Exine Formation Research Articles

Secretory COPII Protein SEC31B Is Required for Pollen Wall Development.

The pollen wall protects pollen grains from abiotic and biotic stresses. During pollen wall development, tapetal cells play a vital role by secreting proteins, signals, and pollen wall material to ensure microspore development. But the regulatory mechanism underlying the secretory pathway of the tapetum is largely unknown. Here, we characterize the essential role of the Arabidopsis (Arabidopsis thaliana) COPII protein SECRETORY31B (SEC31B) in pollen wall development and the secretory activity of tapetal cells. The sporophyte-controlled atsec31b mutant exhibits severe pollen and seed abortion. Transmission electron microscopy observation indicates that pollen exine formation in the atsec31b mutant is disrupted significantly. AtSEC31B is a functional COPII protein revealed by endoplasmic reticulum (ER) exit site localization, interaction with AtSEC13A, and retarded ER-Golgi protein trafficking in the atsec31b mutant. A genetic tapetum-specific rescue assay indicates that AtSEC31B functions primarily in the tapetum. Moreover, deletion of AtSEC31B interrupted the formation of the ER-derived tapetosome and altered the location of the ATP-BINDING CASSETTE TRANSPORTER9 protein in the tapetum. Therefore, this work demonstrates that AtSEC31B plays a vital role in pollen wall development by regulating the secretory pathway of the tapetal cells.

Role of Lipid Metabolism in Plant Pollen Exine Development.

Pollen plays important roles in the life cycle of angiosperms plants. It acts as not only a biological protector of male sperms but also a communicator between the male and the female reproductive organs, facilitating pollination and fertilization. Pollen is produced within the anther, and covered by the specialized outer envelope, pollen wall. Although the morphology of pollen varies among different plant species, the pollen wall is mainly comprised of three layers: the pollen coat, the outer exine layer, and the inner intine layer. Except the intine layer, the other two layers are basically of lipidic nature. Particularly, the outer pollen wall layer, the exine, is a highly resistant biopolymer of phenylpropanoid and lipidic monomers covalently coupled by ether and ester linkages. The precise molecular mechanisms underlying pollen coat formation and exine patterning remain largely elusive. Herein, we summarize the current genetic, phenotypic and biochemical studies regarding to the pollen exine development and underlying molecular regulatory mechanisms mainly obtained from monocot rice (Oryza sativa) and dicot Arabidopsis thaliana, aiming to extend our understandings of plant male reproductive biology. Genes, enzymes/proteins and regulatory factors that appear to play conserved and diversified roles in lipid biosynthesis, transportation and modification during pollen exine formation, were highlighted.

Using RNA-seq to Profile Gene Expression of Spikelet Development in Response to Temperature and Nitrogen during Meiosis in Rice (Oryza sativa L.).

Rice reproductive development is sensitive to high temperature and soil nitrogen supply, both of which are predicted to be increased threats to rice crop yield. Rice spikelet development is a critical process that determines yield, yet little is known about the transcriptional regulation of rice spikelet development in response to the combination of heat stress and low nitrogen availability. Here, we profiled gene expression of rice spikelet development during meiosis under heat stress and different nitrogen levels using RNA-seq. We subjected plants to four treatments: 1) NN: normal nitrogen level (165 kg ha-1) with normal temperature (30°C); 2) HH: high nitrogen level (264 kg ha-1) with high temperature (37°C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and normal temperature. The de novo transcriptome assembly resulted in 52,250,482 clean reads aligned with 76,103 unigenes, which were then used to compare differentially expressed genes (DEGs) in the different treatments. Comparing gene expression in samples with the same nitrogen levels but different temperatures, we identified 70 temperature-responsive DEGs in normal nitrogen levels (NN vs NH) and 135 DEGs in high nitrogen levels (HN vs HH), with 27 overlapping DEGs. We identified 17 and seven nitrogen-responsive DEGs by comparing changes in nitrogen levels in lower temperature (NN vs HN) and higher temperature (NH vs HH), with one common DEG. The temperature-responsive genes were principally associated with cytochrome, heat shock protein, peroxidase, and ubiquitin, while the nitrogen-responsive genes were mainly involved in glutamine synthetase, amino acid transporter, pollen development, and plant hormone. Rice spikelet fertility was significantly reduced under high temperature, but less reduced under high-nitrogen treatment. In the high temperature treatments, we observed downregulation of genes involved in spikelet development, such as pollen tube growth, pollen maturation, especially sporopollenin biosynthetic process, and pollen exine formation. Moreover, we observed higher expression levels of the co-expressed DEGs in HN vs HH compared to NN vs NH. These included the six downregulated genes (one pollen maturation and five pollen exine formation genes), as well as the four upregulated DEGs in response to heat. This suggests that high-nitrogen treatment may enhance the gene expression levels to mitigate aspects of heat-stress. The spikelet genes identified in this study may play important roles in response to the combined effects of high temperature and high nitrogen, and may serve as candidates for crop improvement.

Comparative Transcriptome Analysis of Recessive Male Sterility (RGMS) in Sterile and Fertile Brassica napus Lines.

The recessive genetic male sterility (RGMS) system plays a key role in the production of hybrid varieties in self-pollinating B. napus plants, and prevents negative cytoplasmic effects. However, the complete molecular mechanism of the male sterility during male-gametogenesis in RGMS remains to be determined. To identify transcriptomic changes that occur during the transition to male sterility in RGMS, we examined the male sterile line WSLA and male fertile line WSLB, which are near-isogenic lines (NILs) differing only in the fertility trait. We evaluated the phenotypic features and sterility stage using anatomical analysis. Comparative RNA sequencing analysis revealed that 3,199 genes were differentially expressed between WSLA and WSLB. Many of these genes are mainly involved in biological processes related to flowering, including pollen tube development and growth, pollen wall assembly and modification, and pollen exine formation and pollination. The transcript profiles of 93 genes associated with pollen wall and anther development were determined by quantitative RT-PCR in different flower parts, and classified into the following three major clades: 1) up-regulated in WSLA plants; 2) down-regulated in WSLA plants; and 3) down-regulated in buds, but have a higher expression in stigmas of WSLA than in WSLB. A subset of genes associated with sporopollenin accumulation were all up-regulated in WSLA. An excess of sporopollenin results in defective pollen wall formation, which leads to male sterility in WSLA. Some of the genes identified in this study are candidates for future research, as they could provide important insight into the molecular mechanisms underlying RGMS in WSLA.

RNA-seq reveals differentially expressed genes of rice (Oryza sativa) spikelet in response to temperature interacting with nitrogen at meiosis stage

BackgroundRice (Oryza sativa) is one of the most important cereal crops, providing food for more than half of the world’s population. However, grain yields are challenged by various abiotic stresses such as drought, fertilizer, heat, and their interaction. Rice at reproductive stage is much more sensitive to environmental temperatures, and little is known about molecular mechanisms of rice spikelet in response to high temperature interacting with nitrogen (N).ResultsHere we reported the transcriptional profiling analysis of rice spikelet at meiosis stage using RNA sequencing (RNA-seq) as an attempt to gain insights into molecular events associated with temperature and nitrogen. This study received four treatments: 1) NN: normal nitrogen level (165 kg ha−1) with natural temperature (30 °C); 2) HH: high nitrogen level (330 kg ha−1) with high temperature (37 °C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and natural temperature, respectively. The de novo assembly generated 52,553,536 clean reads aligned with 72,667 unigenes. About 10 M reads were identified from each treatment. In these differentially expressed genes (DEGs), we found 151 and 323 temperature-responsive DEGs in NN-vs-NH and HN-vs-HH, and 114 DEGs were co-expressed. Meanwhile, 203 and 144 nitrogen-responsive DEGs were focused in NN-vs-HN and NH-vs-HH, and 111 DEGs were co-expressed. The temperature-responsive genes were principally associated with calcium-dependent protein, cytochrome, flavonoid, heat shock protein, peroxidase, ubiquitin, and transcription factor while the nitrogen-responsive genes were mainly involved in glutamine synthetase, transcription factor, anthocyanin, amino acid transporter, leucine zipper protein, and hormone. It is noted that, rice spikelet fertility was significantly decreased under high temperature, but it was more reduced under higher nitrogen. Accordingly, numerous spikelet genes involved in pollen development, pollen tube growth, pollen germination, especially sporopollenin biosynthetic process, and pollen exine formation were mainly down-regulated under high temperature. Moreover, the expression levels of co-expressed DEGs including 5 sporopollenin biosynthetic process and 7 pollen exine formation genes of NN-vs-NH were lower than that of HN-vs-HH. Therefore, these spikelet genes may play important roles in response to high temperature with high nitrogen and may be good candidates for crop improvement.ConclusionsThis RNA-seq study will help elucidate the molecular mechanisms of rice spikelet defense response to high temperature interacting with high nitrogen level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2141-9) contains supplementary material, which is available to authorized users.

Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction.

Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development.

Data for proteomic profiling of Anthers from a photosensitive male sterile mutant and wild-type cotton (Gossypium hirsutum L.).

Cotton is an important economic crop, used mainly for the production of textile fiber. Using a space mutation breeding technique, a novel photosensitive genetic male sterile mutant CCRI9106 was isolated from the wild-type upland cotton cultivar CCRI040029. To study the male sterile mechanisms of CCRI9106, histological and iTRAQ-facilitated proteomic analyses of anthers were performed. This data article contains data related to the research article titled iTRAQ-Facilitated Proteomic Profiling of Anthers From a Photosensitive Male Sterile Mutant and Wild-type Cotton (Gossypium hirsutum L.)[1]. This research article describes the iTRAQ-facilitated proteomic analysis of the wild-type and a photosensitive male sterile mutant in cotton. The report indicated that exine formation defect is the key reason for male sterility in mutant plant. The information presented here represents the tables and figures that detail the processing of the raw data obtained from iTRAQ analysis.

ITRAQ-facilitated proteomic profiling of anthers from a photosensitive male sterile mutant and wild-type cotton (Gossypium hirsutum L.)

Male sterility is a common phenomenon in flowering plants, and it has been successfully developed in several crops by taking advantage of heterosis. Cotton (Gossypium hirsutum L.) is an important economic crop, used mainly for the production of textile fiber. Using a space mutation breeding technique, a novel photosensitive genetic male sterile mutant CCRI9106 was isolated from the wild-type upland cotton cultivar CCRI040029. To use CCRI9106 in cotton hybrid breeding, it is of great importance to study the molecular mechanisms of its male sterility. Here, histological and iTRAQ-facilitated proteomic analyses of anthers were performed to explore male sterility mechanisms of the mutant. Scanning and transmission electron microscopy of the anthers showed that the development of pollen wall in CCRI9106 was severely defective with a lack of exine formation. At the protein level, 6121 high-confidence proteins were identified and 325 of them showed differential expression patterns between mutant and wild-type anthers. The proteins up- or down-regulated in MT anthers were mainly involved in exine formation, protein degradation, calcium ion binding,etc. These findings provide valuable information on the proteins involved in anther and pollen development, and contribute to elucidate the mechanism of male sterility in upland cotton.

Transcriptional profiling analysis of genic male sterile–fertile Capsicum annuum reveal candidate genes for pollen development and maturation by RNA-Seq technology

To identify genes involved in the pollen development and maturation, genome-wide transcriptional profiling of the flower buds from fertile and sterile plants of the genic male sterile–fertile line 114AB of Capsicum annuum was performed by the RNA-Seq approach. The results indicated that there were numerous changes in gene expression due to the gene mutation in sterile plants. A total of 668 differential expressed genes were identified. Among them, 562 were up-regulated and 106 were down-regulated genes in the fertile plants compared to those of the sterile plants, including 202 and 2 genes specifically accumulated in fertile and sterile plants, respectively. Hundreds of genes potentially involved in pollen development were identified from the genes specifically expressed in the fertile plants, including pollen coat protein, lipid binding protein, lipid transfer protein, pectin methylesterase, male sterility-related protein, anther-specific protein, pectate lyase gene and so forth. Subsequent analysis of expression patterns for a subset of these genes were verified by semi-quantitative RT-PCR and quantitative RT-PCR. Furthermore, the up-regulated genes were predicted to be associated with intracellular membrane-bounded organelle, cytoplasmic vesicle, oxidoreductase activity, metal ion binding, pollen wall assembly, pollen exine formation, starch and sucrose metabolism, and other processes through Gene ontology and KEGG enrichment analysis. Taken together, this study provides a list of candidate genes and their expression patterns for pollen development and maturation in pepper.

Radioautographic visualization of incorporation of lipid precursors into anthers of Muscari comosum (L.) Mili.

Incorporation of the following lipid precursors: DL-mevalonic acid-2 <sup>3</sup>H, <sup>3</sup>H palmitic acid and acetic acid-<sup>3</sup>H sodium salt, into the anther cells of <i>Muscari comosum</i> (L.) Mili. has been investigated. These lipid precursors have been demonstrated to incorporate into pollen mother cells, pollen grains and the tapetum at the consecutive developmental stages. Ali used isotopes are incorporated into the cytoplasm of these cells and in the case of pollen grain the radioactivity of pollen grain wall (mainly composed of sporopellenin) is noticed. The highest radioactivity of pollen grain wall is observed after acetic acid, the lower one after palmitic acid, whereas the lowest uptake of lipid precursor occurs after mevalonic acid. In comparison with tetrad stage the distinct inrease of the cytoplasmic radioactivity of tapetum, which appears to1 accompany the labelling of pollen grain wall, seems to indicate the participation of tapetum in the formation of exine. A possible role of Ubisch bodies in the formation of pollen grain wall is discussed.

ABCG26-mediated polyketide trafficking and hydroxycinnamoyl spermidines contribute to pollen wall exine formation in Arabidopsis.

Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the outer pollen wall (exine) are contributed by surrounding sporophytic tapetal cells. Because the biosynthesis and development of the exine occurs in the innermost cell layers of the anther, direct observations of this process are difficult. The objective of this study was to investigate the transport and assembly of exine components from tapetal cells to microspores in the intact anthers of Arabidopsis thaliana. Intrinsically fluorescent components of developing tapetum and microspores were imaged in intact, live anthers using two-photon microscopy. Mutants of ABCG26, which encodes an ATP binding cassette transporter required for exine formation, accumulated large fluorescent vacuoles in tapetal cells, with corresponding loss of fluorescence on microspores. These vacuolar inclusions were not observed in tapetal cells of double mutants of abcg26 and genes encoding the proposed sporopollenin polyketide biosynthetic metabolon (ACYL COENZYME A SYNTHETASE5, POLYKETIDE SYNTHASE A [PKSA], PKSB, and TETRAKETIDE α-PYRONE REDUCTASE1), providing a genetic link between transport by ABCG26 and polyketide biosynthesis. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides, raising the possibility that they are incorporated into the exine prior to pollen coat deposition. We propose a model where ABCG26-exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with the trafficking of additional constituents, prior to tapetum programmed cell death.

An RNA-seq transcriptome analysis of floral buds of an interspecific Brassica hybrid between B. carinata and B. napus.

Interspecific hybridizations promote gene transfer between species and play an important role in plant speciation and crop improvement. However, hybrid sterility that commonly found in the first generation of hybrids hinders the utilization of interspecific hybridization. The combination of divergent parental genomes can create extensive transcriptome variations, and to determine these gene expression alterations and their effects on hybrids, an interspecific Brassica hybrid of B. carinata × B. napus was generated. Scanning electron microscopy analysis indicated that some of the hybrid pollen grains were irregular in shape and exhibited abnormal exine patterns compared with those from the parents. Using the Illumina HiSeq 2000 platform, 39,598, 32,403 and 42,208 genes were identified in flower buds of B. carinata cv. W29, B. napus cv. Zhongshuang 11 and their hybrids, respectively. The differentially expressed genes were significantly enriched in pollen wall assembly, pollen exine formation, pollen development, pollen tube growth, pollination, gene transcription, macromolecule methylation and translation, which might be associated with impaired fertility in the F1 hybrid. These results will shed light on the mechanisms underlying the low fertility of the interspecific hybrids and expand our knowledge of interspecific hybridization.

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. 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-014-1666-8) contains supplementary material, which is available to authorized users.

Rice CYP703A3, a cytochrome P450 hydroxylase, is essential for development of anther cuticle and pollen exine.

Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum-expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3-2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3-2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in-chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7-hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in-chain hydroxylation of lauric acid required for the development of male organ in higher plants.

Cysteine Protease 51 (CP51), an anther-specific cysteine protease gene, is essential for pollen exine formation in Arabidopsis

Cysteine proteases play important roles in intracellular protein degradation, programmed cell death and responses to environmental stimuli in plant. Although subclassification and biochemical analysis of major plant papain-like cysteine proteases (PLCPs) have been studied, the biological function of many PLCPs remained unknown. In this study, we identified a PLCP gene Cysteine Protease 51 (CP51) which participates in exine formation and anther development in Arabidopsis thaliana. Promoter-GUS fusion detection showed its specific expression in anthers at stages 9–12. RNA interference (RNAi) transgenic plants with reduced CP51 transcriptional levels exhibited a male sterile phenotype with aborted microspores, shortened siliques and fewer or no seeds. Cytological analysis indicated that pollen abortion occurred due to defective pollen exine and the tapetum degraded earlier during the transition from the uninucleated stage to the binucleated stage. Scanning electron microscopy demonstrated that aborted microspores lacked complete or normal reticulate exine, and the intine membrane was extruded in the pollens of CP51-RNAi plants. Transmission electron microscopy further revealed that the tapetum degeneration was initiated early and that normal tectum connections to the bacula were missing in anthers of CP51-RNAi plants. Taken together, these results suggested that CP51 critically mediates tapetum stability and pollen exine formation.

The Arabidopsis Exine Formation Defect (EFD) gene is required for primexine patterning and is critical for pollen fertility

Exine, the outermost layer of a pollen grain, has important roles in protecting microspore cytoplasm and determining species-specific interactions between pollen and stigma. The molecular mechanism underlying pollen exine formation, however, remains largely unknown. Here, we report the characterization of an Arabidopsis male-sterile mutant, efd, which exhibits male sterility in first-forming flowers. The Exine Formation Defect (EFD) gene is strongly expressed in microsporocytes, tetrads and the tapetum, and encodes a nuclear-localized de novo DNA methyltransferase. Detailed observations revealed that EFD is involved in both callose wall and primexine formation during microsporogenesis. Microspores in tetrads are not well separated in efd due to an abnormal callose wall. Its plasma membrane undulation appears normal, but primexine patterning is impaired. Primexine matrix establishment and sporopollenin accumulation at specific positions are disturbed, and thus exine formation is totally blocked in efd. We confirmed that EFD is required for pollen exine formation and male fertility via the regulation of callose wall and primexine formation. We also found that positional sporopollenin accumulation is not involved in regulating membrane undulation, but is related to the complete separation of tetrad microspores during primary exine patterning.

A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development

The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning.

The Arabidopsis CALLOSE DEFECTIVE MICROSPORE1 Gene Is Required for Male Fertility through Regulating Callose Metabolism during Microsporogenesis

During angiosperm microsporogenesis, callose serves as a temporary wall to separate microsporocytes and newly formed microspores in the tetrad. Abnormal callose deposition and dissolution can lead to degeneration of developing microspores. However, genes and their regulation in callose metabolism during microsporogenesis still remain largely unclear. Here, we demonstrated that the Arabidopsis (Arabidopsis thaliana) CALLOSE DEFECTIVE MICROSPORE1 (CDM1) gene, encoding a tandem CCCH-type zinc finger protein, plays an important role in regulation of callose metabolism in male meiocytes and in integrity of newly formed microspores. First, quantitative reverse transcription PCR and in situ hybridization analyses showed that the CDM1 gene was highly expressed in meiocytes and the tapetum from anther stages 4 to 7. In addition, a transfer DNA insertional cdm1 mutant was completely male sterile. Moreover, light microscopy of anther sections revealed that microspores in the mutant anther were initiated, and then degenerated soon afterward with callose deposition defects, eventually leading to male sterility. Furthermore, transmission electron microscopy demonstrated that pollen exine formation was severely affected in the cdm1 mutant. Finally, we found that the cdm1 mutation affected the expression of callose synthesis genes (CALLOSE SYNTHASE5 and CALLOSE SYNTHASE12) and potential callase-related genes (A6 and MYB80), as well as three other putative β-1,3-glucanase genes. Therefore, we propose that the CDM1 gene regulates callose metabolism during microsporogenesis, thereby promoting Arabidopsis male fertility.

Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development.

Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu(2+) and Fe(2+)) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4',6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in cassava. This is the first ever detailed report of microspore reprogramming at the tetrad stage and the first report of microspore embryogenesis induction in cassava with detailed evidence.

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.