Deficiency of very long chain alkanes biosynthesis causes humidity‐sensitive male sterility via affecting pollen adhesion and hydration in rice

Pollen adhesion and hydration are the earliest events of the pollen–stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect‐pollinated plants. Here, we characterized the role of OsGL1‐4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1‐4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1‐4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography–mass spectrometry analysis suggested that the humidity‐sensitive male sterility of osgl1‐4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.     

A single point mutation in Ms44 results in dominant male sterility and improves nitrogen use efficiency in maize

Application of nitrogen fertilizer in the past 50 years has resulted in significant increases in crop yields. However, loss of nitrogen from crop fields has been associated with negative impacts on the environment. Developing maize hybrids with improved nitrogen use efficiency is a cost‐effective strategy for increasing yield sustainably. We report that a dominant male‐sterile mutant Ms44 encodes a lipid transfer protein which is expressed specifically in the tapetum. A single amino acid change from alanine to threonine at the signal peptide cleavage site of the Ms44 protein abolished protein processing and impeded the secretion of protein from tapetal cells into the locule, resulting in dominant male sterility. While the total nitrogen (N) content in plants was not changed, Ms44 male‐sterile plants reduced tassel growth and improved ear growth by partitioning more nitrogen to the ear, resulting in a 9.6% increase in kernel number. Hybrids carrying the Ms44 allele demonstrated a 4%–8.5% yield advantage when N is limiting, 1.7% yield advantage under drought and 0.9% yield advantage under optimal growth conditions relative to the yield of wild type. Furthermore, we have developed an Ms44 maintainer line for fertility restoration, male‐sterile inbred seed increase and hybrid seed production. This study reveals that protein secretion from the tapetum into the locule is critical for pollen development and demonstrates that a reduction in competition between tassel and ear by male sterility improves grain yield under low‐nitrogen conditions in maize.     

Programmed cell death and hybrid incompatibility

We propose a new theory to explain developmental aberrations in plant hybrids. In our theory, hybrid incompatibilities arise from imbalances in the mechanisms that cause male sterility in hermaphroditic plants. Mitochondria often cause male sterility by killing the tapetal tissue that nurtures pollen mother cells. Recent evidence suggests that mitochondria destroy the tapetum by triggering standard pathways of programmed cell death. Some nuclear genotypes repress mitochondrial male sterility and restore pollen fertility. Normal regulation of tapetal development therefore arises from a delicate balance between the disruptive effects of mitochondria and the defensive countermeasures of the nuclear genes. In hybrids, incompatibilities between male-sterile mitochondria and nuclear restorers may frequently upset the regulatory control of programmed cell death, causing tapetal abnormalities and male sterility. We propose that hybrid misregulation of programmed cell death may also spill over into other tissues, explaining various developmental aberrations observed in hybrids.     

芝麻核雄性不育系ms86-1微粉发生的细胞学观察

芝麻(Sesamum indicum)核雄性不育系ms86-1姊妹交后代表现为可育、部分不育(即微粉)及完全不育(简称不育)3种类型。不同育性类型的花药及花粉粒形态差异明显。Alexander染色实验显示微粉植株花粉粒外壁为蓝绿色, 内部为不均一洋红色, 与可育株及不育株花粉粒的染色特征均不相同。为探明芝麻微粉发生机理, 在电子显微镜下比较观察了可育、微粉、不育类型的小孢子发育过程。结果表明, 可育株小孢子母细胞减数分裂时期代谢旺盛, 胞质中出现大量脂质小球; 四分体时期绒毡层细胞开始降解, 单核小孢子时期开始出现乌氏体, 成熟花粉时期花粉囊腔内及花粉粒周围分布着大量乌氏体, 花粉粒外壁有11–13个棱状凸起, 表面存在大量基粒棒, 形成紧密的覆盖层。不育株小孢子发育异常显现于减数分裂时期, 此时胞质中无脂质小球出现, 细胞壁开始积累胼胝质; 四分体时期绒毡层细胞未见降解; 单核小孢子时期无乌氏体出现; 成熟花粉时期花粉囊腔中未发现正常的乌氏体, 存在大量空瘪的败育小孢子, 外壁积累胼胝质, 缺乏基粒棒。微粉株小孢子在减数分裂时期可见胞质内有大量脂质小球, 四分体时期部分绒毡层发生变形, 单核小孢子时期有部分绒毡层开始降解; 绒毡层细胞降解滞后为少量发育进程迟缓的小孢子提供了营养物质, 部分小孢子发育为正常花粉粒; 这些花粉粒比较饱满, 表面有少量颗粒状突起, 但未能形成覆盖层, 花粉囊腔中及小孢子周围存在少量的乌氏体。小孢子形成的育性类型与绒毡层降解是否正常有关。     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map‐based cloning and sequence analysis, we identified a 1,459‐bp deletion in an adenosine triphosphate (ATP)‐binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very‐long‐chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice 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.     

Magnesium Transporter 5 plays an important role in Mg transport for male gametophyte development in Arabidopsis

During anther development the male gametophyte develops inside the locule and the tapetal cells provide all nutrients for its development. Magnesium Transporter 5 (MGT5) is a member of the MGT family and has dual functions of Mg export and import. Here, we show that male gametophyte mitosis and intine formation are defective in a mgt5 mutant. The transient expression of GFP‐MGT5 revealed that MGT5 is localized in the plasma membrane. These findings suggest that in the male gametophyte MGT5 plays a role in importing Mg from the locule and that Mg is essential for male gametophyte development. The expression of MGT5 in the knockout ABORTED MICROSPORES (AMS) mutant (AMS being an essential regulator of tapetum) is tremendously reduced. Chromatin immunoprecipitation and mobility shift assay experiments demonstrated that AMS can directly bind the promoter of MGT5. An immunoelectron microscopy assay revealed that MGT5‐His is localized to the plasma membrane of the tapetum. These findings suggest that AMS directly regulates MGT5 in the tapetum and thus induces export of Mg into the locule. The mgt5 plant exhibits severe male sterility while the expression of MGT5 under the tapetum‐specific promoter A9 partly rescued mgt5 fertility. mgt5 fertility was restored under high‐Mg conditions. These findings suggest that the mgt5 tapetum still has the ability to export Mg and that a sufficient supply of Mg from the tapetum can improve the importation of Mg in the mgt5 male gametophyte. Therefore, MGT5 plays an important role in Mg transport from the tapetum to the microspore.     

Brassinosteroid‐mediated reactive oxygen species are essential for tapetum degradation and pollen fertility in tomato

Phytohormone brassinosteroids (BRs) are essential for plant growth and development, but the mechanisms of BR‐mediated pollen development remain largely unknown. In this study, we show that pollen viability, pollen germination and seed number decreased in the BR‐deficient mutant d^{^im}, which has a lesion in the BR biosynthetic gene DWARF (DWF), and in the bzr1 mutant, which is deficient in BR signaling regulator BRASSINAZOLE RESISTANT 1 (BZR1), compared with those in wild‐type plants, whereas plants overexpressing DWF or BZR1 exhibited the opposite effects. Loss or gain of function in the DWF or BZR1 genes altered the timing of reactive oxygen species (ROS) production and programmed cell death (PCD) in tapetal cells, resulting in delayed or premature tapetal degeneration, respectively. Further analysis revealed that BZR1 could directly bind to the promoter of RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1), and that RBOH1‐mediated ROS promote pollen and seed development by triggering PCD and tapetal cell degradation. In contrast, the suppression of RBOH1 compromised BR signaling‐mediated ROS production and pollen development. These findings provide strong evidence that BZR1‐dependent ROS production plays a critical role in the BR‐mediated regulation of tapetal cell degeneration and pollen development in Solanum lycopersicum (tomato) plants.     

Microscopy of a cytoplasmic male-sterile soybean from an interspecific cross between Glycine max and G. soja (Leguminosae)

Cytoplasmic male sterility has been found independently in soybean three times since 1995, but no microscopic investigation has been published. The purpose of this microscopic study was to establish the developmental sequence leading to sterility in a cytoplasmic male-sterile soybean line that has been found to be stable under all environmental conditions tested and to demarcate the temporal and spatial parameters that result in degeneration of the microspores and pollen grains. Light microscopy showed an abnormal development and/or premature degeneration of the tapetum after meiosis II, but some pollen grains persisted until after microspore mitosis. The pollen grains never completely filled with reserves. Premature formation of the endothecium also was evident. Histochemical staining for water-insoluble carbohydrates revealed an abnormal pattern of starch deposition in anther walls that coincided with lack of pollen filling. Electron microscopy showed degeneration of the inner mitochondrial membrane in the tapetal cells as the first detectable change leading to cell degeneration. Subsequently, the tapetal endoplasmic reticulum exhibited atypical concentric rings. Pollen grains displayed mitochondria with unusually enlarged inner mitochondrial spaces, degraded plastids, a rudimentary intine, and no starch or lipid reserves. Results link mitochondrial degeneration, premature formation of the endothecium, and energy deprivation to male sterility.     

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radishand has been introduced into rapeseed (Brassica napus) by protoplastfusion. As with all CMS systems, it involves a constitutivelyexpressed mitochondrial gene which induces male sterility tootherwise hermaphroditic plants (so they become females) anda nuclear gene named restorer of fertility that restores pollenproduction in plants carrying a sterility-inducing cytoplasm.A correlative approach using light and electron microscopy wasapplied to define what stages throughout development were affectedand the subcellular events leading to the abortion of the developingpollen grains upon the expression of the mitochondrial protein.Three central stages of development (tetrad, mid-microsporeand vacuolate microspore) were compared between fertile, restored,and sterile plants. At each stage observed, the pollen in fertileand restored plants had similar cellular structures and organization.The deleterious effect of the sterility protein expression startedas early as the tetrad stage. No typical mitochondria were identifiedin the tapetum at any developmental stage and in the vacuolatemicrospores of the sterile plants. In addition, some strikingultrastructural alterations of the cell's organization werealso observed compared with the normal pattern of development.The results showed that Ogu-INRA CMS was due to premature celldeath events of the tapetal cells, presumably by an autolysisprocess rather than a normal PCD, which impairs pollen developmentat the vacuolate microspore stage, in the absence of functionalmitochondria. Key words: Brassica napus, cell death, light and electron microscopy, mitochondria, plastids, pollen development, Ogu-INRA cytoplasmic male sterility, transgenic-restored plants, tapetum Received 30 September 2007; Revised 11 December 2007 Accepted 20 December 2007     

Induction of male sterility in transgenic chrysanthemums (Chrysanthemum morifolium Ramat.) by expression of a mutated ethylene receptor gene, Cm-ETR1/H69A, and the stability of this sterility at varying growth temperatures

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most popular ornamental flowers in the world, and many agronomic traits have recently been introduced to chrysanthemum cultivars by gene transformation. Concerns have been raised, however, regarding transgene flow from transgenic plants to wild plants. In early studies, ethylene receptor genes have been used for genetic modification in plants, such as flower longevity and fruit ripening. Recently, overexpression of ethylene receptor genes from melon (CmETR1/H69A) caused delayed tapetum degradation of the anther sac and a reduction in pollen grains. We therefore introduced the ethylene receptor gene into chrysanthemums to induce male sterility and prevent transgene flow via pollen. The chrysanthemum cultivar Yamate shiro was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA105, carrying the binary vector pBIK102H69A, which contains the CmETR1/H69A gene. A total of 335 shoots were regenerated from 1,282 leaf discs on regeneration medium (26.1%). The presence of the Cm-ETR1/H69A gene was confirmed in all of the regenerated plantlets by Southern blot analysis. These genetically modified (GM) plants and their non-GM counterparts were grown in a closed greenhouse and flowered at temperatures between 10 and 35°C. In 15 of the 335 GM chrysanthemum lines, the number of mature pollen grains was significantly reduced, particularly in three of the lines (Nos. 91, 191 and 324). In these three lines, pollen grains were not observed at temperatures between 20 and 35°C but were observed at 10 and 15°C, and mature pollen grains were formed only at 15°C. In northern blot analyses, expression of the CmETR1/H69A gene was suppressed at low temperatures. This phenomenon was observed as a result of both the suppression of CmETR1/H69A expression at low temperatures and the optimal growth temperature of chrysanthemums (15–20°C). Furthermore, the female fertility of these three GM lines was significantly lower than that of the non-GM plants. Thus, the mutated ethylene receptor is able to reduce both male and female fertility significantly in transgenic chrysanthemums, although the stability of male and/or female sterility at varying growth temperatures is a matter of concern for its practical use.     

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis

In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. In this study, we identified that the Arabidopsis Transmembrane Protein 18 (AtTMEM18) gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins (TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen‐rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18‐GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility. Furthermore, expression of the rice TMEM18‐homologous protein (OsTMEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.     

Rice No Pollen 1 (NP1) is required for anther cuticle formation and pollen exine patterning

Angiosperm male reproductive organs (anthers and pollen grains) have complex and interesting morphological features, but mechanisms that underlie their patterning are poorly understood. Here we report the isolation and characterization of a male sterile mutant of No Pollen 1 (NP1) in rice (Oryza sativa). The np1‐4 mutant exhibited smaller anthers with a smooth cuticle surface, abnormal Ubisch bodies, and aborted pollen grains covered with irregular exine. Wild‐type exine has two continuous layers; but np1‐4 exine showed a discontinuous structure with large granules of varying size. Chemical analysis revealed reduction in most of the cutin monomers in np1‐4 anthers, and less cuticular wax. Map‐based cloning suggested that NP1 encodes a putative glucose‐methanol‐choline oxidoreductase; and expression analyses found NP1 preferentially expressed in the tapetal layer from stage 8 to stage 10 of anther development. Additionally, the expression of several genes involved in biosynthesis and in the transport of lipid monomers of sporopollenin and cutin was decreased in np1‐4 mutant anthers. Taken together, these observations suggest that NP1 is required for anther cuticle formation, and for patterning of Ubisch bodies and the exine. We propose that products of NP1 are likely important metabolites in the development of Ubisch bodies and pollen exine, necessary for polymerization, assembly, or both.     

Lectin receptor kinase OsLecRK‐S.7 is required for pollen development and male fertility

Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination. Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture. The mutant gene encodes a novel plasma membrane‐localized legume‐lectin receptor kinase that we named OsLecRK‐S.7. OsLecRK‐S.7 was expressed at different levels in all tested tissues and throughout anther development. In vitro kinase assay showed OsLecRK‐S.7 capable of autophosporylation. Mutation in s13283 (E560K) and mutation of the conserved ATP binding site (K418E) both knocked out the kinase activity. Mass spectrometry showed Thr₃₇₆, Ser₃₇₈, Thr₃₈₆, Thr₄₀₃, and Thr₆₅₇ to be the autophosphorylation sites. Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees, but did not abolish the gene function in fertility complementation. oslecrk‐s.7 mutant plant overexpressing OsLecRK‐S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers, and these phenotypes were abolished by E560K or K418E mutation. The results indicated that OsLecRK‐S.7 was a key regulator of pollen development.