Molecular Plant  •  Volume 7  •  Number 4  •  Pages 751–754  •  April 2014

LETTER TO THE EDITOR

Annexin5 Is Essential for Pollen Development in Arabidopsis Dear Editor, Pollen development is a post-meiotic process that produces mature pollen grains from microspores and can be regarded as an ideal model for the study of important plant physiological processes such as reproduction, cellular differentiation, cell fate determination, signal transduction, membrane transport, and fusion and polar growth. The regulation of pollen development is a complicated biological process that is crucial for sexual reproduction in flowering plants (Yamamoto et al., 2003; Durbarry et al., 2005; Twell, 2011; Cheung et al., 2013). The annexin family is a class of proteins that can bind to the membrane, phospholipids, and actin in a Ca2+-dependent manner. The members of this family share an evolutionarily conserved structure that can be found in a wide variety of eukaryotic cells (Gerke and Moss, 2002; Moss and Morgan, 2004; Gerke et al., 2005). In recent years, annexins from many different plants were isolated, and their functions were studied. As with annexins in animals, those in plants have conserved the function of the protein family and also participate in many significant physiological activities (Lee et al., 2004; Laohavisit and Davies, 2011). They play multiple roles in numerous cellular events by acting as a putative ‘linker’ between Ca2+ signaling, the actin cytoskeleton, and the membrane, which are required for pollen development and pollen tube growth. However, the function of plant annexins in pollen remains poorly understood. By analyzing the Arabidopsis information Resource database (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb. cgi?primaryGene=AT1G68090&modeInput=Absolute), we found that, of the eight annexin isoforms in Arabidopsis, annexin 5 (Ann5, At1g68090) was predicted to be specifically expressed in mature pollen, suggesting a putative role of Ann5 in pollen development. Tissue RT–PCR was performed to verify this prediction. As shown in Figure  1A, Ann5 was strongly expressed in open flowers and cotyledons and barely detected in other tissues. In particular, almost no Ann5 transcript was detected in the early flower bud, demonstrating stage-specific transcriptional activity in floral tissues. The promoter activity of Ann5 was then investigated using the β-glucuronidase (GUS) reporter gene. In vegetative tissues, Ann5 was mainly expressed in cotyledons, and its distribution was strictly restricted to the tips of cotyledons at the later stage of cotyledon development (Supplemental Figure 1). In reproductive tissues, Ann5 was only expressed in the anthers of mature buds (Figure 1B) and in mature pollen grains and tubes (Figure  1C). To further confirm the precise temporal

expression pattern of Ann5 during pollen development, we used Ann5Pro–YFP-NLS (nuclear located sequence) and Ann5Pro–GUS reporters and then demonstrated that both the yellow fluorescent protein (YFP) signal and GUS activity were only detected in the bicellular and tricellular stages, not in uninucleate microspores (Figure 1D and Supplemental Figure 2). In addition, the Ann5–GFP fusion protein was diffusely localized to the cytoplasm of pollen grains and tubes (Supplemental Figure 3). Because no T-DNA insertion mutants were identified for Ann5 in the Arabidopsis Biological Resource Center, the RNA interference (RNAi) approach was used to knock down the Ann5 transcript level and investigate the in vivo function of Ann5 in Arabidopsis. The coding sequence or 3′-UTR of Ann5 were used to generate inverted repeat constructs driven by the Lat52 promoter (Twell et al., 1990) (named Lat52–cDNAi and Lat52–UTRi) and the Ann5 promoter (named Ann5Pro– UTRi), respectively. GFP cDNA RNAi was used as control (named Lat52–GFPi and Ann5Pro–GFPi) (Supplemental Figure  4A). Of the 80 Ann5 RNAi lines generated, 63 lines produced approximately 20%–40% abortive pollen grains; this phenotype was stable over five consecutive generations. To ascertain whether the reduced expression of Ann5 in pollen grains was related to the pollen sterile phenotype, we detected the amounts of Ann5 transcript in RNAi lines pollen by RT–PCR. The results demonstrated that the expression of Ann5 was significantly decreased in all Ann5 RNAi lines but not in the control lines (Supplemental Figure 4B and 4E). Subsequently, we directly observed the pollen grains from the wild-type and RNAi lines. Unlike the well-developed pollen in wild-type plants, some smaller, collapsed pollen grains were observed in RNAi lines, providing evidence of pollen grain abortion. Alexander’s staining was then used to distinguish clearly aborted and non-aborted pollen. Smaller, misshapen, and pale-green pollen grains that could be easily distinguished from wild-type pollen were observed in the Ann5 RNAi lines. The pollen lethality effect was studied in greater detail using scanning electron microscopy (SEM). Unlike regular pollen grains in wild-type plants, some pollen from Ann5 RNAi plants appeared shrunken and collapsed © The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst171, Advance Access publication 22 December 2013 Received 4 December 2013; accepted 15 December 2013

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Figure 1.  Down-Regulation of Ann5 in the RNAi Lines Aborts Pollen Development. (A) Expression pattern of Ann5 in various tissues and organs of Arabidopsis as determined by RT–PCR. C, 3-day-old cotyledon; R, root; S, stem; L, leaf; OF, open flower; EB, early bud. Total RNA was extracted from individual tissues of 6-week-old plants. EF4A was used as an internal positive control. (B, C) Histochemical staining for GUS activity in transgenic plants carrying the GUS gene driven by the Ann5 promoter sequence in Columbia (Col) wild-type inflorescence (B), mature pollen, and pollen tube (C). GUS activity was detected in pollen grains and pollen tubes. The black arrows indicate early buds. Bars = 1 mm in (B) and 20 μm in (C).

Letter to the Editor   

(Figure 1E and Supplemental Figure 4C and 4D). Furthermore, the phenotypic differences were fully in accordance with the differences in transcript levels between the Ann5 RNAi lines. Taken together, these findings indicate that the pollen abortive phenotype occurring in transgenic RNAi populations correlated with the down-regulation of Ann5 function. To further confirm that Ann5 knockdown was responsible for the aborted pollen genotype in RNAi lines, a complementation construct PBI121–Ann5Pro–Ann5 (named Ann5Pro– Ann5) was introduced into the Lat52–UTRi-1 lines. In the Lat52–UTRi-1 homozygous plant background, 17 of 20 transformed lines complemented the pollen lethality phenotype. We demonstrated that the smaller and misshapen pollen phenotype was fully rescued in the Ann5Pro–Ann5-1 and 3 recovery lines. However, the percentage of aborted pollen from the Ann5Pro–Ann5-2 recovery line was considerably higher than those of the Ann5Pro–Ann5-1 and 3 complemented plants, because of the lower expression levels from the complementing Ann5, suggesting an association between the expression level of Ann5 and the aborted pollen genotype (Supplemental Figure 5). All of these data demonstrate that the defects observed in pollen grains of RNAi mutants resulted from the knockdown in Ann5 expression and that Ann5 is essential for normal pollen development. To determine the developmental stage at which the pollen grains in Ann5 RNAi plants were aborted, we examined the process of microsporogenesis and microgametogenesis in wild-type and Ann5Pro–UTRi-1 lines. The fluorochrome 4’-6-diamidino2-phenylindole (DAPI) was used to determine the occurrence of pollen mitosis following the staining of pollen nuclei. As shown in Figure 1F, no obvious difference was observed in microspore morphology between wild-type and Ann5Pro–UTRi-1 plant anthers, which formed normal free microspores with brightly stained nuclei at the uninucleate stage. At the late microspore stage, all of the individual microspores in the wild-type plants generated two daughter nuclei consisting of a large vegetative nucleus and dense generative nucleus that showed two distinctive DAPI staining patterns, indicating that the plants were entering the bicellular stage. In contrast, 20% of the pollen grains in Ann5Pro–UTRi-1 anthers exhibited a smaller,

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misshapen, and empty morphology with only diffuse DAPI staining. At the trinucleate stage, all two-celled pollen grains in wild-type plants had completed pollen mitosis II, giving rise to three-celled pollen grains containing one large vegetative cell and two dense sperm cells. In contrast, 26% of the Ann5Pro– UTRi-1 pollen grains completely collapsed at this stage, were devoid of cytoplasmic content, and had no detectable DAPI staining. In addition, as described in Supplemental Table  1, aborted pollen grains were observed in both bicellular and tricellular pollen from the Lat52–UTRi-1/2 and Ann5Pro–cDNAi-1 lines. The above results indicate that the stage at which pollen abortion occurs in RNAi lines is in accordance with Ann5 expression, which also initiated at the bicellular stage. The ultrastructural changes of the aborted pollen grains in the Ann5 RNAi lines were further investigated using transmission electron microscopy (TEM). The highly synchronized and well-developed pollen from wild-type anthers at the bicellular stage contained well-defined structures (Figure  1Ga). However, the ultrastructural analysis of the Ann5Pro–UTRi-1 pollen revealed a strikingly abnormal phenotype. As pollen development progressed, the cytoplasm became grainy and gradually detached. Moreover, a higher amount of electrondense material accumulated in the shrinking cytoplasm than in the wild-type cytoplasm, and condensed at random sites. The shapes of nuclei and most organelles were irregular, and fewer vacuoles were present (Figure  1Gb). Thereafter, the plasma membrane lost integrity, and many breaks were observed in the membrane surface. After a gradual course of autolysis, the pollen exhibited a large-scale loss of cytoplasm and disintegration of the basic organelle structure. Only debris from degraded pollen was found, and no recognizable membrane systems were detected (Figure 1Gb–d). Finally, most pollen grains did not show defined structures in TEM images, as they were collapsed and contained little or no cytoplasm (Figure 1Ge). However, the exine walls and intine layer of Ann5Pro–UTRi-1 pollen maintained nearly normal morphology throughout all stages of pollen development, even until the pollen completely collapsed, with a long, stringy shape at stage 13 of anther development (Supplemental Figure 6).

(D) Temporal expression profile of Ann5 in transgenic Arabidopsis Ann5Pro–YFP-NLS lines at the early microspore, late microspore, bicellular, and tricellular stages of pollen development. Pollen grains from homozygous transgenic plants were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Yellow fluorescent protein (YFP), DAPI fluorescence, and bright-field signals are displayed in the top, middle, and bottom panels, respectively. Vegetative nuclei are indicated by arrowheads and generative nuclei by arrows. The YFP signal accumulated beginning in the bicellular pollen grain stage in transgenic lines. Bars = 5 μm. (E) The observations of the pollen lethal phenotype in wild-type and pCAMBIA1300–Lat52–UTRRNAi-1 (designated as Lat52–UTRi) plants. Brightfield, Alexander’s viability staining, and scanning electron microscopy (SEM) images are shown in the left, middle, and right panels, respectively. Arrowheads indicate the representative collapsed pollen grains. Bars = 20 μm. (F) Comparison of the progression of mitotic division between wild-type and Ann5Pro–UTRi-1 plants. The uninucleate microspores, bicellular, and tricellular pollen released from anthers in wild-type and Ann5Pro–UTRi-1 plants were identified by nuclear DAPI staining. We visualized pollen under a light microscope with or without a UV fluorescent filter. Representative pollen from anthers at different developmental stages is shown. Vegetative nuclei are indicated by arrowheads and generative nuclei by arrows. Sterile pollen grains (red asterisks) were observed in Ann5Pro– UTRi-1 anthers in both the bicellular and tricellular stages. Bars = 20 μm. (G) TEM micrographs of pollen cross-sections from wild-type and Ann5Pro–UTRi-1 anthers during the bicellular stage. (a) TEM cross-sections of pollen from wild-type. (b–e) Progression of pollen abortion in Ann5Pro–UTRi-1 lines. Arrowheads indicate plasmolysis and red asterisks point to breaks in the plasma membrane. VN, vegetative nucleus; GN, generative nucleus; V, vesicle; Ex, exine; In, intine. Bars = 2 μm.

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 Letter to the Editor

In conclusion, these data suggest that Ann5 is predominantly expressed in pollen after the bicellular stage, when the pollen ceased further development and eventually became deformed and degraded caused by the down-regulation of the function of Ann5. The phenotype of severely sterile pollen grains could be rescued by a wild-type copy of Ann5. Our results provided reliable genetic evidence to functionally characterize Ann5 during pollen development.

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This work was supported by the National Basic Research Program (2014CB954200–03), the National Natural Science Foundation of China (30800079 and 31270326), the Program for New Century Excellent Talents in University (NCET-13– 0264), and the Natural Science Foundation for Distinguished Yong Scholars of Gansu Province (2013GS10064) to Y.X.

Acknowledgments We thank Dr Haiyun Ren (Beijing Normal University) and Dr Heng Liu (Lanzhou University) for experimental assistance, Dr Jia Li (Lanzhou University) for supplying the high-speed centrifuge, Liping Guan for helping with confocal microscopy, and Wenliang He for technical assistance. No conflict of interest declared.

Jingen Zhua, Shunjie Yuana, Guo Weia, Dong Qiana, Xiaorong Wua, Honglei Jiaa, Mengyuan Guib, b Wenzhe Liu , Lizhe Ana,1, and Yun Xianga,1

a MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China b School of Life Sciences, Northwest University, Xi’an 710069, China 1 To whom correspondence should be addressed. L.A. E-mail [email protected], fax 0086-9318912561, tel. 0086-9318915588; Y.X. E-mail [email protected], fax 0086-9318912561, tel. 0086-18809319776

References Cheung, A.Y., Palanivelu, R., Tang, W.H., Xue, H.W., and Yang, W.C. (2013). Pollen and plant reproduction biology: blooming from East to West. Mol. Plant. 6, 995–997. Durbarry, A., Vizir, I., and Twell, D. (2005). Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression. Plant Physiol. 137, 297–307. Gerke, V., and Moss, S.E. (2002). Annexins: from structure to function. Physiol Rev. 82, 331–371. Gerke, V., Creutz, C.E., and Moss, S.E. (2005). Annexins: linking Ca2+ signalling to membrane dynamics. Nat. Rev. Mol. Cell Biol. 6, 449–461. Laohavisit, A., and Davies, J.M. (2011). Annexins. New Phytol. 189, 40–53. Lee, S., Lee, E.J., Yang, E.J., Lee, J.E., Park, A.R., Song, W.H., and Park, O.K. (2004). Proteomic identification of annexins, calciumdependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell. 16, 1378–1391. Moss, S.E., and Morgan, R.O. (2004). The annexins. Genome Biol. 5, 219. Twell, D. (2011). Male gametogenesis and germline specification in flowering plants. Sexual Plant Reproduction. 24, 149–160. Twell, D., Yamaguchi, J., and McCormick, S. (1990). Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis. Development. 109, 705–713. Yamamoto, Y., Nishimura, M., Hara-Nishimura, I., and Noguchi, T. (2003). Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana. Plant Cell Physiol. 44, 1192–1201.

Annexin5 is essential for pollen development in Arabidopsis.

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