Activation tagging of an Arabidopsis SHI-RELATED SEQUENCE gene produces abnormal anther dehiscence and floral development

The tapetum is a layer of cells covering the inner surface of pollen sac wall. It contributes to anther development by providing enzymes and materials for pollen coat biosynthesis and nutrients for pollen development. At the end of anther development, the tapetum is degenerated, and the anther is dehisced, releasing mature pollen grains. In Arabidopsis, several genes are known to regulate tapetum formation and pollen development. However, little is known about how tapetum degeneration and anther dehiscence are regulated. Here, we show that an activation-tagged mutant of the S HI-R ELATED S EQUENCE 7 (SRS7) gene exhibits disrupted anther dehiscence and abnormal floral organ development in addition to its dwarfed growth with small, curled leaves. In the mutant hypocotyls, cell elongation was reduced, and gibberellic acid sensitivity was diminished. Whereas anther development was normal, its dehiscence was suppressed in the dominant srs7-1D mutant. In wild-type anthers, the tapetum disappeared at anther development stages 11 and 12. In contrast, tapetum degeneration was not completed at these stages, and anther dehiscence was inhibited, causing male sterility in the mutant. The SRS7 gene was expressed mainly in the filaments of flowers, where the DEFECTIVE-IN-ANTHER-DEHISCENCE 1 (DAD1) enzyme catalyzing jasmonic acid (JA) biosynthesis is accumulated immediately before flower opening. The DAD1 gene was induced in the srs7-1D floral buds. In fully open flowers, the SRS7 gene was also expressed in pollen grains. It is therefore possible that the abnormal anther dehiscence and floral development of the srs7-1D mutant would be related with JA.     

MYB类转录因子对花药和花粉发育的调控途径

花药发育和花粉形成的各个步骤由众多基因控制,一些转录因子通过调控花药发育相关基因的表达,是功能性花粉形成的关键因子。MYB类转录因子作为植物中最大的转录因子家族,是其中非常重要的一类转录因子。该文结合近年来国内外有关被子植物花粉发育相关MYB转录因子在花药发育和花粉形成的调控途径,包括绒毡层发育、胼胝质的沉积和降解、光合产物的运输、花药的开裂以及雄配子体形成过程中所起的重要作用等方面的研究进展,重点对MYB类转录因子通过形成对绒毡层发育、同化物分配、苯丙烷物质代谢等相关靶位基因的控制网络,转录调控植物花粉发育和花药开裂过程等研究进行综述讨论。     

高等植物叶绿素生物合成的联乙烯还原酶及编码基因研究进展

联乙烯还原酶(DVR)将各种叶绿素中间物质的8-乙烯基转化为乙基,是叶绿素生物合成必不可少的一个关键酶。迄今已在高等植物中检测到5种DVR活性。水稻和玉米的重组DVR蛋白能将联乙烯叶绿素a、叶绿素酸酯a、原叶绿素酸酯a、镁原卟啉Ⅸ单甲酯和镁原卟啉Ⅸ分别转化为相应的单乙烯物质,从而证实了这5种DVR活性。在高等植物中各种DVR活性是由一个基因编码的具有广谱底物专化性的DVR蛋白所催化,但来源于不同物种的DVR蛋白的催化活性可能具有极显著的差异,并且即使是同一个DVR蛋白,对不同的联乙烯底物也可能具有显著不同的催化活性。在此基础上,提出了"源于一个联乙烯还原酶的叶绿素生物合成多分支路径"假说。该文对近年来国内外有关高等植物叶绿素生物合成途径中联乙烯中间物质与联乙烯还原酶活性、联乙烯还原酶基因的克隆及重组酶活性检测、联乙烯还原酶的数目与叶绿素生物合成的多分支路径等方面的研究进展进行综述,并讨论了有待进一步探讨的若干问题。     

An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development

The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.     

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.     

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理, 将甘蓝型油菜(Brassica napus L.)和白菜(B. campestris L. ssp. chinensis Makino)杂交并连续回交6代获得白菜OguCMS, 在与保持系花药细胞学比较的基础上, 运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片断, 利用RACE克隆得到该片断的cDNA全长, 命名为BcMYBogu(GenBank 登录号: EF127861), 对其氨基酸序列和表达特征进行研究。结果表明, 白菜OguCMS绒毡层在四分体后增生, 高度液泡化, 导致小孢子花粉外壁异常, 细胞质同外壁分离并降解; 花药变白; BcMYBogu具有典型的MYB DNA结合域—W残基和SH[AL]QKY[RF]基序; 系统进化分析显示BcMYBogu与AtMYB26, AtMYB32和AtMYB4等聚类在同一分枝; RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达, 但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。     

OsABCG15 encodes a membrane protein that plays an important role in anther cuticle and pollen exine formation in rice

Key message

An ABC transporter gene ( OsABCG15 ) was proven to be involved in pollen development in rice. The corresponding protein was localized on the plasma membrane using subcellular localization.

Abstract

Wax, cutin, and sporopollenin are important for normal development of the anther cuticle and pollen exine, respectively. Their lipid soluble precursors, which are produced in the tapetum, are then secreted and transferred to the anther and microspore surface for polymerization. However, little is known about the mechanisms underlying the transport of these precursors. Here, we identified and characterized a member of the G subfamily of ATP-binding cassette (ABC) transporters, OsABCG15, which is required for the secretion of these lipid-soluble precursors in rice. Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence. This osabcg15 mutant failed to produce any viable pollen and was completely male sterile. Histological analysis indicated that osabcg15 exhibited an undeveloped anther cuticle, enlarged middle layer, abnormal Ubisch body development, tapetum degeneration with a falling apart style, and collapsed pollen grains without detectable exine. OsABCG15 was expressed preferentially in the tapetum, and the fused GFP-OsABCG15 protein was localized to the plasma membrane. Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine. This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.     

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.     

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.