RiceAntherNet: a gene co‐expression network for identifying anther and pollen development genes

In plants, normal anther and pollen development involves many important biological events and complex molecular regulatory coordination. Understanding gene regulatory relationships during male reproductive development is essential for fundamental biology and crop breeding. In this work, we developed a rice gene co‐expression network for anther development (RiceAntherNet) that allows prediction of gene regulatory relationships during pollen development. RiceAntherNet was generated from 57 rice anther tissue microarrays across all developmental stages. The microarray datasets from nine rice male sterile mutants, including msp1‐4, ostdl1a, gamyb‐2, tip2, udt1‐1, tdr, eat1‐1, ptc1 and mads3‐4, were used to explore and test the network. Among the changed genes, three clades showing differential expression patterns were constructed to identify genes associated with pollen formation. Many of these have known roles in pollen development, for example, seven genes in Clade 1 (OsABCG15, OsLAP5, OsLAP6, DPW, CYP703A3, OsNP1 and OsCP1) are involved in rice pollen wall formation. Furthermore, Clade 1 contained 12 genes whose predicted orthologs in Arabidopsis have been reported as key during pollen development and may play similar roles in rice. Genes in Clade 2 are expressed earlier than Clade 1 (anther stages 2–9), while genes in Clade 3 are expressed later (stages 10–12). RiceAntherNet serves as a valuable tool for identifying novel genes during plant anther and pollen development. A website is provided ( https://www.cpib.ac.uk/anther/riceindex.html ) to present the expression profiles for gene characterization. This will assist in determining the key relationships between genes, thus enabling characterization of critical genes associated with anther and pollen regulatory networks.     

水稻花粉发育的分子机理

水稻的小孢子母细胞在花粉囊中进行减数分裂产生小孢子,小孢子进一步发育成花粉粒。当花粉成熟时,花粉粒从花粉囊中释放出来进行受精。分子生物学的研究已经发现了一些参与这一过程的基因,包括控制花粉囊组织的分化、小孢子母细胞的减数分裂、小孢子的发育和花药的开裂等。本文旨在总结水稻花粉发育过程及其调控分子机制的研究进展。     

水稻花粉发育的分子机理

水稻的小孢子母细胞在花粉囊中进行减数分裂产生小孢子, 小孢子进一步发育成花粉粒。当花粉成熟时, 花粉粒从花粉囊中释放出来进行受精。分子生物学的研究已经发现了一些参与这一过程的基因, 包括控制花粉囊组织的分化、小孢子母细胞的减数分裂、小孢子的发育和花药的开裂等。本文旨在总结水稻花粉发育过程及其调控分子机制的研究进展。     

A rice ��-1,3-glucanase gene Osg1 is required for callose degradation in pollen development

Plant β-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative β-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these β-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution.     

Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice

In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1-like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1-green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.     

调控拟南芥花粉发育突变体zy1511的基因定位及特征分析

为了进一步研究花药花粉发育过程,我们通过EMS诱变,筛选到拟南芥雄性不育突变体zy1511。遗传分析表明,zy1511为隐性单位点突变。细胞学观察表明．突变体花药中小孢子从四分体释放出后绒毡层并没有开始退化,花药发育后期绒毡层依然部分存在。说明突变体花药绒毡层退化比野生型的要迟,因此,小孢子不能发育成正常花粉粒。利用图位克隆的方法将zv1511定位于第一条染色体上分子标记F25P12和T8L23之间134．kb的区间内。本项工作为zy1511基因的克隆及对花粉发育功能分析奠定了基础。目前尚未见到该区间内雄性不育基因的报道。因此,zy1511是控制花粉发育的尚未发现的关键基因。     

水稻P0491E01基因调控花药发育的功能分析(简报)

高等植物的花药发育是包含基因不同程度相互作用的复杂发育过程,一般可分为两个阶段。第一个是花药的形态建成阶段,在这一阶段中细胞与组织发生分化,小孢子母细胞进行减数分裂形成四分     

Effects of chilling on male gametophyte development in rice

Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic anthers. Anthers observed during tetrad release also have callose (1,3-beta-glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity.     

PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.     

水稻OsMS2基因在花药发育中的功能分析

拟南芥MS2（MALE STERILITY2）是一个调控花药花粉发育的关键基因。水稻OsMS2（Os03g07140）基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比,转基因植株营养生长阶段正常,但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察,结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟,小孢子壁的形成出现异常。扫描电镜观察结果显示,小孢子壁光滑,不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

低温预处理影响水稻花药培养效率的机理初探

低温预处理延缓药壁中层和绒毡层的降解,促进表皮层和药室内壁层的发育,延缓花药过氧化物酶同工酶活性的增强。处理期间花药可溶性蛋白质、淀粉酶同工酶潜带发生明显变化。处理期间花药的~3H-TdR渗入和花粉的发育、分裂,表明花粉存在合成和充实活动。绒毡层和花粉间存在囊泡,表皮层和药室内壁层之间存在多泡体的穿壁运动,说明低温处理中药壁向花粉输送雄核发育所需的物质。在进入正常培养初期,经过低温处理的花药药壁仍有表皮层和药室内壁层的发育,多细胞花粉出现提早、数量增加,花粉退化延缓。而未经处理的花药药壁各层均迅速降解,花粉大量退化。     

Post-meiotic deficient anther1 (PDA1) encodes an ABC transporter required for the development of anther cuticle and pollen exine in rice

The tapetum of the anther locule encloses the male reproductive cells and plays a supportive role for normal pollen development. However, the underlying mechanism remains less understood. Previously, we identified a complete recessive male sterile mutant, post-meiotic deficient anther1 (pda1), with abnormal postmeiotic tapetal development. In this study we comprehensively characterized pda1. Chemical analysis uncovered that pda1 anther had significant lower levels of cutin monomers and cuticular waxes. PDA1 gene encodes an ATP-binding cassette (ABC) half-transporter, namely OsABCG15, which is conserved from algae to higher plants. In situ RNA hybridization assay showed that PDA1 is strongly expressed in tapetal cells, and weakly in microspores during the anther development. Additionally, the expression of two pollen exine biosynthetic genes CYP704B2 and CYP703A3 was dramatically reduced in pda1 mutant anthers. Altogether, these observations suggest that the tapetum-expressed ABC transporter PDA1 plays a crucial role in secreting lipidic precursors from the tapetum to developing microspores and the anther epidermis.     

水稻OsMS2基因在花药发育中的功能分析

拟南芥MS2(MALE STERILITY 2)是一个调控花药花粉发育的关键基因。水稻OsMS2(Os03g07140)基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比, 转基因 植株营养生长阶段正常, 但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察, 结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟, 小孢子壁的形成出现异常。扫描电镜观察结果显示, 小孢子壁光滑, 不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

The Post-meiotic Deficicent Anther1 （PDA1） gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice,we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a γ-ray-treated rice mutant library.Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene.The pda1 mutant anther seemed smaller with white appearance.Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis.However,the pda1 mutant displayed obvious defects in postmeiotic tapetal development,abnormal degeneration occurred in the tapetal cells at stage 9 of anther development.Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant,causing no obvious pollen exine formation.RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB,OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther.Using map-based cloning approach,the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F2 population.The physical distance between HLF610 and HLF627 was about 194 kb.This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.