SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth

The exocyst, a complex of eight proteins, contributes to the morphogenesis of polarized cells in a broad range of eukaryotes. In these organisms, the exocyst appears to facilitate vesicle docking at the plasma membrane during exocytosis. Although we had identified orthologs for each of the eight exocyst components in Arabidopsis (Arabidopsis thaliana), no function has been demonstrated for any of them in plants. The gene encoding one exocyst component ortholog, AtSEC8, is expressed in pollen and vegetative tissues of Arabidopsis. Genetic studies utilizing an allelic series of six independent T-DNA mutations reveal a role for SEC8 in male gametophyte function. Three T-DNA insertions in SEC8 cause an absolute, male-specific transmission defect that can be complemented by expression of SEC8 from the LAT52 pollen promoter. Microscopic analysis shows no obvious abnormalities in the microgametogenesis of the SEC8 mutants, and the mutant pollen grains appear to respond to the signals that initiate germination. However, in vivo assays indicate that these mutant pollen grains are unable to germinate a pollen tube. The other three T-DNA insertions are associated with a partial transmission defect, such that the mutant allele is transmitted through the pollen at a reduced frequency. The partial transmission defect is only evident when mutant gametophytes must compete with wild-type gametophytes, and arises in part from a reduced pollen tube growth rate. These data support the hypothesis that one function of the putative plant exocyst is to facilitate the initiation and maintenance of the polarized growth of pollen tubes.     

花粉蛋白质组学研究进展

花粉是高度退化的生物体（雄配子体）,在植物有性生殖过程中具有重要作用。解析花粉发育、花粉-柱头识别、萌发和花粉管生长等细胞学过程的分子机制是当前研究的热点问题之一。近年来,应用高通量的蛋白质组学技术平台,对水稻、拟南芥和裸子植物花粉的蛋白质组学研究揭示了花粉中表达蛋白质的功能类群特征。花粉中参与细胞壁代谢、蛋白质代谢、细胞骨架动态和信号转导的蛋白质被高度代表,并且近1／4蛋白质有多个同工型。本文综述了花粉蛋白质组学的研究进展。     

花粉蛋白质组学研究进展

花粉是高度退化的生物体(雄配子体), 在植物有性生殖过程中具有重要作用。解析花粉发育、花粉-柱头识别、萌发和花粉管生长等细胞学过程的分子机制是当前研究的热点问题之一。近年来, 应用高通量的蛋白质组学技术平台, 对水稻、拟南芥和裸子植物花粉的蛋白质组学研究揭示了花粉中表达蛋白质的功能类群特征。花粉中参与细胞壁代谢、蛋白质代谢、细胞骨架动态和信号转导的蛋白质被高度代表, 并且近1/4蛋白质有多个同工型。本文综述了花粉蛋白质组学的研究进展。     

COPII genes SEC31A/B are essential for gametogenesis and interchangeable in pollen development in Arabidopsis

In eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A-mCherry and SEC31B-GFP exhibited a high co-localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a-1/+sec31b-3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.     

Arabidopsis SNARE protein SEC22 is essential for gametophyte development and maintenance of Golgi-stack integrity

Membrane traffic contributes to plant growth and development. However, the functional significance of SNARE proteins involved in membrane fusion of the early secretory pathway has not been explored with respect to plant development. Here we analyze the Arabidopsis v-SNARE SEC22. Loss of SEC22 function impairs gametophyte development, as indicated by reciprocal crosses between wild-type plants and plants heterozygous for T-DNA insertions in the SEC22 gene. sec22 mutant pollen becomes abnormal during the bicellular stage, eventually giving rise to degenerated pollen grains. Most mutant embryo sacs fail to support embryogenesis and display unfused polar nuclei in their central cell. Immunolocalization by both light and electron microscopy revealed an association of mutant-complementing Myc-tagged SEC22 with the central and peripheral endoplasmic reticulum (ER). Ultrastructural analysis of developing sec22 mutant pollen demonstrated Golgi fragmentation and consumption. As a consequence, the plasma membrane-targeted syntaxin SYP124 was retained in the ER. Our results suggest that SEC22 plays an essential role in early secretory traffic between the ER and the Golgi.     

Spatial and temporal expression of actin depolymerizing factors ADF7 and ADF10 during male gametophyte development in Arabidopsis thaliana

The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization.     

The peroxin loss-of-function mutation abstinence by mutual consent disrupts male-female gametophyte recognition

In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.     

SNAIL1 is essential for female gametogenesis in Arabidopsis thaliana

Two yeast Brix family members Ssf1 and Ssf2,involved in large ribosomal subunit synthesis, are essential for yeast cell viability and mating efficiency. Their putative homologs exist in the Arabidopsis genome; however, their role in plant development is unknown. Here, we show that Arabidopsis thaliana SNAIL1(At SNAIL1), a protein sharing high sequence identity with yeast Ssf1 and Ssf2, is critical to mitosis progression of female gametophyte development.The snail1 homozygous mutant was nonviable and its heterozygous mutant was semi-sterile with shorter siliques.The mutation in SNAIL1 led to absence of female transmission and reduced male transmission. Further phenotypic analysis showed that the synchronic development of female gametophyte in the snail1 heterozygous mutant was greatly impaired and the snail1 pollen tube growth, in vivo, was also compromised. Furthermore, SNAIL1 was a nucleolarlocalized protein with a putative role in protein synthesis.Our data suggest that SNAIL1 may function in ribosome biogenesis like Ssf1 and Ssf2 and plays an important role during megagametogenesis in Arabidopsis.     

Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana

Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen has been elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 80% and pollen tube lengths of hundreds of microns, with both Columbia and Landsberg erecta (Ler) ecotypes. We found that pollen germination and tube growth were dependent on pollen density in both liquid and solid medium. Pollen germination rates were not substantially affected by flower or plant age. The quartet1 mutation negatively affected pollen germination, especially in the Ler ecotype. This protocol will facilitate functional analyses of insertional mutants affecting male gametophyte function, and should allow detailed gene expression analyses during pollen tube growth. Arabidopsis thaliana can now be included on the list of plant species that are suitable models for physiological studies of pollen tube elongation and tip growth.     

An exocyst complex functions in plant cell growth in Arabidopsis and tobacco

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.     

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.

sRNA, PARE, and AGO-IP sequencing uncovered the role of miRNAs during pollen development, showing that miRNAs transition from regulating genes involved in development to transposable elements.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.     

拟南芥温度诱导脂质运载蛋白TIL1参与雌配子体发育

雌配子体的正常发育是种子形成的前提条件之一,拟南芥温度诱导的脂质运载蛋白编码基因TIL1突变使胚珠败育,结实率下降明显。基因表达分析表明T-DNA插入使得TIL1基因敲除,突变体TIL1基因功能缺失;互交实验、Alexander染色、花粉离体培养和胚珠透明实验结果表明till-1突变体雄配子体发育正常、雌配子体胚囊发育有缺陷;通过遗传互补实验证明外源克隆的TIL1基因能恢复突变体的败育表型,并确定了TIL1基因主要在胚珠的胚囊中表达。实验结果表明TIL1基因参与了植物雌配子体发育这一重要的生理过程。