no primexine and plasma membrane undulation is … · no primexine and plasma membrane undulation...

NO PRIMEXINE AND PLASMA MEMBRANEUNDULATION Is Essential for Primexine Deposition andPlasma Membrane Undulation during Microsporogenesisin Arabidopsis1[OA]

Hai-Shuang Chang2, Cheng Zhang2, Yu-Hua Chang, Jun Zhu, Xiao-Feng Xu, Zhi-Hao Shi, Xiao-Lei Zhang,Ling Xu, Hai Huang, Sen Zhang3, and Zhong-Nan Yang3*

College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China (H.-S.C.,Y.-H.C., J.Z., X.-F.X., Z.-H.S., X.-L.Z., S.Z., Z.-N.Y.); Department of Biology, East China Normal University,Shanghai 200062, China (C.Z., L.X.); and National Key Laboratory of Plant Molecular Genetics, Institute ofPlant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai 200032, China (H.H.)

Primexine deposition and plasma membrane undulation are the initial steps of pollen wall formation. However, little is knownabout the genes involved in this important biological process. Here, we report a novel gene, NO PRIMEXINE AND PLASMAMEMBRANE UNDULATION (NPU), which functions in the early stage of pollen wall development in Arabidopsis (Arabidopsisthaliana). Loss of NPU function causes male sterility due to a defect in callose synthesis and sporopollenin deposition, resultingin disrupted pollen in npu mutants. Transmission electronic microscopy observation demonstrated that primexine depositionand plasma membrane undulation are completely absent in the npu mutants. NPU encodes a membrane protein with twotransmembrane domains and one intracellular domain. In situ hybridization analysis revealed that NPU is strongly expressedin microspores and the tapetum during the tetrad stage. All these results together indicate that NPU plays a vital role inprimexine deposition and plasma membrane undulation during early pollen wall development.

The pollen wall plays a pivotal role in pollen-stigmarecognition, pollen hydration, and pollen protectionon the stigma and pollen tube elongation (Zinkl et al.,1999; Edlund et al., 2004; Scott et al., 2004). The pollenwall is organized in a highly complex manner, withthree major layers: exine, intine, and tryphine (Heslop-Harrison, 1971; Piffanelli et al., 1998). The exine is theouter layer of the pollen wall and includes the sexine(tectum and bacula) and nexine (foot layer). Located

between the exine layer and plasma membrane, theintine is mainly comprised of cellulose, pectin, andproteins (Brett and Waldron, 1990). Tryphine is se-creted from the tapetal cells and deposited onto theexine layer during the later stages of pollen develop-ment.

Pollen wall development is a complicated process inwhich a set of elaborate and coordinated mechanismsare carried out by both the microspores and tapetum(Blackmore et al., 2007). It is initiated along with thetermination of meiosis (Paxson-Sowders et al., 1997;Piffanelli et al., 1998). Close to the end of meiosis, themicrospores are enclosed by the callose wall, whichserves as a mold for primexine patterning and alsoprotects the meiocytes and tetrads (Waterkeyn andBienfait, 1970; Worrall et al., 1992; Dong et al., 2005). Atthe tetrad stage, the primexine is deposited betweenthe plasma membrane and the inner callose wall(Fitzgerald and Knox, 1995; Paxson-Sowders et al.,1997), and the plasma membrane starts to invaginateand form the undulations that are common to variousspecies (Dahl, 1986; Dickinson and Sheldon, 1986;Takahashi, 1989; Fitzgerald and Knox, 1995). On thepeaks of the undulations, probaculae are deposited ina well-regulated fashion and eventually form themature pollen exine layer (Paxson-Sowders et al.,1997). Therefore, the primexine deposition and timely

1 This work was supported by the National Natural ScienceFoundation of China (grant nos. 30925007 and 30971553) and theNational Basic Research Program of China (grant no. 2007CB947600)and by the Leading Academic Discipline Project of Shanghai Mu-nicipal Education Commission (grant no. J50401). This work wasalso supported by the National Key Laboratory of Plant MolecularGenetics of China.

2 These authors contributed equally to the article.3 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] authors responsible for distribution of materials integral to

the findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org)are: Sen Zhang ([email protected]) and Zhong-Nan Yang([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.111.184853

264 Plant Physiology�, January 2012, Vol. 158, pp. 264–272, www.plantphysiol.org � 2011 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon August 3, 2019 - Published by Downloaded from

Copyright © 2012 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

undulation of the plasma membrane both play impor-tant roles in early pollen wall formation (Rowley andSkvarla 1975; Takahashi and Skvarla, 1991; Fitzgeraldand Knox, 1995).At present, a number of mutants have been identi-

fied that are involved in primexine formation andmicrospore membrane undulation in Arabidopsis(Arabidopsis thaliana). In the no exine formation1(nef1)mu-tant, the primexine is coarsely developed, and someparts of the plasma membrane of the microspore aredisrupted at the later stages. The NEF1 protein ispredicted to be a plastid integral membrane protein(Ariizumi et al., 2004). In the mutant defective in exineformation1 (dex1) the primexine deposition is delayedand reduced in thickness, and the undulation of themicrospore plasma membrane is scant (Paxson-Sowderset al., 1997, 2001). As a result, the exine pattern is notformed properly, and the pollen wall does not form. TheDEX1 protein is predicted to be a membrane-associatedprotein that has at least two calcium-binding ligands(Paxson-Sowders et al., 2001). In the ruptured pollengrain1 (rpg1) mutant, the primexine is irregularly de-posited, and as a result only a spotted, irregular exinelayer forms. The microspores are ruptured, with cyto-plasmic leakage. The RPG1 protein is a membraneprotein of the MtN3/saliva family that functions as asugar transporter (Guan et al., 2008; Chen et al., 2010).These reported proteins are helpful in understandingthe importance of primexine deposition and plasmamembrane undulation on a molecular basis.In this article, we report on the gene NO PRIMEX-

INE AND PLASMA MEMBRANE UNDULATION(NPU), which encodes a transmembrane protein inArabidopsis. The callose synthesis is severely disrup-ted, and the absence of primexine deposition andplasma membrane undulation leads to the defects ofmicrosporogenesis and male sterility in the npu mu-tants. Functional analysis of NPU indicates that itplays an important role in primexine deposition andplasma membrane undulation during pollen wall for-mation in Arabidopsis.

RESULTS

The npu-1 Mutant Exhibits a Male Sterility Phenotype

The npu-1mutantwas isolated from a pool of T-DNA-tagged lines (Li et al., 2005). Themutant plants exhibitedreduced fertility with normal vegetative growth (Fig.1A). The average seed yield of the mutant plants wasapproximately 0.43% of the wild type. Backcrossingwith wild-type pollen grains resulted in F1 plants withnormal fertility. This result indicated that male fertilitywas hampered and female fertilitywas unaffected in thenpu-1mutant. The progeny of the self-pollinated hetero-zygous plants segregated wild-type and mutant phe-notypes at a 3:1 (215:74) ratio, which indicates that thephenotype of npu-1 is controlled by a single recessivelocus.

Molecular Cloning of the NPU Gene

To identify the corresponding gene of the npu-1mu-tant, we performed thermal asymmetric interlaced(TAIL)-PCR (Liu et al., 1995). A genomic DNA frag-ment that flanked the left border of T-DNA wasobtained. Sequencing of the TAIL-PCR products indi-cated that the T-DNA was inserted into the seventhexon of a predicted open reading frame of At3g51610(Fig. 2A). PCR analysis using T-DNA and genome-specific primers showed that all of the mutant plantsanalyzed (n . 80) were homozygous for the insertion(data not shown). Therefore, the phenotype of the npu-1mutants is evidently linked with the T-DNA insertion.To ensure that NPU is in fact At3g51610, a complemen-tation experiment was conducted. A 4.7-kb DNA frag-ment containing the genomic sequence of NPU, asequence 1.5-kb upstream from the translation initia-tion codon and 800 bp downstream from the stopcodon, was cloned fromwild-type Arabidopsis and in-troduced into the homozygous mutant plants. A totalof 40 transgenic plants were obtained and all of themexhibited normal fertility (Fig. 1A). This demonstratedthat At3g51610 is the NPU gene, and the 4.7-kb ge-nomic DNA fragment contains sufficient informationfor normal NPU function.

We also obtained two additional npu mutant alleles.The T-DNA-tagged line SALK-062174 (npu-2) wasobtained from the SIGnAL collection at the Arabidop-sis Biological Resource Center (Fig. 1A). PCR analysisconfirmed the T-DNA insertion into the second exon ofNPU (Fig. 2A). The npu-3 mutant was screened froman ethyl methanesulfonate-induced population in ourlaboratory. Sequence analysis revealed a point muta-tion from G to A, in 246th base after the start codon ofthe NPU genomic sequence in the mutant. This tran-sition causes a premature termination at the secondexon of At3g51610 (Fig. 2A). The phenotypes of bothnpu-2 and npu-3were similar to npu-1 (Fig. 1A), exceptthat they are completely male sterile.

Microsporogenesis of the npu Mutant Is Disruptedafter Meiosis

To analyze the male fertility defects of the npumutant, Alexander staining (Alexander, 1969) wasperformed. As shown in Figure 1B, wild-type pollengrains were stained purple. In the locules of the npu-1mutant, therewere a few purple-stained pollen grains.However, no normal pollen was observed in the loculesof either the npu-2 or npu-3 mutant. These observationsare in agreement with their fertility patterns, in that npu-1is partially sterile, and npu-2 and npu-3 are completelysterile.

We then performed anther cross sections to compareanther development in the wild-type and npu-3 plants(Fig. 3, A–L). According to the 14 well-ordered an-ther development stages in Arabidopsis reported bySanders et al. (1999), the anther development in npu-3was similar to that of the wild type from stage 1 to

The NPU Gene

Plant Physiol. Vol. 158, 2012 265 www.plantphysiol.orgon August 3, 2019 - Published by Downloaded from



stage 6. However, at stage 7, the tetrads of npu-3seemed to be slightly different from that of wild type.The microspores were more closely wrapped insidethe tetrad of npu-3 mutant than that of wild type (Fig.3, A and B). At stage 8, the wild-type microspores wereangular in shape, while the mutant microspores wereround in appearance (Fig. 3, C and D). At stage 9, thewild-type microspores became vacuolated and devel-oped the basis of a regular exine wall (Fig. 3E), whilethe npu-3 microspores began to degenerate (Fig. 3F).

During stages 10 and 11, most microspores in npu-3degenerated (Fig. 3, H and J), while the wild-typelocules were filled with well-shaped microspores (Fig.3, G and I). At stage 12, the microspores of the wildtype were released from the locules following antherdehiscence (Fig. 3K). However, the microspores in npu-3 were completely aborted (Fig. 3L). These resultsshowed that the microspore development started to beabnormal from stage 7. To further examine the defectof microspore development at stage 7, aniline blue was

Figure 1. Characterization of the npumutants. A,The wild type (Columbia [Col] and Landsbergerecta [Ler]), the npu allele mutant, and theNPU-complemented plants. B, Alexander staining ofthe wild-type and npu anthers. SEM examinationof dehiscent anthers and pollen grains of wild-type (C and E) and npu-3 (D and F) plants. C andD, Dehiscent anthers. The wild-type anther con-tains numerous pollen grains, while the npu-3anther is filled with degenerated microspores.Bars = 100 mm. E, Wild-type pollen grains with aregular reticulate exine pattern. Bar = 5 mm. F,Ruptured npu-3 microspores of stages 9 and 10with defective exine pattern formation. Bar =2 mm.

Figure 2. Characterization of theNPU gene. A, Gene structure of NPU and the T-DNA insertion. Black boxes, exons; gray lines,introns; light-gray lines, untranslated regions. npu-1, npu-2, and npu-3 are allele mutant lines. Left (LB) and right (RB) borders ofT-DNA sequences are shown. DNF and DNR, The left border and right border of the DNA fragment. B, Predicted proteinstructure of NPU. Gray box, plasma membrane; black bar, transmembrane regions. C. Subcellular localization of 35S:GFPinflorescence in transgenic wild-type protoplast. Green fluorescence indicates the localization of 35S:GFP protein. Bar = 20 mm.D, Subcellular localization of NPU:GFP inflorescence in transgenic wild-type protoplast. Green fluorescence indicates thelocalization of NPU:GFP protein; red fluorescence is the autofluorescence of chloroplasts. Bar = 20 mm.

Chang et al.

266 Plant Physiol. Vol. 158, 2012 www.plantphysiol.orgon August 3, 2019 - Published by Downloaded from



used to stain callose. The result showed that the callosewall between microspores in the tetrads of npu-3 wasmuch thinner than that of the wild type (Fig. 3M). Itwas reported that CalS5 was related with callosesynthesis, and A6 was proposed to be related withcallose dissolution in Arabidopsis (Hird et al., 1993;Dong et al., 2005). The expression of CalS5 was largelydown-regulated in npu-3, while the expression level ofA6 was similar to that of the wild type (Fig. 3, N andO). This demonstrated that NPU affected the callosesynthesis in anther development.

Primexine Deposition or Microspore Plasma MembraneUndulation Was Not Observed in npu-3

To further elucidate the cause of pollen degenerationin npu plants, we compared the ultrastructure of themicrospore development in the wild-type and npu-3plants by scanning electron microscopy (SEM) andtransmission electron microscopy (TEM). As shown inFigure 1C, the wild-type anther was filled with maturepollen grains after dehiscence, and the pollen grains

were plump in shape. By contrast, broken pollen andpollen remnants were observed in the npu-3 anther(Fig. 1D). The mature wild-type pollen grains werecovered with orderly reticulate pollen exine (Fig. 1E).Nevertheless, the reticulate exine pattern was seldomobserved on the surface of the npu-3 pollen grains (Fig.1F). These results indicate that the exine pattern for-mation and the sporopollenin deposition were defec-tive in npu-3 pollen.

To further clarify the details of abnormal exine de-velopment of npu-3 pollen, we performed TEM obser-vation. At the early tetrad stage in the wild type, boththe primexine deposition and regular plasma mem-brane undulation were observed (n = 25), and theprobaculae was observed growing on the membraneundulation peaks (Fig. 4, A and C). By contrast, neitherprimexine deposition nor regular undulation of themicrospore membrane occurred around the micro-spores of npu-3 (n = 28), and the globular sporopollen-nin was observed on the surface of plasma membrane(Fig. 4, B and D). After the microspores released fromtetrads, the primexine was still visible, and the exine

Figure 3. Anther development, callose wall, and expression analyses of A6 and CalS5 in wild-type and npu-3 mutant plants. Aand B, Anthers of tetrad stage. npu-3 tetrads (A) seemed to be closely compacted compared with those of the wild type (B). C andD, Anthers at stage 8. npu-3 microspores (C) are round compared with wild-type microspores (D). E and F, Anthers at stage 9.npu-3microspores began to be degenerated (F). G and H, Anthers at stage 10. Cytoplasm of npu-3microspores was disintegrated(H). I and J, Anthers at stage 11. Most npu-3 microspores were degenerated (J). K and L, Anthers at stage 12. Remnants ofmicrospores were observed in npu-3 anther locule (L). M, Callose wall of npu-3 mutant was thinner around the microsporescompared with that of the wild type. N, Semiquantitative RT-PCR analyses of A6 and CalS5 in wild-type and npu-3 floral buds.TUB, TUBULIN expression as a control. O, Real-time PCR analysis of CalS5 in wild-type and npu-3 floral buds. E, epidermis; En,endothecium; MSp, microspore; PG, pollen grain; RM, remnants of microspores; T, tapetum; Tds, tetrads. Bars = 10 mm.

The NPU Gene




layer formed a regular pattern in the wild type (Fig. 4E).By contrast, the exine pattern was not observed in thenpu-3 mutants, and the globular sporopollenin wasrandomly deposited on the plasmamembrane (Fig. 4F).Later, the primexine disappeared and the typical exinepattern successfully formed, including the tectum andthe foot layer in the wild type (Fig. 4G). However, muchsporopollenin was accumulated irregularly on the pol-len surface of the npu-3 mutant plants instead of thetypical exine pattern (Fig. 4H). These TEM observationsshowed that primexine was not deposited on theplasma membrane and the undulation did not formin npu-3 during microsporogenesis, which resulted inthe altered exine pattern formation in the npu-3 mu-tants.

NPU Encodes an Unknown Protein Localized to the

Plasma Membrane

The NPU gene encodes a functionally unknownprotein of 230 amino acids that is predicted to localizeto the plasma membrane. Residues 35 to 177 werepredicted to be on the cytoplasmic side of the mem-brane with residues 1 to 14 and 201 to 230 beingextracellular. To confirm the subcellular localization ofthe NPU protein, we fused this gene with GFP drivenby the Cauliflower mosaic virus 35S promoter. Theconstruct was transformed to the protoplast of thewild type. As shown in Figure 2C, the GFP signalcould only be detected on the plasma membrane. Thisresult confirmed that NPU is a plasma membraneprotein.

Pfam database (http://www.sanger.ac.uk/Software/Pfam/) analysis showed that there was only one copyof the NPU gene in Arabidopsis. Furthermore, homo-logs of the NPU protein were identified in various

plant species by BLASTp and tBLASTn searches in theNational Center for Biotechnology Information data-base (Fig. 5A) and The Institute for Genomic ResearchFunctional Genome Database (Fig. 5B). No significantsequence similarity was found outside the plant king-dom (Fig. 5). The homologs of NPU showed strongconservation in plants, including a rice (Oryza sativa)protein with 70% identity, a grape (Vitis vinifera) pro-tein with 81% identity, a poplar (Populus trichocarpa)protein with 77% identity, a maize (Zea mays) proteinwith 69% identity, a castor bean (Ricinus communis)protein with 75% identity, a sorghum protein (Sorghumbicolor) with 70% identity, a moss (Physcomitrella patensssp.) protein with 38% identity, and a green algae(Chlamydomonas reinhardtii) with 38% identity to NPUprotein. However, the function of these proteins ispresently unknown.

NPU Is Highly Expressed in Microspores andTapetal Cells

We performed semiquantitative reverse transcrip-tion (RT)-PCR to analyze the expression levels in roots,stems, leaves, inflorescences, and 10-d-old seedlings.The results showed that NPU was expressed in all ofthese organs, with the strongest expression in buds(Fig. 6A). To obtain the precise expression pattern ofNPU during anther development, RNA in situ hybrid-ization experiments were performed. The hybridiza-tion signal was first detected in the microsporocytesand tapetal cells at stage 5 (Fig. 6B) and became strongerat stage 6 (Fig. 6C). The signal was the strongest intetrads and tapetal cells at stage 7 (Fig. 6D) and de-creased significantly during stages 8 and 9 in develop-ing microspores and tapetal cells (Fig. 6, E and F). Theseresults were in accordance with the TEM observations

Figure 4. Ultrastructure of the pollen wall devel-opment in wild-type (A, C, E, and G) and npu-3(B, D, F, and H) plants. A and C, Tetrad stage.Primexine was deposited between the plasmamembrane and the inner callose wall, and theprobaculae was growing on the peaks of theundulation. Arrowheads show the microsporemembrane undulation. B and D, Tetrad stage.Arrow indicates the absence of primexine depo-sition and microspore membrane undulation. Eand F, Released microspore stage. Baculae andtectum were formed in wild-type microspores; nobaculae or tectum was formed on npu-3 micro-spores, only globular sporopollenin. G and H,Stage 9 microspore. Sporopollenin was randomlyaccumulated around the degenerated npu-3 mi-crospores. Ba, baculae; CW, callose wall; DMsp,degenerated microspore; Msp, microspore; Pe,primexine; Pm, plasma membrane; Pb, probacu-lae; SP, spotted sporopollenin; Tc, tectum. Bars =1 mm.

Chang et al.




of npu-3 and together suggested that NPU functionsmainly at the tetrad stage.

DISCUSSION

npu Is a Novel Male Sterile Mutant with a Defect in

Sporopollenin Deposition

In Arabidopsis, many male-sterile mutants havebeen reported. In this study, we identified a newmale-sterile mutant of npu. Of the three alleles, theweak allele npu-1 exhibits reduced male fertility. How-

ever, both npu-2 and npu-3 are completely male sterile.This shows that the NPU gene is essential for malefertility. In npu-1, T-DNAwas inserted in the 3# termi-nus of this gene, while the T-DNA insertion in npu-2and the point mutation in npu-3 were located in the 5#terminus (Fig. 2A). The position of the T-DNA inser-tion in this gene was consistent with their fertilityphenotype. Cytological analysis revealed that malesterility is caused by the defect in pollen wall forma-tion (Fig. 3). Callose synthesis, callose dissolution,sporopollenin synthesis, and deposition play impor-tant roles in pollen wall formation during antherdevelopment (Worrall et al., 1992; Dong et al., 2005;

Figure 5. Phylogenetic analysis of NPU and homologous proteins. A, Multiple alignments of NPU and its homologs. Black bars,putative transmembrane regions; thin black line, predicted intracellular region; black curved sweeping lines, predictedextracellular domains. Protein sequence of NPU homologs in plants are as follows: Sh, Sorghum bicolor, SORBI-DRAFT_09g005000; Zm, Zea mays, LOC100278212; Os, Oryza sativa, Os05g0168400; Rc, Ricinus communis,XP_002532862.1; Pp, Populus trichocarpa, XP_002323065.1; Vv, Vitis vinifera, XP_002270471.1; Ph, Physcomitrella patens,XP_001784831.1; Ch, Chlamydomonas reinhardtii, XP_001690235.1. B, Unrooted phylogenetic tree of NPU and its homo-logous proteins. Protein sequences of NPU and its homologs were analyzed with the neighbor-joining method by MEGA 4.0software. The numbers at the nodes represent percentage bootstrap values based on 1,000 replications.

The NPU Gene




de Azevedo Souza et al., 2009). Our research showedthat callose synthesis and sporopollenin depositionwere abnormal in npumutants. The high expression ofNPU at stage 7 (Fig. 6D), the defective tetrads in thenpu mutants (Fig. 4, G and I), and the largely down-regulated expression of CalS5 in npu buds indicate thatNPU functions at the early stages of pollen wallformation. Besides, primexine deposition and plasmamembrane undulation were defective in npu mutants,which led to the defect in sporopollenin deposition(Fig. 4). This further confirms that NPU is responsiblefor the early stages of pollen wall formation.

Primexine and Plasma Membrane Undulation

Fitzgerald and Knox (1995) reported the events inearly pollen wall development using rapid freeze-substitution technology. Themicrospores in the tetradswere initially surrounded by a callose wall after mei-osis. After the primexine matrix had assembled be-tween the microspore plasma membrane and callosewall, the plasma membrane gradually undulated andformed crypts. Several mutants were reported to affectthe early events of pollen wall development. In thedex1 mutant, the primexine matrix assembly is bothreduced and delayed, and the plasma membrane un-dulation of dex1 fails to form (Paxson-Sowders et al.,1997, 2001). In the nef1 mutant, the primexine matrixassembly appears to be coarser than that of the wildtype, and the undulation of the nef1microspore plasmamembrane appears to be abnormal (Ariizumi et al.,2004). NPU is a critical protein in primexine matrixassembly, as the primexine matrix is completely absentin npu mutants (Fig. 4).

Southworth and Jernstedt (1995) proposed a Ten-segrity model to explain the processes of exine pat-terning. After the primexine matrix is secreted frommicrospores, the hydrated primexine matrix exertsosmotic pressure on the microspore cell surface, re-sulting in a change in the cytoskeletal tension. Subse-

quently, the plasma membrane begins to undulate andthe exine layer is assembled. In npu-3, the primexinematrix is completely absent and plasma membrane isnot undulated (Fig. 4). Based on this model, we believethat the plasmamembrane undulation is dependent onthe primexinematrix during primexine formation. Dueto the lack of primexine matrix, the osmotic pressure isnot exerted on the microspore surface, and the plasmamembrane fails to undulate in the npu mutants.

The Putative Role of NPU

The NPU protein was predicted to have two extra-cellular regions, two transmembrane regions, and oneintracellular region (Fig. 2B). Its subcellular localiza-tion in the plasma membrane was confirmed in ourresearch (Fig. 2C). NPU functions sporophytically inanther development because the wild type and npu-3male sterile phenotype segregated in F2 populationwith 3:1. It is in agreement with its expression attranscription level in microsporocytes and tapetal cellsin anther development (Fig. 6D). In the knockoutmutant of NPU (npu-3), primexine could not form(Fig. 4). It was reported that the synthesis, secretion,and siting of primexine were all controlled by individ-ual microspores (Fitzgerald and Knox, 1995; Perez-Munoz et al., 1993a, 1993b). Therefore, the NPU proteinis likely to be located on themembrane of microspore. Itis believed that primexine is composed of polysaccha-ride material (Heslop-Harrison, 1968). Another mem-brane integral protein RPG1, which is involved in theexine patterning in Arabidopsis, has recently beendemonstrated to be a sugar transporter (Guan et al.,2008; Chen et al., 2010). Therefore, the NPU proteinmayalso function as a sugar transporter on the microsporemembrane, which is responsible for transporting thepolysaccharide material essential for primexine matrixformation.

Callose is composed of a polysaccharide (b-1,3glucan), and CalS5 was reported to be responsible for

Figure 6. Expression analysis of NPU. A, Semi-quantitative RT-PCR of RNA isolated from varioustissues with NPU and b-TUBULIN-specificprimer sets. Inf, Inflorescence; R, root; S, stem;L, leaf; B, buds; Sdl, seedling. B, In situ hybridi-zation of the NPU transcript in a stage 5 antherwith an antisense probe. C, In situ hybridizationof the NPU transcript in a stage 6 anther with anantisense probe. D, In situ hybridization of theNPU transcript in a stage 7 anther with an anti-sense probe. F, In situ hybridization of the NPUtranscript in a stage 8 anther with an antisenseprobe. G, In situ hybridization of the NPU tran-script in a stage 9 anther with an antisense probe.H, In situ hybridization of the NPU transcript in astage 7 anther with a sense probe.

Chang et al.




the synthesis of callose deposited at the primary cellwall of meiocyte and tetrads (Dong et al., 2005). In situhybridization results also show that NPU initiallyexpresses at meiocytes with its highest expression attetrad stage (Fig. 6). The expression pattern of NPU issimilar to Cals5 in anther development. It was reportedthat plant cells maintain a relatively stable level ofsugar concentration, and a high or low level of sugarmay inhibit or activate some other gene expressions(Koch, 1996). In the npumutant, the block of primexinematrix material might lead to a high level of sugarinside microspores/microsporocytes, which inhibitsthe expression of Cals5 in the mutant. However, theremight be other mechanisms that demonstrate how theknockout of NPU affects Cals5 expression. The down-regulated expression of CalS5 further affects the cal-lose synthesis, as shown in Figure 3. Therefore, the lowcallose accumulation in the npumutant might be a sideeffect of the block of primexine matrix formation.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) plants used in this study are in the

ecotype Columbia-0 background. The npu mutant was isolated from a

population of T-DNA-tagged transformants provided by Dr. Zuhua He (Li

et al., 2005). Plants were grown under long-day conditions (16 h of light/8 h of

dark) in an approximately 22�C growth room. The herbicide Basta was used to

monitor the segregation of the T-DNA inserts.

Microscopy

Plants were photographed with a Nikon digital camera (Coolpix 4500).

Flower images were taken using an Olympus dissection microscope with an

Olympus digital camera (BX51). Alexander solution was used as described

(Alexander, 1969). Plant material for the semithin sections was prepared and

embedded in Spurr’s resin as previously described (Owen and Makaroff,

1995). For SEM examination, fresh stamens and pollen grains were coated

with 8 nm of gold and observed under a JSM-840 microscope (JEOL). For TEM

observation, Arabidopsis floral buds from the inflorescence were fixed and

embedded as described (Zhang et al., 2007).

Protein Structure Prediction and Phylogenetic Analysis

The SOSUI, TMHMM, and SMART programs were run to predict the

transmembrane, intracellular, and extracellular regions, respectively, of NPU.

The NPU protein sequence was used to search for NPU homologs using the

BLASTp and tBLASTn programs. Multiple sequence alignment of full-length

protein sequences was performed using ClustalX 2.0 and was displayed using

BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). Phylo-

genetic trees were constructed and tested byMEGA 4.0 based on the neighbor-

joining method (http://www.megasoftware.net/).

Identification and Complementation of the NPU Gene

The identification of the T-DNA insertion was performed using primers

that specifically amplify the BAR gene of T-DNA: Bar-F (5#-GCACCATCGT-

CAACCACTAC-3#) and Bar-R (5#-TGCCAGAAACCCACGTCAT-3#). For

TAIL-PCR, T-DNA left border primers AtLB1 (5#-ATACGACGGATCG-

TAATTTGTC-3#), AtLB2 (5#-TAATAACGCTGCGGACATCTAC-3#), and

AtLB3 (5#-TTGACCATCATACTCATTGCTG-3#) and genomic DNA of the

mutant plants were used. The TAIL-PCR procedure and arbitrary degenerate

primers were as described (Liu et al., 1995). Close linkage of the T-DNA

insertion site and mutant phenotype were analyzed with primer AtLB3 and

plant-specific primers, LP (5#-GACTAGGTAATTGATATTGAACC-3#) and RP

(5#-GGTTATGTTAATAGTGGCTTTG-3#). A DNA fragment of 4.7 kb, including

1.5-kb upstream and 800-bp downstream sequences, was amplified using KOD

polymerase (Takara Biotechnology) with primers CMP-F (5#-GAATTCATTTA-

GAAACAACGACCAGCAT-3#) and CMP-R (5#-GTCGACGGGTAAGAGATC-

CTAACACGG-3#). The fragment was verified by sequencing and was cloned

into a pCAMBIA1300 binary vector (Cambia). The plasmids were transformed

into Agrobacterium tumefaciens GV3101 and then introduced into the homozy-

gous mutant plants. Seeds were selected using 20 mg/L hygromycin for trans-

formants that were fertile with a homozygous background. The primers used

for the verification of the background of the transformants were AtLB3 and

plant-specific primers CMPV-F (5#-ATGAAAACCCTTCAACGTCTAT-3#) andCMPV-R (5#-TGTCTGTCTGGTGGCATTACTA-3#).

Expression Analysis

The full-length cDNA of the wild-type plants without the stop codon was

cloned for the GFP fusion with the primers GFP-F (5#-CCCGGGATGGCGGG-

CATGGCTGCGGTG-3#) and GFP-R (5#-GGTACCCCATCCCCCTTGC-

CAAATTCTCC-3#). The cDNA was cloned into the pMON530 vector with

enhanced GFP. The primers used for the expression analysis of CalS5 and A6

genes were as follows: CalS5 primers, forward, 5#-ATTATTGCAGCTGCTA-

GAGATG-3#, and reverse, 5#-CTTGTTCAGAGGTTCTGGCTT-3#; A6 primers,

forward, 5#-TACCTAAACCGACGAACA-3#, and reverse, 5#-ATGCCAATA-

AATGGAGAC-3#. Semiquantitative RT-PCR with 30 cycles was used to an-

alyze the expression level of theNPU gene. The primers were as follows: RT-F,

5#-CAGGATTACGACCGTGGAAC-3#, and RT-R, 5#-CATTCATGCTGCTGC-

TCTCC-3#. The DIG (for digoxigenin) RNA labeling kit (Roche) and PCR DIG

probe synthesis kit (Roche) were used for the RNA in situ hybridization

experiment. An NPU-specific cDNA fragment of 304 bp was amplified and

cloned into the pSK vector. Antisense and sense DIG-labeled probes were

prepared as described (Zhu et al., 2008).

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers SORBIDRAFT_09g005000, LOC100278212,

Os05g0168400,XP_002532862.1, XP_002323065.1,XP_002270471.1, XP_001784831.1,

and XP_001690235.1.

ACKNOWLEDGMENTS

We thank Dr. Zu-hua He from the Shanghai Institute of Plant Physiology

and Ecology for providing the T-DNA-tagged lines and Hui-qi Zhang and

Nai-ying Yang from Shanghai Normal University for their help with SEM and

TEM. We also thank Pacific Edit for reviewing the manuscript prior to

submission.

Received August 5, 2011; accepted November 15, 2011; published November

18, 2011.

LITERATURE CITED

Alexander MP (1969) Differential staining of aborted and nonaborted

pollen. Stain Technol 44: 117–122

Ariizumi T, Hatakeyama K, Hinata K, Inatsugi R, Nishida I, Sato S, Kato

T, Tabata S, Toriyama K (2004) Disruption of the novel plant protein

NEF1 affects lipid accumulation in the plastids of the tapetum and exine

formation of pollen, resulting in male sterility in Arabidopsis thaliana.

Plant J 39: 170–181

Blackmore S, Wortley AH, Skvarla JJ, Rowley JR (2007) Pollen wall

development in flowering plants. New Phytol 174: 483–498

Brett C, Waldron K (1990) Physiology and biochemistry of plant cell walls.

In M Black, J Chapman, eds, Topics in Plant Physiology 2. Unwin

Hyman, London, pp 72–127.

Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, GuoWJ,

Kim JG, Underwood W, Chaudhuri B, et al (2010) Sugar transporters

for intercellular exchange and nutrition of pathogens. Nature 468:

527–532

Dahl AO (1986) Observations on pollen development in Arabidopsis under

gravitationally controlled environments. In S Blackmore, IK Ferguson,

eds, Pollen and Spores: Form and Function. Academic Press, London,

pp 49–60.

The NPU Gene




de Azevedo Souza C, Kim SS, Koch S, Kienow L, Schneider K, McKim

SM, Haughn GW, Kombrink E, Douglas CJ (2009) A novel fatty Acyl-

CoA Synthetase is required for pollen development and sporopollenin

biosynthesis in Arabidopsis. Plant Cell 21: 507–525

Dickinson HG, Sheldon JM (1986) The generation of patterning at the

plasma membrane of the young microspores of Lilium. In S Blackmore,

IK Ferguson, eds, Pollen and Spores: Form and Function. Academic

Press, London, pp 1–17.

Dong X, Hong Z, Sivaramakrishnan M, Mahfouz M, Verma DP (2005)

Callose synthase (CalS5) is required for exine formation during micro-

gametogenesis and for pollen viability in Arabidopsis. Plant J 42: 315–328

Edlund AF, Swanson R, Preuss D (2004) Pollen and stigma structure and

function: the role of diversity in pollination. Plant Cell (Suppl) 16: S84–S97

Fitzgerald MA, Knox RB (1995) Initiation of primexine in freeze substituted

microspores of Brassica campestris. Sex Plant Reprod 8: 99–104

Guan YF, Huang XY, Zhu J, Gao JF, Zhang HX, Yang ZN (2008) RUP-

TURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is

crucial for exine pattern formation and cell integrity of microspores in

Arabidopsis. Plant Physiol 147: 852–863

Heslop-Harrison J (1968) The pollen grain wall. The succession of events in

the growth of intricately patterned pollen walls is described and

discussed. Science 161: 230–237

Heslop-Harrison J (1971) Wall pattern formation in angiosperm microspo-

rogenesis. Symp Soc Exp Biol 25: 277–300

Hird DL, Worrall D, Hodge R, Smartt S, Paul W, Scott R (1993) The anther-

specific protein encoded by the Brassica napus and Arabidopsis thaliana A6

gene displays similarity to beta-1,3-glucanases. Plant J 4: 1023–1033

Koch KE (1996) Carbohydrate-modulated gene expression in plants. Annu

Rev Plant Physiol Plant Mol Biol 47: 509–540

Li ZM, Zhang HK, Cao JS, He ZH (2005) [Construction of an activation

tagging library of Arabidopsis and cloning for mutant genes]. Zhi Wu

Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 31: 499–506

Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation

and mapping of Arabidopsis thaliana T-DNA insert junctions by ther-

mal asymmetric interlaced PCR. Plant J 8: 457–463

Owen HA, Makaroff CA (1995) Ultrastructure of microsporogenesis and

microgametogenesis inArabidopsis thaliana (L.) Heynh. ecotypeWassilewskija

(Brassicaceae). Protoplasma 185: 7–21

Paxson-Sowders DM, Dodrill CH, Owen HA, Makaroff CA (2001) DEX1,

a novel plant protein, is required for exine pattern formation during

pollen development in Arabidopsis. Plant Physiol 127: 1739–1749

Paxson-Sowders DM, Owen HA, Makaroff CA (1997) A comparative

ultrastructural analysis of exine pattern development in wild-type

Arabidopsis and a mutant defective in pattern formation. Protoplasma

198: 53–65

Perez-Munoz CA, Jernstedt JA, Webster BD (1993a) Pollen wall develop-

ment in Vigna vexillata I. Characterization of wall layers. Am J Bot 80:

1183–1192

Perez-Munoz CA, Jernstedt JA, Webster BD (1993b) Pollen wall development

in Vigna vexillata: II. Ultrasmactural studies. Am J Bot 80: 1193–1202

Piffanelli P, Ross JHE, Murphy DJ (1998) Biogenesis and function of the

lipidic structures of pollen grains. Sex Plant Reprod 11: 65–80

Rowley JR, Skvarla JJ (1975) The glycocalyx and initiation of exine

spinules on microspores of Canna. Am J Bot 62: 479–485

Sanders PM, Bui AQ,Weterings K, McIntire KN, Hsu Y-C, Lee PY, Truong

MT, Beals TP, Goldberg RB (1999) Anther developmental defects in

Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11: 297–322

Scott RJ, Spielman M, Dickinson HG (2004) Stamen structure and func-

tion. Plant Cell (Suppl) 16: S46–S60

Southworth D, Jernstedt JA (1995) Pollen exine development precedes

microtubule rearrangement in Vigna unguiculata (Fabaceae): a model for

pollen wall patterning. Protoplasma 187: 79–87

Takahashi M (1989) Development of the echinate pollen wall in Farfugium

japonicum (Compositae: Senecioneae). J Plant Res 102: 219–234

TakahashiM, Skvarla JJ (1991) Exine pattern formation by plasmamembrane in

Bougainvillea spectabilis (Nyctaginaceae). Am J Bot 78: 1063–1069

Waterkeyn L, Bienfait A (1970) On a possible function of the callosic

special cell wall in Ipomoea purpurea (L.) Roth. Grana 10: 13–20

Worrall D, Hird DL, Hodge R, Paul W, Draper J, Scott R (1992) Premature

dissolution of the microsporocyte callose wall causes male sterility in

transgenic tobacco. Plant Cell 4: 759–771

Zhang ZB, Zhu J, Gao JF, Wang C, Li H, Li H, Zhang HQ, Zhang S, Wang

DM, Wang QX, et al (2007) Transcription factor AtMYB103 is required

for anther development by regulating tapetum development, callose

dissolution and exine formation in Arabidopsis. Plant J 52: 528–538

Zhu J, Chen H, Li H, Gao JF, Jiang H, Wang C, Guan YF, Yang ZN (2008)

Defective in Tapetal development and function 1 is essential for anther

development and tapetal function for microspore maturation in Arabi-

dopsis. Plant J 55: 266–277

Zinkl GM, Zwiebel BI, Grier DG, Preuss D (1999) Pollen-stigma adhesion

in Arabidopsis: a species-specific interaction mediated by lipophilic

molecules in the pollen exine. Development 126: 5431–5440

Chang et al.




no primexine and plasma membrane undulation is … · no primexine and plasma membrane undulation...

Documents