Phosphorylation of a NAC transcription factor by ZmCCaMK regulates abscisic acid-induced antioxidant defense in maize

Outcomes• Functional analysis reveals that ZmNAC84 is essential for

ABA-induced antioxidant defense in a ZmCCaMK-dependent manner.

• Moreover, overexpression of ZmNAC84 in tobacco canimprove drought tolerance, and alleviate drought-inducedoxidative damage of transgenic plants.

Zhu et al. (2016) ”Phosphorylation of a NAC transcription factor by ZmCCaMK regulates abscisic acid-induced antioxidant defense in maize.” Plant Physiol. DOI: http:/ / dx. doi. org/ 10. 1104/ pp. 16. 00168

Background• Calcium/calmodulin-dependent protein kinase (CCaMK) has

been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance ofplants to drought stress. CCaMK is also central to theinteractions with symbiotic microbes.

• However, its downstream molecular events are poorlyunderstood.

Significance• These results define a mechanism for ZmCCaMK function in ABA-

induced antioxidant defense, where ABA-produced H2O2 first inducesexpression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK,and subsequently the activated ZmCCaMK phosphorylatesZmNAC84 at S113, thereby inducing antioxidant defense byactivating downstream genes.

Approach• Here, we identify a NAC transcription factor, ZmNAC84,

in maize, which physically interacts with ZmCCaMK invitro and in vivo.

CCaMK is a key signalling component in plants.CCaMK is involved in symbiosis with mycorrhizal fungi andnitrogen fixing bacteria. It is also involved in the response to abioticstress. How the kinase can have these multiple function sis notunderstood at the molecular level. Figure is from Oldroyd (2013,Nat Rev Microbiol).

CCaMK phosphorylates the transcription factorZmNAC84. The transcription factor is known to directlyactivate a number of stress response genes. Serine-113 isphospphorylated by CCaMK and this activates the transcriptionfactor. When Ser-113 is mutated to Ala, the signal transductionis inhibited. When Ser-113 is mutated to the phosphorylationmimim Asd, the activation is constitutive.

Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase Required for Rice Male Reproduction

Outcomes• Compared with the wild type (WT), dpw2 anthers have

increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase.

• In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors.

Pollen development is impacted indpw2 mutants. The Pollen in dpw2-1(E) and dpw2-2 (F) are collapsedcompared to the wild-type pollen (D).Scale bars are 10 µm.

Xu et al. (2016) “Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase Required for Rice Male Reproduction.” Plant Physiol. DOI: 10.1104/pp.16.00095

Background• Aliphatic and aromatic lipids are both essential structural components

of the plant cuticle, an important interface between the plant and environment and critical for drought tolerance.

• Although crosslinks between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin, root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive.

Significance• DPW2 plays a fundamental role in pollen development via the

biosynthesis of key components of the anther cuticle and pollen wall.

DPW2 is a feroloyl: ω-hydroxy fatty acidtransferase. Heterologouslyexpressed protein wasincubated with feruloyl-CoAand ω-hydroxy palmitic acid.The product ester wasidentified by massspectrometry. The enzume canalso use sinapoyl-CoA but notcoumarouyl-CoA or caffeoyl-CoA. C15 and C17 ω-hydroxyfatty acids are also goodsubstrates, but longer orshorter compounds are not.

Approach• In this study, we characterized the rice male sterile

mutant, defective pollen wall 2 (dpw2), which showed anabnormal anther cuticle, a defective pollen wall, andcomplete male sterility.

DPW2 is specifically expressed inanthers. Rice plants weretransformed with PPW2promoter-GUSconstructs and stained for expressionat different time points ofdevelopment.

Characterizing strain variation in engineered E. coli using a multi-omics based workflow.

Brunk et al. (2016) “Characterizing strain variation in engineered E. coli using a multi-omics based workflow”, Cell Systems, DOI: 10.1016/j.cels.2016.04.004

Background• Understanding complex metabolic interactions in engineered

microbes is one of the major challenges in biofuel R&D.• The development of omics technologies, such as metabolomics and

proteomics, and systems biology has dramatically enhanced ourability to understand biological phenomena, but the interpretation oflarge omics data into meaningful ‘knowledge’ is still very challenging.

Significance• By integrating the strengths of synthetic biology (metabolic

engineering) and systems biology, we can drive the transition fromvision to conception of a designed working phenotype.

• We shared this workflow as an open-source tool in the form ofiPython notebooks, and it will allow anyone in microbial engineeringfield to apply this workflow to their system

Approach• We present a workflow that integrates various omics data and

genome-scale models (Fig 1)• We obtained and analyzed large omics data set from eight

engineered strains producing three different biofuels (Fig 2) for theworkflow demonstration

Fig 1. Overall workflow presented in this work

Fig 2. Biofuels pathways used for this work

Outcomes• From the workflow, we identified the roles of candidate genes,

pathways, and biochemical reactions in observed experimentalphenomena, which helps studying the effects of biofuel production in amicrobial host.

• We finally used this approach to facilitate the construction of a mutantstrain with improved productivity

A genetic screen identifies a requirement for cysteine-rich–receptor-like kinases in rice NH1 (OsNPR1)-mediated immunity

Outcomes• We identify the snim1 mutant and demonstrate that cysteine-rich-receptor-

like kinase CRK6 and CRK10 complement the snim1 mutant.• Silencing of CRK6 and CRK10 individually recreates the snim1 phenotype;

a crk10 frameshift mutant displays compromised immunity.• Elevated NH1 levels enhance CRK10 expression while reduced NH1

levels decrease CRK10 expression.• Rice TGA2.1 protein binds to the CRK10 promoter.

Chern et al. (2016) “A genetic screen identifies a requirement for cysteine-rich–receptor-like kinases in rice NH1 (OsNPR1)-mediated immunity.” PLoS Genetics, DOI: 10.1371/journal.pgen.1006049

Background• Systemic acquired resistance, mediated by Arabidopsis NPR1

and rice NH1, confers broad-spectrum immunity to diverse pathogens.

• NPR1 and NH1 interact with TGA transcription factors to activate downstream signaling; the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined.

Significance• These experiments demonstrate a requirement for CRKs in NH1-mediated

immunity and establish a molecular link between NH1 and induction of CRK10 expression.

• CRKs are newly identified genes that can be used to engineer biofuel crops for enhanced disease resistance and sustainability.

Approach• Forward genetics approach to identify rice mutants (snim) that

block the NH1-mediated immunity.• Comparative genome hybridization to identify the gene(s).

NH1ox snim1

Xoo induceddisease lesions

A

NH1ox snim1

BTH inducedlesion mimics

B1) The snim1 mutant is compromised in immunity to Xoo and BTH-induced necrotic lesion formation. Two representative leaves each are displayed for the NH1ox parent and the snim1 mutant in (A) and (B). Inoculation with Xoo was carried out with the scissor-dip method. (A) Xoo-induced, long water-soaked disease lesions in the snim1 mutant. (B) Lesion mimic necrotic spots. The necrotic spots (marked with arrowheads) developed one week after application of 1 mM BTH.

BTH

CRK10 gene Immunity

NH1

TGA

CRK10

Plasmamembrane

2) Working model:

Tissue-specific distribution of hemicelluloses in six different sugarcane hybrids as related to cell wall recalcitrance

Outcomes• Evaluation of the digestibility of sugarcane polysaccharides by

commercial enzymes indicated that the cell wall recalcitrance variedconsiderably along the internode regions and in the sugarcanehybrids.

• Pith regions of the hybrids with high MLG and low-lignin contentsreached up to 85 % cellulose conversion after 72 h of hydrolysis,without any pretreatment.

The six sugarcane hybrids differ substantially in theircell wall composition. Here is shown mixed-linkage glucancontent. Lignin, cellulose crystallinity, hemicellulose conposiition,etc., were also analyzed biochemically and by immunofluoresceneimaging (not shown). Example of immunofluorescence labeling ofxylan in interface (upper) and pith (lower) is shown to the right.

Costa et al. (2016) ”Tissue-specific distribution of hemicelluloses in six different sugarcane hybrids as related to cell wall recalcitrance.” Biotechnology for Biofuels, DOI:10.1186/s13068-016-0513-2

Background• Grasses are lignocellulosic materials useful to supply the billion-

tons annual requirement for renewable resources that aim toproduce transportation fuels and a variety of chemicals.

• However, the polysaccharides contained in grass cell walls arepresent in a recalcitrant composite. Deconstruction of these cellwalls is still a challenge.

Significance• The collective characteristics of the internode regions were related to

the varied recalcitrance found in the samples.• Components such as lignin and GAX were critical for the increased

recalcitrance, but low cellulose crystallinity index, high MLG contents,and highly substituted GAX contributed to the generation of a lessrecalcitrant material.

Saccharification differs between the sugarcane hybrids.Biomass samples were extracted to remove sucrose, milled anddigested with Ctec2 for 72 hrs at 45 C.

Approach• Six different sugarcane hybrids were used as model

grasses to evaluate the tissue-specific distribution ofhemicelluloses and the role of these components in cellwall recalcitrance.

From Sugars to Wheels: The Conversion of Ethanol to 1,3-Butadiene over Metal-Promoted Magnesia-Silicate Catalysts

Outcomes• A process for the conversion of ethanol to 1,3-BD, which uses our catalyst

and accounts for separations and product logistics• High 1,3-BD selectivity (60-75%) in a one-step process, with minimal

conversion of ethanol to butenes and ethylene, and no production of diethyl ether and acetylene.

• Net carbon-negative process relative to petroleum (155% GHG reduction)

Shylesh et al. (2016) “From Sugars to Wheels: The Conversion of Ethanol to 1,3-Butadiene over Metal-Promoted Magnesia-Silicate Catalysts”, ChemSusChem, doi: 10.1002/cssc.201600195

Background• 1,3-Butadiene (1,3-BD) is a high-value chemical intermediate produced from

naphtha crackers and used mainly as a monomer for the production of synthetic rubbers

• 1,3-BD prices have fluctuated in recent years, reaching $4740/ton• 1,3-BD can be produced renewably by chemically converting bio-based

ethanol, but reported yields have been low (10-30% for MgO-SiO2 catalysts)

Significance• One-step, low-carbon bio-based 1,3-BD production process capable of

competing with fossil-based pathways, particularly when market conditions necessitate energy-intensive on-purpose 1,3-BD production

Simplified process flow diagram that depicts the various steps in the conversion of ethanol to 1,3-BD.

Approach• Development and investigation of a new catalyst and process for the

one-step conversion of ethanol to 1,3-BD• Chemical process and life-cycle GHG modeling of pathways using

Au/MgO-SiO2 catalysts starting from corn grain-, sugarcane-, and corn stover-derived ethanol

Life-cycle GHG Emissions per kg of 1,3-BD.

Fractional pretreatment of raw and calcium oxalate-extracted agave bagasse using ionic liquid and alkaline hydrogen peroxide

Outcomes• IL pretreatment had more pronounced impact on cellulose crystallinty for

all samples and conditions studied• Calcium oxalate extraction significantly improved saccharification yields

after IL pretreatment• Calcium oxalate extraction significantly reduced saccharification yields

after AHP pretreatment

Perez-Pimienta et al. (2016) “Fractional pretreatment of raw and calcium oxalate-extracted agave bagasse using ionic liquid and alkaline hydrogen peroxide.” Biomass and Bioenergy, DOI:10.1016/j.biombioe.2016.05.001.

Background• Agave bagasse (AGB) is a promising bioenergy feedstock in

Mexico and Central America• Previous studies have shown that the presence of calcium oxalate

can adversely impact pretreatment efficiency

Significance• Highlights the importance of understanding of all components found in

plant cell walls and how they impact deconstruction• Ionic liquids generated highest sugar yields observed

Impact of ionic liquid (IL) and alkaline hot peroxide (AHP) pretreatments on (top) cellulose crystalllinity index and (bottom) sugar yields from intact (AGB) and extracted (EAB) agave bagasse.

Approach• Compared alkaline hot peroxide (AHP) and ionic liquid (IL)

pretreatment before and after calcium oxalate extraction• Monitored crystallinity index and sugar yields as a function of process

severity