The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development

In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.     

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.     

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.     

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.     

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.     

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.     

Anther wall layers control pollen sugar nutrition inLilium

Summary Anthers ofLilium were for the first time investigated at the ultrastructural level in order to appreciate the possible ways of sugar transport in the microsporangium. Our results have shown that the cells of the outer anther wall layers and the cell of the connective were interconnected by plasmodesmata, thus allowing assimilates to travel through the symplasmic pathway from the vascular bundle to the most internal middle layer (ML 1). ML 1 was devoid of cell communication throughout pollen development. Tapetal cells were also lacking plasmodesmata on their external face towards ML 1, but adjacent tapetal cells developed lateral junctions: the tapetum could represent a syncytium. Sugars destinated to pollen in the loculus have then to cross the ML 1 and the tapetal layers by the apoplasmic pathway; it is suggested that these two envelopes could be involved in the control of sugar transport from the outer anther wall layers to the locular fluid. Before microspore mitosis, the tapetum degenerated but ML 1 remained structurally unchanged. During pollen development, the guard cells of stomata were lacking cell communication, and preserved their starch content, which could be the sign of photosynthesis within the anther wall. In order to check whether these structural disconnections in anther tissues corresponded to physiological barriers, isolated pollen and stamens were cultivated during the anther maturation phase, on a medium containing increasing concentrations of sucrose (0 M, 1/6 M, 1/2 M, 1 M). After 7 days of culture, isolated pollen was engorged with starch grains and was unable to germinate, whereas in cultivated stamens, pollen did not contain any starch grain: sporophytic tissues, however, accumulated abnormal amylaceous reserves. These results strongly suggest that the anther wall layers, in particular ML 1, starve pollen with sugars during its maturation. They are acting as a physiological buffer storing nutriment surplus in starch grains.Abbreviations ML 1 middle layer 1 - ML 2 middle layer 2 - PAS periodic acid Schiff - PATAg periodic acid thiosemicarbazide silver nitrate     

花药培养过程栽培稻花粉不育杂种F1与亲本台中65的细胞学研究

利用活体压片、半薄及超薄切片技术,对栽培稻(Oryza sativa L.)花粉不育杂种F1及育性正常的亲本台中65离体培养前后的小孢子及花药壁进行细胞学研究.结果表明:与台中65相比,杂种F1的花粉小孢子在发育至单核中-晚期,出现比例较高的胞质凝聚小孢子和少量星型小孢子,正常小孢子和淀粉化小孢子比例降低.在离体条件下,胞质凝聚小孢子、星型小孢子、正常小孢子、液泡化小孢子、淀粉化小孢子的发育主要沿着胞质凝聚败育过程、孢子体发育过程、配子体发育过程、液泡化过程和淀粉化过程进行;药壁组织在离体条件下,杂种F1比台中65的绒毡层降解速度快,中层膨大程度高.杂种F1与台中65在离体培养下小孢子发育及药壁细胞学的差异主要是受到S-a座位内等位基因互作及离体培养环境的影响.     

Sphingadienine-1-phosphate levels are regulated by a novel glycoside hydrolase family 1 glucocerebrosidase widely distributed in seed plants

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice.     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile (PSGMS) rice ( Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.     

Tapetum Dimorphism and Its Histochemical Study in Sweet Pepper (Capsicum annuum L. Var. grossum)

In sweet pepper, the portion of tapetum toward the interior of the anther comprising large cells is derived from cells of connective of anther whereas the remaining tapetum on the outside of the anther comprising comparatively small cells is derived from the parietal layer. Those ceils of the former prosessing large vacuoles and large nuclei are stained weaker than the cells of the latter by methyl green-pyronin and mercuric-bromophenol blue staining. Large spherical grains which contain acid phosphatase appear in the vacuoles in both kinds of tapetum at sporogenesis stage. During meiosis of pollen mother cells, DNA, RNA and protein sysntheses increase in tapetum. The tapetum derived from connective accumulates more DNA than that derived from parietal layer. The activity of acid phosphatase becomes higher in tapetum when it degenerates. The degeneration of two kinds of tapetum is similar. There are no starch grains in tapetum through its whole course of development.     

灯盏花雄性不育种质鉴定与花药发育的细胞学研究

对云南泸西栽培灯盏花群体进行调查,发现了灯盏花雄性不育种质个体,其出现频率约为1.06×10-4.对所发现的灯盏花不育株形态特征及其花药发育过程进行了观察,并对花粉活力进行鉴定.结果显示:(1)灯盏花不育株根、茎、叶形态与正常可育植株基本相似,管状花小,花丝短,花药瘦小,无花粉粒散出或花粉无活力.(2)灯盏花在其花药发育的小孢子母细胞时期、四分体时期、小孢子时期和单核早期,由于绒毡层细胞液泡化、提前解体,不能为小孢子或花粉发育提供所需物质,导致小孢子母细胞和四分体解体,产生无花粉的花药;或小孢子和单核花粉胞内降解,形成不同形状和外壁纹饰的败育花粉.研究认为,灯盏花花药绒毡层异常是其花粉败育的主要原因.