RFP tagged version of AtDCP1 and AtPAB2 under control of their endogenous promoters were selected to study PBs and SGs respectively. Our experiments conducted in young seedlings root tips indicate that both structures response to selected stresses with different dynamics. These first studies show that these selected lines provide useful cytosolic RNPs dynamic reporter lines and stress sensors in Arabidopsis. Abstract No. 91 Deep Sequencing Analysis of Brassica napus Lines Selected for Low Respiration and High Yield Byzova Marina (1,2)*, Martens Cindy(1,2), Woloszynska Magdalena (1,2), Verwulgen Tom (1,2), De Block Marc (3), Van Lijsebettens Mieke (1,2) (1) Department of Plant Systems Biology, VIB, 9052 Gent, Belgium, (2) Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium, (3) Bayer CropScience N.V., 9052 Gent, Belgium Plants utilize sophisticated epigenetic regulatory mechanisms to coordinate changes in gene expression to respond rapidly and reversibly to the environment. Moreover, one of the most complex quantitative traits in plants, yield, has been demonstrated to possess an epigenetic component that correlates with energy homeostasis [1]. The respiration rate was used as a selection marker for recurrent artificial selection in isogenic Brassica napus doubled haploid Simon populations. Selected lines with a low respiration rate had an increased seed production while those with high respiration were characterized by a decreased seed yield. All selected lines were found to be genetically identical but epigenetically different. Epigenetic states as well as the agronomic and physiological characteristics of the lines were stably transmitted for over eight generations [2]. To provide more insight into regulatory and metabolic pathways associated with transcriptional and epigenetic states, we have performed RNA deep sequencing analyses on polysomal RNA of the selected low- and high-respiration lines. To analyze expression data of tetraploid B. napus lines, a bioinformatics pipeline was developed based on sequencing genome information of Brassica oleracea and Brassica rapa that form together the components of the B. napus genome. SNPs between the B. oleracea and B. rapa orthologs were used to assign the B. napus RNAseq reads to the correct transcripts. Differential expression testing was done using the R package EdgeR. In house developed bioinformatics tools as CORNET, PLAZA and BINGO were used for identification of key regulatory pathways and genes. Additionally, mitochondrial genomes of the selected lines have been sequenced which excluded mitochondrial DNA polymorphisms as a cause of the differences in plant performance. [1]. De Block M, Van Lijsebettens M. Curr Opin Plant Biol. 2011, 14: 275-82 [2]. Hauben et al., Proc Natl Acad USA 2009, 106: 20109-14 Abstract No. 92 Ribosome Number is Negatively Related to Biomass Accumulation in Arabidopsis in a Stable Diurnal Growth Regime Ishihara,Hirofumi,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Pyl,Eva-Theresa,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Schulze,Waltraud,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Obata,Toshihiro,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm Fernie,Alisdair,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Suplice,Ronan,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Stitt,Mark,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm,


Ribosome production and protein synthesis require a huge investment of energy and resources in all cells. The aim of this work is to investigate whether ribosome production and protein synthesis could be significant factors in determining plant growth rates. Quantitative RT-PCR analysis of rRNA spiked with eight artificial RNAs was used for absolute quantification of ribosomes. Ribosome abundances were measured in 20 Arabidopsis accessions that have different growth rates. Most unexpectedly, ribosome abundances at the end of the night (EN), but not the end of day (ED), were negatively correlated with biomass (R = -0.63). As this result showed that plant growth is not increased by having higher ribosome abundance, we reasoned that higher biomass production might instead be due to greater efficiency of ribosome use. The percentage of ribosomes engaged in protein synthesis was estimated by polysome loading analysis of the 20 accessions harvested at ED and EN. Ribosome abundance in polysomes at EN was highly negatively correlated with biomass (R = -0.87). The estimated rates of both night-time and daily (24 h) protein synthesis were substantially higher in small accessions than in large accessions, but total protein did not differ significantly. This suggests that the small accessions have a higher rate of protein turnover, and consume more energy to synthesise proteins during the night than in large accessions. Therefore, a new method to measure global protein turnover rates from selected Arabidopsis accessions grown in soil was developed. The plants were labeled with 13CO2 for 24 hours and harvested at the ED and EN during the pulse, and at 1 day and 4 days after the end of pulse. Labeling of free amino acids and protein amino acid residues was measured by GCMS for estimation of protein turnover rates. Abstract No. 93 Characterization of RNA Silencing Amplification and tasiRNA Pathways in Arabidopsis thaliana Ishihara,Hirofumi,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Pyl,Eva-Theresa,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Schulze,Waltraud,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Obata,Toshihiro,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm Fernie,Alisdair,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Suplice,Ronan,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Stitt,Mark,Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, In plants, gene silencing mediated by small RNAs (sRNAs) can be amplified. This mechanism (also known as transitivity) is dependent on RNA-DEPENDENT RNA POLYMERASEs (RDRs), which convert the sRNA target transcript into double stranded RNA (dsRNA). The dsRNA is then processed by DICER LIKE (DCL) enzymes, resulting in the production of secondary sRNAs, which will re-enforce silencing or target new sequence-related transcripts. Trans-acting small interfering RNAs (tasiRNAs) are a plant-specific class of sRNAs, which production is dependent on this amplification mechanism. For most of the tasiRNA families, production is triggered when the precursor transcript is cleaved by a miRNA that is 22 nt long and/or asymmetric in relation to the miRNA/miRNA* duplex. In contrast, most plant miRNAs are mainly 21 nt long/symmetric and cannot trigger production of secondary sRNAs. Nonetheless, it is not clear how 22 nt/asymmetric miRNAs can initiate transitivity. It is likely that ARGONAUTE1 (AGO1), the main effector of the silencing pathway, when loaded with 22 nt/asymmetric miRNAs would assume a different conformation, which would allow recruitment of RDRs. Here we report our efforts to better understand the biogenesis of tasiRNAs, and consequently the amplification process in plants. We have used a combination of molecular biology and biochemistry to identify new components, which are involved in


the mechanism of silencing amplification. Abstract No. 94 iDiffIR: Identifying Differential Intron Retention from RNA-seq, with Applications from Arabidopsis Hamilton, Michael, Computer Science Department, Colorado State University, Fort Collins, CO, USA Reddy, Anireddy SN, Department of Biology, Colorado State University, Fort Collins, CO, USA Ben-Hur, Asa, Computer Science Department, Colorado State University, Fort Collins, CO, USA The increasing availability of RNA-seq data coupled with the interest of elucidating cell-, tissue-, and condition-specific alternative splicing (AS) has created a need for the development of computational tools that model and identify differential splicing from the alignment of short reads. While the prevalence of intron retention (IR) in plants is well-known, computational approaches that quantify differential AS aren often exon-centric. Motivated by these challenges, we developed iDiffIR, a fast, intron-centric method that identifies genome-wide, differential IR from RNA-seq data with or without biological replicates. Our method uses an interpretable statistic that provides biologically meaningful results and identifies more differential IR events than other available methods. The iDiffIR software package and a comprehensive tutorial are freely available at http://combi.cs.colostate.edu/idiffir. To demonstrate the effectiveness of our approach, we applied iDiffIR on two publicly-available Arabidopsis RNA-seq datasets. Using data from a recent study on the effects of splicing by PTB homologs, we detected around 1500 differential IR events from ? 1300 genes in response to over-expressed or knockdown PTB-1&2 mutants. Also among the significant events, we identified novel differential IR in PTB1 and PTB3, providing evidence that PTBs auto- and cross-regulate IR. In addition, we recover an experimentally-verified, differential IR event that is not annotated in gene models, underscoring iDiffIR's ability to detect biologically-significant, novel events. Finally, from a transcriptome-wide analysis of the effects of DNA methylation on immune response, we identify around 500 differential IR events in DNA methyltransferase (MET1) mutants. Among these, about 75% showed lower levels of IR in the mutant, therefore suggesting a role of DNA methylation in IR. A GO analysis of genes containing differential IR events revealed significant enrichment of defense-related categories. This result complements original work where methylation mutants were shown to exhibit increased immunity to P. syringae, suggesting a role of IR in plant immunity. Abstract No. 95 Gene Silencing as a Negative Component in the Formation of Heterosis in Maize Thiemann, Alexander, University of Hamburg, Biocenter Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststrasse 18, 22609 Hamburg, Germany Edelmann, Susanne, University of Hamburg, Biocenter Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststrasse 18, 22609 Hamburg, Germany Scholten, Stefan, University of Hamburg, Biocenter Klein Flottbek, Developmental Biology and Biotechnology, Ohnhorststrasse 18, 22609 Hamburg, Germany Heterosis describes the superior agronomic performance of heterozygous F1 plants compared to their homozygous parental inbred lines. Heterosis has been intensively used in plant breeding since the early 20th century and has since then led to an enormous increase in crop trait performance. However, despite of its great agricultural importance the molecular and genetic mechanisms underlying the phenomenon are yet not fully understood. An epigenetic contribution to heterosis has been recently discussed. Comparisons between inbred parents and their corresponding hybrids in a variety of


different plant species revealed differences in DNA methylation and also in the abundance of certain groups of small RNAs. Nevertheless a direct involvement of small RNAs in heterosis formation has not yet been demonstrated. By analyzing transgenic maize plants in vitro with a chemically inducible expression of a viral gene silencing suppressor (P1/HC-Pro) of Wheat streak mosaic virus (WSMV) we are able to directly link small RNAs to the formation of heterosis. Gene silencing suppressors, as viral tools for the inhibition of the plant defense, are capable to inhibit the function of small RNAs. The inducible expression system allowed for the comparison of identical genotypes under induced and non-induced conditions. As a result the expression of P1/HC-Pro led to an increase of heterosis for growth in comparison to the non-induced plants. Our initial results suggest a direct link between gene silencing and hybrid superiority. It seems that the functional interference of small RNAs by the viral suppressor causes an increase of the heterosis effect in maize. Abstract No. 96 Maize Ufo1 Modulates Tissue-specific Small RNA Profiles and Locus-specific Gene Expression Lee, Tzuu-fen, Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware Wang, PoHao, Department of Crop and Soil Sciences, Pennsylvania State University, State College, Pennsylvania Chopra, Surinder, Department of Crop and Soil Sciences, Pennsylvania State University, State College, Pennsylvania Meyers, Blake C., Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware pericarp color 1 (p1) regulates flavonoid biosynthetic genes which produce brick-red phlobaphene in maize. Many P1 alleles such as P1-rr (red pericarp, red cob) and P1-wr (white pericarp, red cob) exhibit tissue-specific pigmentation as the result of their differential expression patterns. Unstable factor for orange 1 (Ufo1) is a dominant modifier which regulates the expression levels of certain p1 alleles epigenetically. Compared with the colorless pericarp in P1-wr plants, P1-wr; Ufo1-1 plants exhibit enhanced pigmentation in various tissues, which is associated with hypomethylation of P1-wr allele and increased level of p1 expression. To investigate whether Ufo1 function is associated with small RNA-directed epigenetic regulation, we conducted extensive small RNA and transcriptome profilings in leaf, tassel, young ear, and pericarp of P1wr; Ufo1-1 plants. Our results showed a 1.5 fold reduction in the 24 nt abundance only in the Ufo1-1 pericarp, suggesting that the reduction of 24 nt, silencing small RNAs may contribute to the release of transcriptional repression of genomic loci including P1-wr allele in pericarp. The impact of Ufo1 on small RNA abundance is tissue-specific: while 16% of small RNA-generating loci were repressed by Ufo1 in pericarp, 5% of loci were repressed in leaf. Furthermore, although majority of the Ufo1-impacted small RNA loci were present in more than one tissue type, 20%-40% of Ufo1-repressed loci and 30-70% of Ufo1-induced loci can be found in one tissue only, and may be associated with certain genome loci. RNA-seq results showed that many genes involved in various biological processes were differentially expressed between wild-type and Ufo1-1. In summary, our results indicated that Ufo1 affects small RNA abundance and gene expression regulation in a locus-specific rather than a global fashion. Ufo 1 may function in the maintenance of chromatin and DNA methylation state in a tissue-specific manner. Abstract No. 98 The Dynamic of a Stele-based Regulatory Network During Arabidopsis Development and Response to Stresses Liseron-Monfils,Christophe, Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor NY 11724 Zhang,Lifang, Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor NY 11724 Iossifov, Ivan, Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor NY 11724 Brady,Siobhan,Department of Plant Biology and Genome Center UC Davis, Davis CA 95616


Ware,Doreen, USDA-ARS-NAA Robert W. Holley Center Ithaca NY 14853, Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor NY 11724 The intensification of plant stresses caused by the climate change and an increasing world population are two major challenges for today agriculture. These challenging tasks imply to be able to increase crop yield while decreasing plant susceptibility to stress. One way to reach this goal is to a better understanding of plant responses to stress stimuli. Based on the specific expression of transcription factor in the root stele tissue, we have generated an in-vitro interaction map between transcription factors and the promoter of miRNAs using a Yeast one hybrid system that we developed. We have then extended this Gene Regulatory Network (GRN) to all the validated and predicted targets of miRNAs (based on TAIR data) and the experimentally validated protein-protein interactions from the Arabidopsis Interactome data. Co-expression Correlations were measured by maximal information coefficient allowing the measure of linear and non-linear interactions. As miRNA expressions were not present on the ATH1 array (Genevestigor). We developed a pipeline to predict the behavior of the network 23 stress responses and development tissues, as well as the overlap of the significant interactions between any of these studied conditions. We detected interesting pattern of nonlinearity correlations for certain interaction types. We also used significant interactions to discover putative master regulators in each studied conditions by searching shared cis-elements in significant gene promoters and annotations. We unrevealed master regulator of stresses and relation to plant development. We are currently validating the master regulators found for each condition by using a mutant approach and promoter deletion approaches. This research aimed to be a base to search the stress-development regulation and will be extended to crop plant such as maize. Abstract No. 99 Small RNA Regulatory Networks in Cotton (Gossypium hirsutum) During Reniform Nematode (Rotylenchulus reniformis) Infestation Ruijuan Li* (1), Hongtao Hu (1), Narendra K. Singh (1), Kathy S. Lawrence (2), David B. Weaver (3), and Robert D. Locy (1) (1) Auburn University, Department of Biological Sciences, 101 Rouse Life Sciences Building, Auburn, AL 36849; (2) Auburn University, Department of Entomology and Plant Pathology, 301 Funchess Hall, Auburn, AL 36849; (3) Auburn University, Department of Agronomy and Soil, 201 Funchess Hall, Auburn, AL 36849. Reniform nematode (RN, Rotylenchulus reniformis) is a major soil-born pathogen affecting cotton. Most commercial cotton germplasm is susceptible to RN, but germplasm that appears to be resistant to RN including the RN hypersensitive lines, LONREN-1 and LONREN-2 and resistant line BARBREN-713 have emerged as promising sources of resistance to this important pathogen. The objective of this investigation is to examine the small RNA regulatory networks during RN infestation of susceptible, hypersensitive, and resistant genotypes, and based on changes in the expression of genes in miRNAome, tasiRNAome, and the transcriptome of these respective genotypes, define the role that specific networks may play in resistance and hypersensitivity to RN infestation. Eight small RNA libraries were generated from the roots of susceptible genotypes (DPL90 & SG747, combined to make 1 susceptible sample), two hypersensitive genotypes (LONREN-1 & LONREN-2), and the resistant BARBREN-713 genotype either pre- or post-infestation (combined 1,2,5, & 10 dpi) with RN. Based on similarity to previously reported Viridiplantae miRNAs, 1,423,666 small RNA sequencing reads belonging to 4939 unique small RNA sequences matched to 300 existing miRNAs were identified, leaving with 43,121,079 reads belonging to 9,572,433 unique sequences as unanotated sRNAs. 128 novel miRNAs with hairpin structure precursors were predicted using mireap, and 46956 small RNA sequencing


reads belonging to 211 unique sequences matched to precursor sequences were identified. The expression of known and novel or cotton root specific microRNA were determined. Finally, 679 phased small RNA clusters were generated from non-miRNA small RNA sequences with differentially expressed miRNAs as phase initiators. 15 differentially expressed conserved miRNAs and 17 novel miRNAs were predicted to be phase initiators, and the expression of the predicted tasi-RNAs were examined in the libraries and will be compared between genotypes and treaments. The differential expression of transcripts following nematode infestation in each genotype will be reported. Abstract No. 100 miRNAs Mediate SnRK1-dependent Energy Signaling in Arabidopsis Confraria, Ana (1), Martinho, Cláudia (1), Elias, Alexandre (1), Rubio-Somoza, Ignacio (2), and Baena-González, Elena (1). (1) Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal; (2) Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany. The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response. Abstract No. 101 SCI1, a Tissue-Specific Inhibitor of Cell Proliferation in Plants, Is Probably Also Involved in RNA Processing Strini, Edward J.(1,2); Almeida-Souza, Hebréia O.(1); Lubini, Greice(1,2); DePaoli, Henrique C.(1,2); Bertolino, Lígia T.(1,2); Quiapim, Andréa C.(1); Goldman, Gustavo H.(3); Goldman, Maria Helena S.(1,*). (1) FFCLRP/University of São Paulo (USP), Dept. Biology, Ribeirão Preto – SP, Brazil 14040-901; (2) PPG-FMRP/University of São Paulo (USP), Dept. Genetics, Ribeirão Preto – SP, Brazil 14049-900; (3) FCFRP/University of São Paulo (USP), Dept. Pharmaceutical Science, Ribeirão Preto – SP, Brazil 14040-903. E-mail: [email protected]


The success of plant reproduction depends on the appropriate development of the reproductive organs which involves specific regulatory networks. We have characterized a novel stigma/style expressed gene encoding a small lysine-rich protein with 15 putative phosphorylation sites. Real time RT-PCR and in situ hybridization experiments showed that it is a tissue-specific developmentally regulated gene. The RNAi and overexpression transgenic plants showed stigmas/styles with remarkably enlarged and reduced areas, respectively, which we have demonstrated that occurs due to differences in cell numbers. Based on these phenotypes, this gene was designated SCI1 (Stigma/style Cell-cycle Inhibitor 1). Furthermore, the differences in cell division affected the differentiation timing of the stigmatic papillar cells, showing that their differentiation is coupled to stigma cell divisions. Fluorescence microscopy with SCI1-GFP protein fusion demonstrated its nuclear localization, which is confined to the nucleolus and splicing speckles. To study how SCI1 controls cell proliferation/differentiation, we constructed and screened a yeast two-hybrid stigma/style cDNA library using SCI1 as bait. Additionally, pull down experiments using recombinant SCI1 and stigma/style proteins were performed. These experiments identified several putative interaction proteins: a cyclin-dependent kinase, a MAPK, a calcium-dependent protein kinase, a DEAD-box protein, two 14-3-3 proteins, two phosphatases, some RNA-binding proteins and several unknown proteins. Bimolecular fluorescence complementation and co-immunoprecipitation assays have already confirmed some of these interactions. Taken together, the co-localization of SCI1 with CypRS64 and SRp34, two splicing factors, and the interaction partners identified suggest that SCI1 may be also involved in RNA processing. Financial Support: FAPESP, CNPq, CAPES (BRAZIL). Abstract No. 103 Genome-wide Analysis of miRNA Targets in Brachypodium and Bioenergy Crops Schmidt, Skye, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Jeong, Dong-Hoon, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. *Franke, Karl, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Rymarquis, Linda, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Current address: Monsanto Company, Chesterfield, MO, USA. Park, Sunhee, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Ganssmann, Matthias, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Current address: Eurofins-GAB German, Marcelo, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Current address: Dow AgroSciences LLC, Portand, OR, USA. Accerbi, Monica, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Zhai, Jixian, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Paoli, Emanuele, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Current address: Dow AgroSciences LLC, Portand, OR, USA. Fahlgren, Noah, Donald Danforth Plant Science Center, St. Louis, MO, USA. Fox, Samuel, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, 97322, USA Yen, Yang, Department of Biology and Microbiology, South Dakota State University, Brookings, SD, USA. Garvin, David, USDA-ARS Plant Science Research Unit and University of Minnesota, St. Paul, MN, USA. Mockler, Todd, Donald Danforth Plant Science Center, St. Louis, MO, USA. Carrington, James, Donald Danforth Plant Science Center, St. Louis, MO, USA. Meyers, Blake, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Green, Pamela, Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.


Brachypodium distachyon has emerged as a model system for temperate grasses including wheat, barley, and oat, as well as for biofuel plants such as switchgrass and sorghum. To enhance knowledge and understanding of the small RNA population in Brachypodium, a very large set of small RNAs was cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified not only conserved miRNAs that have not been reported in Brachypodium, but also non-conserved miRNAs that were not found in other plants. A total of 116 miRNAs were identified. To investigate miRNA cleavage function, four PARE (Parallel Analysis of RNA Ends) libraries were constructed from key tissues. These are the first PARE libraries described for Brachypodium and their sequencing resulted in a total of ~70 million raw sequences and ~5 million distinct genome-matched sequences. The result is an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE data and the miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage events in a correspondingly tissue-preferential manner. Using PARE data from each species, we show examples of conserved cleavage of homologous targets in Brachypodium, sorghum and switchgrass. The knowledge gained from this study should provide insight about the roles of miRNAs and the regulation of their targets in Brachypodium and related biofuel plants. Funded by the DOE. Abstract No. 104 mRNA Storage and Translation in Male Gametophyte Hafidh, Said, Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Praha 6, Czech Republic Breznenová, Katarína, Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Praha 6, Czech Republic Bokvaj, Pavel, Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Praha 6, Czech Republic Honys, David, Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Praha 6, Czech Republic Male gametophyte, highly organized haploid flower organ, offers unique chance to analyze development and differentiation of single haploid cell, cell-cell interaction and recognition, cellular polarity and pollen tube tip growth. Posttranscriptional control of gene expression plays a vital role during tobacco pollen maturation and tube growth. Number of pollen mRNAs were shown to be bound to pollen-specific stored ribonucleoprotein particles annotated as EPP complexes. Similarly to the role played in growing mammalian neurons, EPP particles represent pre-loaded complex machinery devoted to mRNA processing, transport, subcellular localization and protein synthesis. Here we performed detailed functional, transcriptomic and proteomic characterization of pollen storage RNP particles in tobacco (Nicotiana tabacum L.). In particular, we aimed to integrate our knowledge on the categorization of translationally regulated transcripts in developing pollen and to identify the mode of action of the translational repression and derepression of mRNAs stored in developing pollen and gradually activated during progamic phase that is likely to involve DEAD-box RNA helicases. Acknowledgement: The authors gratefully acknowledge the financial support from Czech Science Foundation (P501/11/1462, P501/11/P321, P305/12/2611) and Czech Ministry of Education, Youth and Sports (LD13049). Abstract No. 105 Short, Simple, Silencing Using the Trans-acting siRNA Pathway Jacobs, Thomas*, University of Georgia, Athens, GA USA LaFayette, Peter, University of Georgia, Athens, GA USA Lawler, Noah, University of Georgia, Athens, GA USA Vodkin, Lila, University of Illinois, Urbana, IL, USA Parrott, Wayne, University of Georgia, Athens, GA USA


Transgenic gene-silencing is an essential tool for basic genetic research and developing novel agronomic traits. We have adapted a new, highly effective, gene-silencing strategy using the trans-acting small-interfering RNA (ta-siRNA) pathway. A single 22-nt microRNA (miRNA) recognition sequence is fused to a target sequence, and the resulting mRNA transcript is targeted by a miRNA species that induces the production of siRNAs. Prior to our investigation, there were no verified ta-siRNA-inducing miRNA recognition sequences in soybean. We identified six putative miRNA recognition sequences from a soybean hairy root small RNA sequencing dataset, and evaluated the abundance of each of the corresponding miRNAs in various soybean tissue types using publically available data. To test the gene-silencing approach, one miRNA recognition sequence (1514), which was expressed in many tissue types, was fused to the gene targets nodulation factor receptor (NFR), cytochrome P450, and green fluorescent protein (GFP) and transformed into soybean hairy roots. Small RNA sequence data show the production of siRNAs, and quantitative RT-PCR showed approximately 90% down-regulation of the P450 and GFP target genes. The remaining five miRNA recognition sequences were validated by targeting GFP in hairy roots. All of the miRNA recognition sequences reduced GFP expression, and five of the six reduced expression by 80-95%. The 1514 silencing constructs have been stably transformed into soybean, and silencing has been observed in many tissue types. These results demonstrate that the six miRNA recognition sequences identified here will be useful for creating gene-silencing constructs in soybean and potentially other legumes. This simple yet highly effective gene-silencing approach should be applicable to those plant species in which ta-siRNAs have been identified. Abstract No. 106 Effects of Root-knot Nematode Parasitism on Host Gene Silencing Walsh, Ellie (1), Pengue, Gina (1), Carballo, Valentina (2), Marella, Heather (3), McIntyre, Lauren (4), Morse, Allison (4), Koch, Karen (5), Taylor, Chris G. (1); (1) Department of Plant Pathology, The Ohio State University, OARDC, Wooster, OH 44691, USA, (2) Donald Danforth Plant Science Center, St. Louis, MO 63132, USA, (3) Bridgewater State University, 131 Summer Street, Bridgewater, Massachusetts 02325, USA, (4) Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL 32610, USA, (5) Horticultural Sciences Department, University of Florida, Gainesville, FL 32610, USA. Plant-parasitic nematodes cause significant damage to crops worldwide. The root-knot nematode (RKN, Meloidogyne spp.), one of the most damaging nematodes due to its broad host range, establishes intimate feeding sites (giant cells) within the roots of a variety of plants. How plant-parasitic nematodes, such as RKN, cause such dramatic physiological changes whilst evading plant defenses is unknown. Recently it has been demonstrated that a variety of plant pathogens interfere with their host’s gene silencing pathways. This work aims to provide a more refined look into how root-knot nematodes influence their host’s silencing pathways during the course of infection. Interference of gene silencing pathways during nematode invasion was indicated in microarray data sets generated from laser-captured giant cells in A. thaliana roots. Subsets of genes regulated by small RNAs, as well as silencing machinery components that interact with small RNAs, were upregulated during the infection process. Furthermore, results examining the effects of compromising these pathways in A. thaliana and N. tabacum, suggest that these components influence the host's susceptibility to RKN by allowing more adult females to form and increasing fecundity. We have generated multiple transgenic N. tabacum lines expressing a silenced reporter gene to detect the disruption of these pathways. During the course of infection, it is evident that the silenced reporter gene, targeted either by dsRNA or miRNA, is recovered specifically within giant cells. Better insight into this interaction will be invaluable to our


growing understanding of the roles of host gene silencing during parasitic interactions. Abstract No. 109 Dynamic Ribonucleoprotein-mRNA Association During Oxygen Deprivation Sorenson, Reed, University of California, Riverside 4111 Genomics Building Riverside, CA 92521 Bailey-Serres, Julia, University of California, Riverside 4119 Genomics Building Riverside, CA 92521 Abstract Content: Low oxygen stress necessitates a frugal energy budget, limiting ATP synthesis to inefficient anaerobic production, and restricting its consumption by non-essential processes. Protein synthesis is a target of energy-use restriction. Global cellular levels of polyribosomes (multiple ribosome loaded mRNA) decrease when oxygen partial pressure is low. This response is dynamic and reversible, and exhibits a tight correlation with oxygen availability. Genome wide studies of mRNA-association with polysomes have demonstrated that discriminatory translation favors synthesis of proteins that participate in anaerobic metabolism. By contrast, the majority of non-translated mRNAs remain stable during the stress, rapidly returning to polysomes upon reoxygenation. We characterized a ribonucleoprotein complex resembling stress granules of other eukaryotes that aggregate non-translated mRNAs during oxygen deprivation. Arabidopsis thaliana encodes three oligouridylate binding proteins (UBP1s) identified as homologs of Tia-1, a mammalian stress granule marker. Redistribution of GFP-tagged UBP1C and UBP1A from a diffuse cytosolic to granular localization occurred during hypoxia and was rapidly reversed by reoxygenation. The formation of UBP1 granules was inhibited by cycloheximide, indicating a requirement of completion of translational elongation. UBP1C granules contained polyadenylated RNAs. An immunopurification scheme was developed to isolate UBP1C-mRNA complexes from seedlings under control, low-light, hypoxia, and reoxygenation conditions. Microarray analysis identified the mRNAs enriched in UBP1C complexes under control conditions. During oxygen deprivation, these mRNAs remained UBP1C-bound and most other mRNAs increased in UBP1C association, including mRNAs that were stable and translationally repressed. Hypoxia-triggered UBP1C association was rapidly reversed by reoxygenation demonstrating selective and dynamic UBP1C-mRNA binding during oxygen deprivation. This research was supported by NSF grants IOS-0750811 and IOS-1121626. Abstract No. 110 MicroRNA Gene Regulatory Networks in the Arabidopsis Root Identify Highly Connected Transcription Factor Families Zhang, Lifang, Cold Spring Harbor Laboratory Lee, Young Koung, Cold Spring Harbor Laboratory Liseron-Monfils, Christophe, Cold Spring Harbor Laboratory Gaudinier, Allison, UC Davis Brady, Siobhan, UC Davis Ware, Doreen, Cold Spring Harbor Laboratory, USDA-ARS MicroRNAs (miRNAs) play a central role in plant development and in the response to environmental stresses. Here, we present the construction and analyses of a comprehensive Gene Regulatory Network (GRN) controlling miRNA expression in the root. To systematically resolve points of crosstalk between transcription factors (TFs), miRNAs, and their targets, we made use of a gene-centered yeast one-hybrid (Y1H) approach to screen against more than 900 TFs families. With Y1H we had cloned 96 and screened 86 promoters of the approximately 116 conserved miRNAs in plant kingdom in order to fine-map their interactions. We then screened ~50 promoters of Arabidopsis protein-coding genes, including miRNA targets or highly-connected TFs in the network. This network is composed of close to 2000 PDI(Protein-DNA interactions). Here, we are presenting the general topology of the network. There are more than 400 TFs from 42 TFs families in this network. One third of them coming from 23 TFs that have more than 15 targets. They are


concentrated in several TF families such as AP2-EREBP, bZIP, C2H2, ZF-HD, GRAS, NAC, trihelix. Based on these analyses, we identified several sub-modules within the stele network, which represent known miRNA functions as well as predicted regulators of these functions in environmental stress responses. Our root GRN thus provides a framework for modeling developmental and adaptive responses to environmental conditions at the whole-plant level. This will facilitate identification of candidate genes and pathways as control points for germplasm improvement related to global food and energy security. Abstract No. 111 Deep Sequencing Analysis of Transcriptional Response to High Salt in Arabidopsis thaliana and Eutrema parvulum Chalivendra C Subbaiah, Oh Dong-Ha and Dassanayake Maheshi RNA-seq, a rapid, affordable and powerful tool for the high resolution analyses of plant transcriptomes, is useful in extending our efforts to understand biology beyond the boundaries of model organisms. Wild relatives of crops and extremophiles related to tractable model plant species are great genetic resources of stress tolerance and other useful agronomic traits. We are investigating the molecular basis of tolerance to high salt using the recently sequenced genome of the extremophile Eutrema parvulum (synon. Thellungiella parvula), a Brassicaceae member closely related to Arabidposis. Preliminary analysis indicates that copy number variation due to selective tandem genome duplication events may have significantly contributed to the high salt tolerance of this species (Dassanayake et al., 2011). We will discuss how transcriptome evidence supports gene subfunctionalization due to tandem and translocation duplications in E. parvulum. We will also present comparative transcriptome profiling at different salt levels, different time points after salt treatment, and for different tissues from Arabidopsis and E. parvulum. Our analyses indicate that gene expression divergence correlates with the differences in physiological responses to salt stress. Abstract No. 113 MicroRNA Superfamilies Descended From miR390 and Their Roles in Secondary siRNA Biogenesis in Eudicots Rui Xia, Blake C. Meyers, Zhongchi Liu, Eric P. Beers, Songqing Ye, and Zongrang Liu. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA; Appalachian Fruit Research Station, Agricultural Research Service, United States Department of Agriculture, Kearneysville, West Virginia 25430, USA; Alson H Smith Agricultural Research and Extension Center, Department of Horticulture, Virginia Polytechnic Institute and State University, Winchester, Virginia 22602, USA; Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19717, USA; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA. Transacting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA -TAS-PPR-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting


or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. Abstract No. 114 Detection of Putative Splicing Regulatory Elements from the Analysis of Discriminative Motifs Across Plants Hamilton, Michael, Computer Science Department, Colorado State University, Fort Collins, CO, USA Reddy, Anireddy SN, Department of Biology, Colorado State University, Fort Collins, CO, USA Ben-Hur, Asa, Computer Science Department, Colorado State University, Fort Collins, CO, USA Alternative splicing is a widespread phenomenon in plants, which is known to be regulated at several levels in response to a variety of conditions. Whereas recent studies have uncovered condition-specific splicing regulators in metazoans, the plant splicing code remains unexplored for the most part. In this work we present a first step towards this goal, which is the elucidation of putative splicing regulatory elements (SREs). Using gene annotations in 9 plant species (the dicots A. thaliana and G. max; the monocots O. sativa, Z. mays, B. distachyon, and S. bicolor; the moss P. patens; and the algae C. reinhardtii and V. carteri) we identify a collection of elements that are likely to function as splicing suppressors and splicing enhancers by comparing retained introns and their flanking exons with the corresponding regions in introns that are excised. By comparing the distribution of six-mers in the two populations of sequences, we identified elements that exhibit statistically significant over-representation. Specifically, we found multiple motifs over-represented in 3' exons of intron retention events across all species, suggesting the existence of conserved exonic splicing silencers (ESSs). In intronic sequences, conserved AU-rich motifs are present in sequences exhibiting intron excision, i.e., they function as intronic splicing enhancers (ISEs). Some of these ISEs are known to be associated with increased exon inclusion in metazoans. It is interesting to note that while we were able to identify putative ISEs and ESSs, we were unable to detect conserved ISSs and ESEs that function in intron retention. In conclusion, the motifs we detected are most likely functional considering their conservation across phylogenetically divergent photosynthetic eukaryotes and their similarity to known SREs in mammals. Abstract No. 115 Global Analysis of Gene Expression and Alternative Splicing in a Splicing Regulator Mutant: Role of SR45 in Thermotolerance Albaqami Mohammed(1,3), Hamilton Michael (2,3), Rogers Mark (2,3), Palusa Saiprasad G.(1), Xing D. (1), Ali Gul Shad (1), Ben-Hur Asa (2), and Reddy Anireddy S.N.(1) 1Department of Biology, Program in Molecular Plant Biology, Colorado State University, Fort Collins, CO 80523, USA 2Computer Science Department, Colorado State University, Fort Collins, CO, USA. 3Contributed equally Serine/arginine-rich (SR) and SR-like RNA binding proteins are key regulators of pre-mRNA splicing. SR45, one of the SR-like proteins, regulates alternative splicing (AS) of pre-mRNAs of SR genes and multiple developmental processes. However, the role of SR45 in global regulation of AS and gene expression is not known. To address this and to gain insights into


the mode of action of SR45 in controlling diverse processes we compared the transcriptomes of the sr45 mutant and wild type (WT) using high throughput sequencing. We identified over 1300 differentially expressed (DE) and 454 differentially spliced (DS) genes in the mutant. We detected 309 significant, differential intron retention (IR) events from 237 genes. Of these events, ~85% show higher rates of IR in the WT, suggesting a role for SR45 in promoting IR. In addition, we find evidence for 20% more 5' events and 33% more 3' events in WT, suggesting that SR45 plays a role in both 5' and 3' splice-site selection. The top over-represented Gene Ontology (GO) terms in DE and DS genes in the biological processes namespace are hormonal signaling and response to abiotic stresses including heat and in the molecular functions space is sequence-specific DNA binding transcription factors (TF) activity, suggesting that AS of TFs likely mediates some functions of SR45. Based on GO enrichment, we analyzed heat stress responses in the mutant. The sr45 mutant was highly sensitive to high temperature (38oC and 45oC) at different developmental stages. Expression and splicing of several TFs and heat shock proteins implicated in heat tolerance are misregulated in the mutant. In addition, SR45 binds to an intron of a heat shock TF. These results suggest that SR45 is a positive regulator of heat stress response and that the expression and splicing of genes involved in heat signaling might mediate SR45 function in heat tolerance. Abstract No. 116 Developing Chemically Mutagenized Soybean Populations to Analyze Gene Networks in Soybean Zhou Zhou, Shiming Liu, James Anderson, Naoufal Lakhssassi, Khalid Meksem Department of Plant, Soil Science and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901 Soybean cyst nematode (SCN)(Heterodera glycines Ichinohe) is the most economically destructive pathogen in soybean production. Planting resistant cultivars is the primary management method to control SCN, but the nature of genetic resistance is unknown. The chemical mutagen Ethyl Methanesulfonate (EMS) is utilized to induce mutations in soybean populations. The mutagenized population is screened by an efficient reverse genetic strategy known as Targeting Induced Local Lesions IN Genomes (TILLING) for functional gene analyses. The objective of this study is to analyze the role of a gene network in resistance to SCN using the EMS-mutagenized soybean populations developed. Soybean [Glycine max (L.) Merr.] cultivar 'Forrest' seeds were mutagenized with EMS and grown to generate about 3000 M1 plants. M1 plants were self-pollinated to produce M2 plants. Genomic DNA was extracted from young leaves of individual M2 plants and quantified to normalize concentration of DNAs. The DNA samples were then pooled eight-fold in 96-well plates for mutation screening by TILLING. Moreover, 11 phenotypic traits including chlorophyll deficiency, leaf shape, branch architecture, seed color and seed weight were collected from mutant populations and analyzed in this study. Abstract No. 118 Identification, Characterization, and Evolution of puf Genes in Marine Diatoms Alexander, Jessica, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 Perera, Minoli,Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 Ghoshroy, Sohini, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 Thurlow, David, Clark University, Chemistry Department, 950 Main Street, Worcester, MA 01610 Robertson, Deborah, Clark University, Biology Department, 950 Main Street, Worcester, MA 01610 Members of the PUF family of RNA binding proteins are conserved among eukaryotes and mediate the post-transcriptional regulation of gene expression by promoting RNA decay and


repressing translation. This project identified and characterized PUF proteins from three marine diatoms (Thalassiosira pseudonana, Phaeodactylum tricornutum, and Fragilariopsis cylindrus) based on the presence of the well-conserved PUM-HD RNA binding domain. The number of puf genes in diatoms varied, ranging from nine in T. pseudonana to four in P. tricornutum and F. cylindricus. As observed in other eukaryotes, the PUM-HDs were well conserved while regions outside the homology domains were highly variable. Amino acids that make contact with RNA regulatory elements were conserved in several diatom PUF proteins, suggesting these proteins are able to bind mRNA. EST data indicate that diatom puf genes are expressed under a variety of environmental conditions, suggesting they may be important in post-transcriptional gene regulation in response to environmental stimuli. Phylogenetic analyses resulted in a tree that contained 11 well-supported clades. PUFs from diatoms were observed in diatom-specific, heterokont-specific and multi-taxa clades. Our phylogenetic analyses provide evidence for lineage-specific gene-family expansions as well as evidence that some PUFs may have conserved functions in different lineages. Future experiments are aimed at identifying the mRNA targets of diatom PUF proteins in vitro, and further examining the expression and function of PUF proteins under different environmental conditions. Abstract No. 121 Arabidopsis Root miRNA Regulatory Network Supports Functional Classification of Transcriptions Factors Involved in Development and Environmental Response Young Koung Lee1, Lifang Zhang1, Allison Gaudinier2, Christophe Liseron-Monfils1, Christos Noutsos1, Mallorie Taylor-Teeples2, Siobhan M. Brady2, and Doreen Ware1,3, 1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 2Department of Plant Biology and Genome Center, UC Davis, Davis, CA 95616 3USDA-ARS, Robert W. Holley Center, Ithaca, NY 14853 Having the correct gene expressed at the right time and the right place is critical to all aspects of plants development and response to environment. Gene expression is regulated at multiple levels, including transcription and post-transcription. To better understand transcriptional and post-transcriptional gene regulatory networks (GRN), we have utilized a gene centered approach to identify transcriptions factors that control the expression of miRNAs, non-coding genes involved in development and stress. Using the Arabidopsis thaliana root as a model, we have screened a total of 130 promoters consisting of 80 miRNA and 41 protein-coding genes, with a library containing 653 TFs approximately 92% of stele expressed transcription factors (TFs). The resulting networks contained 1059 protein-DNA interactions (PDI). To validate these PDIs in vivo, we have obtained 63 homozygous TF mutants to characterize the expression of these TFs, miRNA or miRNA targets in the presence of a perturbed upstream regulator. Our recent data showed that approximately 85% of the TFs and 60% of the miRNAs tested have molecular phenotypes in TF mutant. Three percent of the mutants displayed a short root phenotype. In addition to genetic analysis, we used tobacco transient assays, and systems-level approaches including genome-wide mRNA-seq and small RNA profiling analysis. Several TFs families were also found to be highly connected, one such example is the Zinc Finger TF family. Genetic analyses of TFs mutants show phenotypes in tissues other than root suggesting the network can be more generally applied beyond root. We further expanded the GRN through integration of protein-protein (PP) and miRNA ?mRNA targets (RR) interactions in public data. We identified several sub-modules, which represent known miRNA functions as well as predicted regulators in development and environmental stress responses. To explore the potential role of miRNA in Nitrogen Use Efficiency (NUE), we used available public expression data, and identified sub-network enriched for genes with differential expression in response to nitrogen. Our results suggest the GRN in Arabidopsis root is highly robust


and buffering, and a core root network possesses transcription factor modules that can be expanded to different functional roles in other tissues. Abstract No. 122 Comprehensive Investigation of microRNAs Enhanced by Analysis of Sequence Variants, Expression Patterns, AGO Loading and Target Cleavage Thatcher , Shawn R., Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Jeong, Dong-Hoon, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Brown, Rebecca S.H., Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Zhai, Jixian, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Park, Sunhee, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Rymarquis, Linda A., Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Meyers, Blake C., Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 Green, Pamela J., Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711 MicroRNAs (miRNAs) are a class of small RNAs which usually function by guiding cleavage of target messenger RNAs. They play major roles in a variety of plant processes such as development, response to pathogens and environmental stress adaptation. To identify new miRNAs and miRNA regulation in Arabidopsis thaliana, 27 small RNA libraries were constructed and sequenced from various tissues, stresses and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. rdr2 was used to enrich the miRNA population, leading to the discovery of new miRNAs arising from both known and new precursors, and increasing the total number of Arabidopsis miRNAs by nearly 10%. Parallel Analysis of RNA Ends (PARE) data provide evidence that the majority of these new miRNAs guide target cleavage. Many libraries represented novel stress/tissue conditions, such as submergence-stressed flowers, enabling identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild type plants. By combining small RNA and target expression analysis with ARGONAUTE (AGO) immunoprecipitation data and global target cleavage data from PARE, a much more comprehensive picture of Arabidopsis miRNAs was obtained. Insights from the study of several particularly interesting miRNA expression patterns, such as the differential expression of a miRNA/ miRNA* pair, were greatly enhanced by this combinatorial approach. Supported by the NSF. Abstract No. 124 A Putative Exosome Component is Essential for Female Gametophyte Development and Required for Proper RNA Decay and Processing in Arabidopsis Kumakura, Naoyoshi, Department of Life Sciences Graduate School of Arts and Sciences, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902 Ohtsuki, Hiroka, Department of Life Sciences Graduate School of Arts and Sciences, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902 Takeda, Atsushi, Graduate School of Life Sciences, Ritsumeikan University, Noji-higashi 1-1-1, Kusatsu, Shiga, 525-8577, Japan Watanabe, Yuichiro, Department of Life Sciences Graduate School of Arts and Sciences, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902 In eukaryotic cells, the 3?-5?-exonucleolytic decay and processing of RNAs are essential in RNA metabolism. The exosome is a conserved protein complex that is responsible for 3?-5? RNA degradation. In yeast and human cells, Rrp44/Dis3 is a catalytic center of RNA exosome and is required for cell viability. Arabidopsis has two RRP44 homologs, RRP44A and RRP44B. RRP44B, a conserved RNase-II protein, localizes cytoplasm and is involved in RNA decay. Although RRP44A is essential for female gametophyte development as previously reported, the detailed role of RRP44A is still obscure. To observe the RRP44A


phenotypes more closely, we collected three T-DNA inserted mutants. Homozygous T-DNA inserted mutants were not obtained in three mutants. T-DNA insertions in RRP44A locus were normally transmitted to next generations via male gametophytes, but not via female gametophytes in all three rrp44a mutants. These data suggest that RRP44A is required for viability, specifically female gametophyte development. To know the molecular function of RRP44A, we established tissue specifically knocked-down mutants by RNAi. rRNA processing intermediates, a hallmark of exosome defects, over-accumulated in rrp44a, but not in rrp44b. In addition, some sets of RNAs accumulated in rrp44a mutants. These and other findings suggest that RRP44A is required for female gametophyte development and for RNA decay as an RNA exosome component. Abstract No. 128 Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos Fang, Yihui, Virginia Tech, Blacksburg VA Collakova, Eva, Virginia Tech, Blacksburg VA Aghamirzaie, Delasa, Virginia Tech, Blacksburg VA Klumas, Curtis, Virginia Tech, Blacksburg VA Tabataba, Farzaneh, Virginia Tech, Blacksburg VA Kakumanu, Akshay, Penn State University, University Park, PA Myers, Elijah, Virginia Tech, Blacksburg VA Heath, Lenwood, Virginia Tech, Blacksburg VA Grene, Ruth, Virginia Tech, Blacksburg VA Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants. Abstract No. 129 De Novo Assembly and Transcriptome Profiling of Salt Stress Shoot-tips of Banana (Musa acuminata) in vitro Cultures Widiyanto, Srinanan M.(1), Ilmawati, Gagas P.N.(1), Diningrat, Diky S.(1), Panchangam, Bhargavi(2), and Carlson, John E.(2) (1)School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung-40132, Indonesia. (2)The Schatz Center for Tree Molecular Genetics, School of Forest Resources, Pennsylvania State University, University Park, PA 16802. Excessive saline condition causes loss of both vegetative potential and reproductive plant growth in banana. In this study, sodium chloride at 50 mM, 100 mM and 150 mM were assessed on in vitro shoot-tip cultures of banana cv Pisang Barangan to examine the effect of salt stress treatment on its growth, rate of shoot multiplication and the freshness. After two periods of four-week in vitro culturing, the auxiliary shoot growth and shoot-multiplication rate were significantly reduced with the addition NaCl at 100 mM and 150 mM. To have a better understanding of the defense response, transcriptome profiles in salt


stress in vitro shoot-tip cultures of banana was characterized and compared to that of the normal growth ones. Two substracted cDNA libraries were constructed from both salt-stress and non salt-stress tissues of shoot-tips of banana. Subtracted cDNA sequencing was completed using Illumina paired-end sequencing technology. After sequence trimming, a total of 3,594,326 sequences in pairs (salt-stress tissues) and 3,508,448 sequences in pairs (non salt-stress tissues) were remained in two libraries. The sequence reads from the two tissues were also combined and assembled de novo, generating 48,971 contigs consisted of 26,272,763 bases with a N50 of 584 bases and an average of 536 bases. Preliminary results of mapping analyses indicated a number of genes were specifically expressed in salt-stress shoot-tip tissues. Abstract No. 130 De Novo Assembly and Transcriptome Profiling of Vegetative and Generative Buds in Teak (Tectona grandis Linn f.) Diningrat, Diky S.(1), Widiyanto, Srinanan M.(1), Pancoro Adi(1), Iriawati(1), Panchangam, Bhargavi(2), and Carlson John E.(2) (1)School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung-40132, Indonesia. (2)The Schatz Center for Tree Molecular Genetics, School of Forest Resources, Pennsylvania State University, University Park, PA 16802. Teak (Tectona grandis Linn f.) is one of the world’s premier hardwood tree species, highly famous for its quality, profile and durability of wood. Teak has a long reproductive cycle and produces low seeds. Both problems are basically related to mechanism flower development. Hence, the determination of the genetic pathways and specific genes involved in teak flowering and flower development could be beneficial for teak productivity improvement. The aim of this study was to initial the characterization of floral transcriptome in teak. The subtracted cDNA libraries of the teak tissues were sequenced using Illumina MiSeq technology and generated 3,778,316 paired-end reads sequences for vegetative bud samples and 3,701,878 paired-end reads sequences for generative bud samples. The sequences from the two tissues combined were Sequencing QC tested, Trimmed and de novo assembled using CLC Genomics Workbench. The sequence reads from the two tissues combined assembled de novo generating the 87,365 transcript contigs consist of 42,435,728 bases with N50 of 498. The transcript contigs were annotated through BlastX alignment and functionally characterized by gene ontology (GO) annotation and KEGG metabolic pathways assignments. This study represents the first exploration of teak floral bud transcriptome and the results may serve to guide further gene expression and functional genomic studies in teak. Abstract No. 139 DEAD-box RNA Helicases Identified by Orbitrap LC-MS/MS are Structural Constituents of Pollen mRNA Storing Granules Hafidh Said*,1 Potešil David,2,3 Zdráhal Zbynek,2,3 and Honys David 1 1) Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Praha 6, Czech Republic 2) CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic 3) National centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic Sequestration from translation of protein coding transcripts has emerged as a widespread phenomenon during gametogenesis. Repressed transcripts are confiscated into mRNA storing granules and only utilized later during development and at early stages of embryogenesis. Here, we have re-analysed proteome of the tobacco pollen aggregated mRNPs granules (EPP complexes) containing translationally sequestered mRNAs, non-coding RNAs and associated protein complexes. We have identified three isoforms of the DEAD-box


RNA helicase family of eukaryotic translation initiation factor 4A (eIF4A)-8,9,13 as structural components of the EPP RNA granules with likely role in ATP-dependent helicase activities. RNA helicases of the DEAD-box and the related DExD/H protein families are universal and are involved in almost all aspects of RNA metabolism. In Arabidopsis, SLOW WALKER3, a DEAD-box RNA helicase play key role during female gametogenesis. Extended analysis of tobacco Agilent chip data led to the identification of additional 49 helicases that are expressed as late as 24 h in pollen tubes grown in vitro. Among them, EMB2733/ESP3, a homologue of yeast PRP2 and related to the DEAH RNA helicases, is essential for embryogenesis and is extensively phosphorylated. The role of these helicases in fostering gametogenesis and embryogenesis development is worth an investigation. To extend the link of helicases key role in gametogenesis and embryogenesis, a recent report has localized two DEAD-box RNA helicases, RH11 and RH37, and four RNA-binding proteins within leaf peroxisomes. Both proteins are highly expressed and accumulated during pollen maturation coinciding with changes in RNA metabolism. Peroxisomes were reported to be important for male-female gametophyte cross-talk. A likely association of some of the helicases and other RNA-binding proteins to peroxisomes opens new challenges. Taken together, helicases identified in this study are likely to be integral for EPP mRNA granules assembly and development of both gametophytes as well as in events pre- and post-fertilization.


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book of abstracts of symposium post-transcriptional gene regulation in plants

Documents

crespi abstract

motoaki seki abstract

sarah assmann abstract

vinay nagarajan abstract

peter moffett abstract

dominique gagliardi

yuda fang abstract

yukako chiba abstract