Ontogenesis in miniature. Pollen wall development in Campanula rapunculoides.

MAIN CONCLUSION: Our findings suggest a reconsideration of pollen wall ontogeny process, entailing examination of physical factors, which enable a new understanding of exine developmental processes as self-formation. The pollen wall, the most complex cell wall in plants, is especially interesting as a model of ontogeny in miniature. By a detailed study of each developmental stage of Campanula rapunculoides pollen wall, we aimed to understand the establishment of complex pollen walls and the underlying developmental mechanisms. Other aim was to compare our current observations with studies in other species to reveal the common principles. We also tried to analyse the reasons for commonalities in ontogenies of exines in remote species. TEM, SEM, comparative methods were used in this study. The sequence of events leading to exine emergence from early tetrad stage to maturity is as follows: the appearance of spherical micelles in the periplasmic space and de-mixing of the mixture in periplasm (condensed and depleted layers); appearance of plasma membrane invaginations and columns of spherical micelles inside condensed layer; appearance of rod-like units, pro-tectum and thin foot layer; the appearance of spiral substructure of procolumellae and of dendritic outgrowths on the tops of procolumellae, of vast depleted zone in aperture sites; formation of the endexine lamellae on the base of laminate micelles; gradual twisting of dendritic outgrowths (macromolecule chains) into clubs on the tops of columellae and into spines; final sporopollenin accumulation. Our observations are consistent with the sequence of self-assembling micellar mesophases. Complex organisation of the exine is established through processes of self-assembly operating together with another physical process-phase separation. After genomic determination of the exine building substances, purely physical processes which are not under direct genomic control play an important role after genomic control of constructive substances. The comparison of the underlying mechanisms of exine development in remote species occurred to be general and similar to crystallisation. Our ontogenetic experience has shown the commonality of pollen wall ontogenies in remote species.

Natural variation of MS2 confers male fertility and drives hybrid breeding in soybean.

A complete and genetically stable male sterile line with high outcrossing rate is a prerequisite for the development of commercial hybrid soybean. It was reported in the last century that the soybean male sterile ms2 mutant has the highest record with seed set. Here we report the cloning and characterization of the MS2 gene in soybean, which encodes a protein that is specifically expressed in the anther. MS2 functions in the tapetum and microspore by directly regulating genes involved in the biosynthesis of secondary metabolites and the lipid metabolism, which is essential for the formation of microspore cell wall. Through comparison of the field performance with the widely used male sterile mutants in the same genetic background, we demonstrated that the ms2 mutant conducts the best in outcrossing rate and makes it an ideal tool in building a cost-effective hybrid system for soybean.

Anatomy and Comparative Transcriptome Reveal the Mechanism of Male Sterility in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge is an important traditional herb. Salvia miltiorrhiza is distributed in the Sichuan province of China (here called SC). Under natural conditions, it does not bear seeds and its sterility mechanism is still unclear. Through artificial cross, there was defective pistil and partial pollen abortion in these plants. Electron microscopy results showed that the defective pollen wall was caused by delayed degradation of the tapetum. Due to the lack of starch and organelle, the abortive pollen grains showed shrinkage. RNA-seq was performed to explore the molecular mechanisms of pollen abortion. KEGG enrichment analysis suggested that the pathways of phytohormone, starch, lipid, pectin, and phenylpropanoid affected the fertility of S. miltiorrhiza. Moreover, some differentially expressed genes involved in starch synthesis and plant hormone signaling were identified. These results contribute to the molecular mechanism of pollen sterility and provide a more theoretical foundation for molecular-assisted breeding.

Secretory lipid transfer protein OsLTPL94 acts as a target of EAT1 and is required for rice pollen wall development.

The plant pollen wall protects the male gametophyte from various biotic and abiotic stresses. The formation of a unique pollen wall structure and elaborate exine pattern is a well-organized process, which needs coordination between reproductive cells and the neighboring somatic cells. However, molecular mechanisms underlying this process remain largely unknown. Here, we report a rice male-sterile mutant (l94) that exhibits defective pollen exine patterning and abnormal tapetal cell development. MutMap and knockout analyses demonstrated that the causal gene encodes a type-G non-specific lipid transfer protein (OsLTPL94). Histological and cellular analyses established that OsLTPL94 is strongly expressed in the developing microspores and tapetal cells, and its protein is secreted to the plasma membrane. The l94 mutation impeded the secretory ability of OsLTPL94 protein. Further in vivo and in vitro investigations supported the hypothesis that ETERNAL TAPETUM 1 (EAT1), a basic helix-loop-helix transcription factor (bHLH TF), activated OsLTPL94 expression through direct binding to the E-box motif of the OsLTPL94 promoter, which was supported by the positive correlation between the expression of EAT1 and OsLTPL94 in two independent eat1 mutants. Our findings suggest that the secretory OsLTPL94 plays a key role in the coordinated development of tapetum and microspores with the regulation of EAT1.

An integral insight into pollen wall development: involvement of physical processes in exine ontogeny in Calycanthus floridus L., with an experimental approach.

We aimed to understand the underlying mechanisms of development in the sporopollenin-containing part of the pollen wall, the exine, one of the most complex cell walls in plants. Our hypothesis is that distinct physical processes, phase separation and micellar self-assembly, underpinexine development by taking the molecular building blocks, determined and synthesised by the genome, through several phase transitions. To test this hypothesis, we traced each stage of microspore development in Calycanthus floridus with transmission electron microscopy and then generated in vitro experimental simulations corresponding to every developmental stage. The sequence of structures observed within the periplasmic space around developing microspores starts with spherical units, which are rearranged into columns to then form rod-like units (the young columellae) and, finally, white line centred endexine lamellae. Phase separation precedes each developmental stage. The set of experimental simulations, obtained as self-assembled micellar mesophases formed at the interface between lipid and water compartments, was the same: spherical micelles; columns of spherical micelles; cylindrical micelles; and laminate micelles, separated by gaps, resembling white-lined lamellae. Thus, patterns simulating structures observed at the main stages of exine development in C. floridus were obtained from in vitro experiments, and hence purely physicochemical processes can construct exine-like patterns. This highlights the important part played by physical processes that are not under direct genomic control and share influence on the emerging ultrastructure with the genome during exine development. These findings suggest that a new approach to ontogenetic studies, including a consideration of physical factors, is required for a better understanding of developmental processes.

Mutation of glucose-methanol-choline oxidoreductase leads to thermosensitive genic male sterility in rice and Arabidopsis.

Thermosensitive genic male sterility (TGMS) lines serve as the major genetic resource for two-line hybrid breeding in rice. However, their unstable sterility under occasional low temperatures in summer highly limits their application. In this study, we identified a novel rice TGMS line, ostms18, of cultivar ZH11 (Oryza sativa ssp. japonica). ostms18 sterility is more stable in summer than the TGMS line carrying the widely used locus tms5 in the ZH11 genetic background, suggesting its potential application for rice breeding. The ostms18 TGMS trait is caused by the point mutation from Gly to Ser in a glucose-methanol-choline (GMC) oxidoreductase; knockout of the oxidoreductase was previously reported to cause complete male sterility. Cellular analysis revealed the pollen wall of ostms18 to be defective, leading to aborted pollen under high temperature. Further analysis showed that the tapetal transcription factor OsMS188 directly regulates OsTMS18 for pollen wall formation. Under low temperature, the flawed pollen wall in ostms18 is sufficient to protect its microspore, allowing for development of functional pollen and restoring fertility. We identified the orthologous gene in Arabidopsis. Although mutants for the gene were fertile under normal conditions (24°C), fertility was significantly reduced under high temperature (28°C), exhibiting a TGMS trait. A cellular mechanism integrated with genetic mutations and different plant species for fertility restoration of TGMS lines is proposed.

ADAPTOR PROTEIN-1 complex-mediated post-Golgi trafficking is critical for pollen wall development in Arabidopsis.

Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum- and/or microspore-localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood. In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans-Golgi network/early endosome (TGN/EE)-localized ADAPTOR PROTEIN-1 complex (AP-1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP-1 in pollen wall formation. The deposition of Acyl-CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP-1µ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP-1 subunits at the TGN/EE. Our observations show that AP-1-mediated post-Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.

bHLH010/089 Transcription Factors Control Pollen Wall Development via Specific Transcriptional and Metabolic Networks in Arabidopsis thaliana.

The pollen wall is a specialized extracellular cell wall that protects male gametophytes from various environmental stresses and facilitates pollination. Here, we reported that bHLH010 and bHLH089 together are required for the development of the pollen wall by regulating their specific downstream transcriptional and metabolic networks. Both the exine and intine structures of bhlh010 bhlh089 pollen grains were severely defective. Further untargeted metabolomic and transcriptomic analyses revealed that the accumulation of pollen wall morphogenesis-related metabolites, including polysaccharides, glyceryl derivatives, and flavonols, were significantly changed, and the expression of such metabolic enzyme-encoding genes and transporter-encoding genes related to pollen wall morphogenesis was downregulated in bhlh010 bhlh089 mutants. Among these downstream target genes, CSLB03 is a novel target with no biological function being reported yet. We found that bHLH010 interacted with the two E-box sequences at the promoter of CSLB03 and directly activated the expression of CSLB03. The cslb03 mutant alleles showed bhlh010 bhlh089-like pollen developmental defects, with most of the pollen grains exhibiting defective pollen wall structures.

Delayed callose degradation restores the fertility of multiple P/TGMS lines in Arabidopsis.

Photoperiod/temperature-sensitive genic male sterility (P/TGMS) is widely applied for improving crop production. Previous investigations using the reversible male sterile (rvms) mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis. In this work, we isolated a restorer of rvms-2 (res3), as the male sterility of rvms-2 was rescued by res3. Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression, revealing a pathway for tapetum secretory function. Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3. The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3. We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation. A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.

IMPERFECTIVE EXINE FORMATION (IEF) is required for exine formation and male fertility in Arabidopsis.

KEY MESSAGE: IEF, a novel plasma plasma membrane protein, is important for exine formation in Arabidopsis. Exine, an important part of pollen wall, is crucial for male fertility. The major component of exine is sporopollenin which are synthesized and secreted by tapetum. Although sporopollenin synthesis has been well studied, the transportation of it remains elusive. To understand it, we analyzed the gene expression pattern in tapetal microdissection data, and investigated the potential transporter genes that are putatively regulated by ABORTED MICROSPORES (AMS). Among these genes, we identified IMPERFECTIVE EXINE FORMATION (IEF) that is important for exine formation. Compared to the wild type, ief mutants exhibit severe male sterility and pollen abortion, suggesting IEF is crucial for pollen development and male fertility. Using both scanning and transmission electron microscopes, we showed that exine structure was not well defined in ief mutant. The transient expression of IEF-GFP driven by the 35S promoter indicated that IEF-GFP was localized in plasma membrane. Furthermore, AMS can specifically activate the expression of promoterIEF:LUC in vitro, which suggesting AMS regulates IEF for exine formation. The expression of ATP-BINDING CASSETTE TRANSPORTER G26 (AGCB26) was not affected in ief mutants. In addition, SEM and TEM data showed that the sporopollenin deposition is more defective in abcg26/ief-2 than that of in abcg26, which suggesting that IEF is involved in an independent sporopollenin transportation pathway. This work reveal a novel gene, IEF regulated by AMS that is essential for exine formation.

Effects of pine pollen wall on gut microbiota and biomarkers in mice with dyslipidemia.

Pinus yunnanensis pollen is rich in various physiological functions. However, whether the pine pollen wall (PW) plays a beneficial role in the body has not been studied. In this work, we have analyzed its effects on the metabolism and gut microbiota of mouse models of dyslipidemia. We found that the intake of pine PW prevents the liver pathologic changes and reduce the concentrations of TNF-α, IL-6, TC, and high-density lipoprotein cholesterol. Moreover, it can regulate bile acid and fat metabolism, SCFAs content, and the structure of the gut microbiota. According to the change of carbohydrate metabolites, we speculated that cellulose should be the main component to play the above beneficial role, and sporopollenin cannot be utilized in the intestine. Therefore, we consider this study of great significance as it gives a description of biological effects of the pine PW and paves the road to its use in health products.

A GDSL-type esterase/lipase gene, GELP77, is necessary for pollen dissociation and fertility in Arabidopsis.

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.

Artificial pollen walls simulated by the tandem processes of phase separation and self-assembly in vitro.

Despite numerous attempts to elucidate the developmental mechanisms responsible for the observed diversity of pollen and spore walls, the processes involved remained obscure until the structures observed during exine development were recognized as a sequence of self-assembling micellar mesophases. To confirm this, a series of in vitro experiments was undertaken in which exine-like patterns were generated in colloidal mixtures by self-assembly, without any genomic participation. The intention was to test whether all the main types of exine structure could be simulated experimentally. Mixtures of substances, analogous to those involved in microspore development, were left undisturbed while water evaporated and self-assembly occurred. We varied the substances, their combinations and concentrations, and the physical constraints to make the experiments closer to the situation in nature. The resulting dry films were then examined using transmission electron microscopy. A variety of microstructures, simulating the full range of exine types, was obtained by micellar self-assembly. Moreover, the signs of related physicochemical process (i.e. phase separation) were also observed. Simple, energy-efficient, physical-chemical interactions, phase separation and self-assembly, are capable of generating exine-like patterns, providing evidence that these processes share control of exine formation with the well-documented program of gene expression.

Defective Pollen Wall 3 (DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

HMS1 interacts with HMS1I to regulate very-long-chain fatty acid biosynthesis and the humidity-sensitive genic male sterility in rice (Oryza sativa).

Environment-sensitive genic male sterility (EGMS) lines are used widely in two-line hybrid breeding in rice (Oryza sativa). At present, photoperiod-sensitive genic male sterility (PGMS) lines and thermo-sensitive genic male sterility (TGMS) lines are predominantly used in two-line hybrid rice, with humidity-sensitive genic male sterility (HGMS) lines rarely being reported. Here, it is shown that HUMIDITY-SENSITIVE GENIC MALE STERILITY 1 (HMS1), encoding a ß-ketoacyl-CoA synthase, plays key roles in the biosynthesis of very-long-chain fatty acids (VLCFAs) and HGMS in rice. The hms1 mutant displayed decreased seed setting under low humidity, but normal seed setting under high humidity. HMS1 catalyzed the biosynthesis of the C26 and C28 VLCFAs, contributing to the formation of bacula and tryphine in the pollen wall, which protect the pollen from dehydration. Under low-humidity conditions, hms1 pollen showed poor adhesion and reduced germination on the stigmas, which could be rescued by increasing humidity. HMS1-INTERACTING PROTEIN (HMS1I) interacted with HMS1 to coregulate HGMS. Furthermore, both japonica and indica rice varieties with defective HMS1 exhibited HGMS, suggesting that hms1 potentially could be used in hybrid breeding. The results herein reveal the novel mechanism of VLCFA-mediated pollen wall formation, which protects pollen from low-humidity stress in rice, and has a potential use in hybrid crop breeding.

MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis.

Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen-stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP-oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

Comparative transcriptome analysis and ChIP-sequencing reveals stage-specific gene expression and regulation profiles associated with pollen wall formation in Brassica rapa.

BACKGROUND: Genic male sterility (GMS) line is an important approach to utilize heterosis in Brassica rapa, one of the most widely cultivated vegetable crops in Northeast Asia. However, the molecular genetic mechanisms of GMS remain to be largely unknown. RESULTS: Detailed phenotypic observation of 'Bcajh97-01A/B', a B. rapa genic male sterile AB line in this study revealed that the aberrant meiotic cytokinesis and premature tapetal programmed cell death occurring in the sterile line ultimately resulted in microspore degeneration and pollen wall defect. Further gene expression profile of the sterile and fertile floral buds of 'Bcajh97-01A/B' at five typical developmental stages during pollen development supported the result of phenotypic observation and identified stage-specific genes associated with the main events associated with pollen wall development, including tapetum development or functioning, callose metabolism, pollen exine formation and cell wall modification. Additionally, by using ChIP-sequencing, the genomic and gene-level distribution of trimethylated histone H3 lysine 4 (H3K4) and H3K27 were mapped on the fertile floral buds, and a great deal of pollen development-associated genes that were covalently modified by H3K4me3 and H3K27me3 were identified. CONCLUSIONS: Our study provids a deeper understanding into the gene expression and regulation network during pollen development and pollen wall formation in B. rapa, and enabled the identification of a set of candidate genes for further functional annotation.

The zinc-finger transcription factor BcMF20 and its orthologs in Cruciferae which are required for pollen development.

Brassica campestris Male Fertility 20 (BcMF20) is a typical zinc-finger transcription factor that was previously isolated from flower buds of Chinese cabbage (Brassica campestris ssp. chinensis). By applying expression pattern analysis, it can be known that BcMF20 was specifically and strongly expressed in tapetum and pollen, beginning from the uninucleate stage, and was maintained during the mature-pollen stage. As BcMF20 was highly conserved in Cruciferae, it can be indicated that this zinc-finger transcription factor is important during the growth of Cruciferae. In this study, 12 C2H2-type zinc-finger TFs which shared high homology with BcMF20 were found from NCBI via BLAST. A new molecular phylogenetic tree was constructed by the comparison between BcMF20 and these 12 C2H2-type zinc-finger TFs with NJ method. By analyzing this phylogenetic tree, the evolution of BcMF20 was discussed. Then, antisense RNA technology was applied in the transgenesis of Arabidopsis thaliana to get the deletion mutants of BcMF20, so that its function during the pollen development can be identified. The results showed: BcMF20 are in the same clade with three genes from Arabidopsis. The inhibition of BcMF20 expression led to smaller amounts of and lower rate in germination of pollen and lower rate in fruit setting in certain transgenetic plants. This also led to the complete collapse of pollen grains. By SEM and TEM, pollen morphology and anther development processes were observed. In the middle uninucleate microspore stage, a relatively thin or even no primexine was formed in microspores. This may result in the malformation of the pollen wall and finally cause the deformity of pollens. Above all, it can be indicated that BcMF20 may act as a part of regulation mechanisms of TAZ1 and MS1. Together they play a role in a genetic pathway in the tapetum to act on proliferation of tapetal cells and keep the normal development of pollens.

The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana.

The sexine layer of pollen grain is mainly composed of sporopollenins. The sporophytic secretory tapetum is required for the biosynthesis of sporopollenin. Although several enzymes involved in sporopollenin biosynthesis have been reported, the regulatory mechanism of these enzymes in tapetal layer remains elusive. ABORTED MICROSPORES (AMS) and MALE STERILE 188/MYB103/MYB80 (MS188/MYB103/MYB80) are two tapetal cell-specific transcription factors required for pollen wall formation. AMS functions upstream of MS188. Here we report that AMS and MS188 target the CYP703A2 gene, which is involved in sporopollenin biosynthesis. We found that AMS and MS188 were localized in tapetum while CYP703A2 was localized in both tapetum and locule. Chromatin immunoprecipitation (ChIP) showed that MS188 directly bound to the promoter of CYP703A2 and luciferase-inducible assay showed that MS188 activated the expression of CYP703A2. Yeast two-hybrid and electrophoretic mobility shift assays (EMSAs) further demonstrated that MS188 complexed with AMS. The expression of CYP703A2 could be partially restored by the elevated levels of MS188 in the ams mutant. Therefore, our data reveal that MS188 coordinates with AMS to activate CYP703A2 in sporopollenin biosynthesis of plant tapetum.

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.