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Summary 483 I. Introduction 483 II. Progress of research on pollen wall development 485 III. The developmental role of the special cell wall 487 IV. Meiosis and the establishment of microspore symmetry 489 V. The origins of the exine during the tetrad stage 490 VI. The free microspore stage to pollen maturation 495 VII. Conclusions 495 Acknowledgements 496 References 496 The outer pollen wall, or exine, is more structurally complex than any other plant cell wall, comprising several distinct layers, each with its own organizational pattern. Since elucidation of the basic events of pollen wall ontogeny using electron microscopy in the 1970s, knowledge of their developmental genetics has increased enormously. However, self-assembly processes that are not under direct genetic control also play an important role in pollen wall patterning. This review integrates ultrastructural and developmental findings with recent models for self-assembly in an attempt to understand the origins of the morphological complexity and diversity that underpin the science of palynology.

Key messageOsMS1 functions as a transcriptional activator and interacts with known tapetal regulatory factors through its plant homeodomain (PHD) regulating tapetal programmed cell death (PCD) and pollen exine formation in rice.The tapetum, a hallmark tissue in the stamen, undergoes degradation triggered by PCD during post-meiotic anther development. This degradation process is indispensable for anther cuticle and pollen exine formation. Previous study has shown that PTC1 plays a critical role in the regulation of tapetal PCD. However, it remained unclear how this occurs. To further investigate the role of this gene in rice, we used CRISPR/Cas9 system to generate the homozygous mutant named as osms1, which showed complete male sterility with slightly yellow and small anthers, as well as invisible pollen grains. In addition, cytological observation revealed delayed tapetal PCD, defective pollen exine formation and a lack of DNA fragmentation according to a TUNEL analysis in the anthers of osms1 mutant. OsMS1, which encodes a PHD finger protein, was located in the nucleus of rice protoplasts and functioned as a transcription factor with transcriptional activation activity. Y2H and BiFC assays demonstrated that OsMS1 can interact with OsMADS15 and TDR INTERACTING PROTEIN2 (TIP2). It has been reported that TIP2 coordinated with TDR to modulate the expression of EAT1 and further regulated tapetal PCD in rice. Results of qPCR suggested that the expression of the genes associated with tapetal PCD and pollen wall biosynthesis, such as EAT1, AP37, AP25, OsC6 and OsC4, were significantly reduced in osms1 mutant. Taken together, our results demonstrate that the interaction of OsMS1 with known tapetal regulatory factors through its PHD finger regulates tapetal PCD and pollen exine formation in rice.

Background During pollen wall formation in flowering plants, a conserved metabolon consisting of acyl-CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide α-pyrone reductase (TKPR), is required for sporopollenin synthesis. Despite this, the precise function of each of these components in different species remains unclear. Results In this study, we characterized the function of OsTKPR1, a rice orthologue of Arabidopsis TKPR1. Loss of function of OsTKPR1 delayed tapetum degradation, reduced the levels of anther cuticular lipids, and impaired Ubisch body and pollen exine formation, resulting in complete male sterility. In addition, the phenylpropanoid pathway in mutant anthers was remarkably altered. Localization studies suggest that OsTKPR1 accumulates in the endoplasmic reticulum, while specific accumulation of OsTKPR1 mRNA in the anther tapetum and microspores is consistent with its function in anther and pollen wall development. Conclusions Our results show that OsTKPR1 is indispensable for anther cuticle development and pollen wall formation in rice, providing new insights into the biochemical mechanisms of the conserved sporopollenin metabolon in flowering plants.

Summary Anther cuticle and pollen exine are two crucial lipid layers that ensure normal pollen development and pollen–stigma interaction for successful fertilization and seed production in plants. Their formation processes share certain common pathways of lipid biosynthesis and transport across four anther wall layers. However, molecular mechanism underlying a trade‐off of lipid‐metabolic products to promote the proper formation of the two lipid layers remains elusive. Here, we identified and characterized a maize male‐sterility mutant pksb, which displayed denser anther cuticle but thinner pollen exine as well as delayed tapetal degeneration compared with its wild type. Based on map‐based cloning and CRISPR/Cas9 mutagenesis, we found that the causal gene (ZmPKSB) of pksb mutant encoded an endoplasmic reticulum (ER)‐localized polyketide synthase (PKS) with catalytic activities to malonyl‐CoA and midchain‐fatty acyl‐CoA to generate triketide and tetraketide α‐pyrone. A conserved catalytic triad (C171, H320 and N353) was essential for its enzymatic activity. ZmPKSB was specifically expressed in maize anthers from stages S8b to S9‐10 with its peak at S9 and was directly activated by a transcription factor ZmMYB84. Moreover, loss function of ZmMYB84 resulted in denser anther cuticle but thinner pollen exine similar to the pksb mutant. The ZmMYB84‐ZmPKSB regulatory module controlled a trade‐off between anther cuticle and pollen exine formation by altering expression of a series of genes related to biosynthesis and transport of sporopollenin, cutin and wax. These findings provide new insights into the fine‐tuning regulation of lipid‐metabolic balance to precisely promote anther cuticle and pollen exine formation in plants. Maize male‐sterility mutant pksb displays dense anther cuticle but thin pollen exine. ZmPKSB has conserved polyketide synthase activities dependent on a catalytic triad (C171, H320 and N353). ZmPKSB is directly activated by a transcription factor ZmMYB84, thus forming a regulatory module (ZmMYB84‐ZmPKSB) that plays synchronous roles in anther cuticle and pollen exine formation required for male fertility. The ZmMYB84‐ZmPKSB regulatory module controls a trade‐off of lipid‐metabolic products individually used for anther cuticle and pollen exine formation.

Summary The pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map‐based cloning and confirmed by genetic analysis (co‐segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de‐esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de‐esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de‐esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de‐esterified homogalacturonan in pollen exine formation.

Two of the Arabidopsis thaliana SEC23 homologs, AtSEC23A and AtSEC23D, are expressed in the tapetum and play essential roles in pollen wall formation, exine patterning, and tapetum development. Abstract The specialized multilayered pollen wall plays multiple roles to ensure normal microspore development. The major components of the pollen wall (e.g. sporopollenin and lipidic precursors) are provided from the tapetum. Material export from the endoplasmic reticulum (ER) is mediated by coat protein complex II (COPII) vesicles. The Arabidopsis thaliana genome encodes seven homologs of SEC23, a COPII component. However, the functional importance of this diversity remains elusive. Here, we analyzed knockout and knockdown lines for AtSEC23A and AtSEC23D, two of the A. thaliana SEC23 homologs, respectively. Single atsec23a and atsec23d mutant plants, despite normal fertility, showed an impaired exine pattern. Double atsec23ad mutant plants were semi-sterile and exhibited developmental defects in pollen and tapetal cells. Pollen grains of atsec23ad had defective exine and intine, and showed signs of cell degeneration. Moreover, the development of tapetal cells was altered, with structural abnormalities in organelles. AtSEC23A and AtSEC23D exhibited the characteristic localization pattern of COPII proteins and were highly expressed in the tapetum. Our work suggests that AtSEC23A and AtSEC23D may organize pollen wall development and exine patterning by regulating ER export of lipids and proteins necessary for pollen wall formation. Also, our results shed light on the functional heterogeneity of SEC23 homologs.

Background Sporopollenin is a major component of the pollen exine pattern. In Arabidopsis, acyl-CoA synthetase5 ( ACOS5 ) is involved in sporopollenin precursor biosynthesis. In this study, we identified its orthologue, OsACOS12 , in rice ( Oryza sativa ) and compared the functional conservation of ACOS in rice to Arabidopsis. Results Sequence analysis showed that OsACOS12 shares 63.9 % amino acid sequence identity with ACOS5. The osacos12 mutation caused by a pre-mature stop codon in LOC_Os04g24530 exhibits defective sexine resulting in a male sterile phenotype in rice. In situ hybridization shows that OsACOS12 is expressed in tapetal cells and microspores at the transcript level. The localization of OsACOS12-GFP demonstrated that OsACOS12 protein is accumulated in tapetal cells and anther locules. OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis. Conclusions OsACOS12 is an orthologue of ACOS5 that is essential for sporopollenin synthesis in rice. ACOS5 and OsACOS12 are conserved for pollen wall formation in monocot and dicot species.

Summary The N6‐methyladenosine (m6A) mRNA modification is crucial for plant development and stress responses. In rice, the male sterility resulting from the deficiency of OsFIP37, a core component of m6A methyltransferase complex, emphasizes the significant role of m6A in male fertility. m6A is reversible and can be removed by m6A demethylases. However, whether mRNA m6A demethylase regulates male fertility in rice has remained unknown. Here, we identify the mRNA m6A demethylase OsALKBH9 and demonstrate its involvement in male fertility regulation. Knockout of OsALKBH9 causes male sterility, dependent on its m6A demethylation activity. Cytological analysis reveals defective tapetal programmed cell death (PCD) and excessive accumulation of microspores exine in Osalkbh9‐1. Transcriptome analysis of anthers shows up‐regulation of genes involved in tapetum development, sporopollenin synthesis, and transport pathways in Osalkbh9‐1. Additionally, we demonstrate that OsALKBH9 demethylates the m6A modification in TDR and GAMYB transcripts, which affects the stability of these mRNAs and ultimately leads to excessive accumulation of pollen exine. Our findings highlight the precise control of mRNA m6A modification and reveal the pivotal roles played by OsALKBH9‐mediated m6A demethylation in tapetal PCD and pollen exine accumulation in rice.

Pollen is encased in a robust wall that shields the male gametophyte from various stresses and aids in pollination. The pollen wall consists of gametophyte-derived intine and sporophyte-derived exine. The exine is mainly composed of sporopollenin, which is biopolymers of aliphatic lipids and phenolics. The process of exine formation has been the subject of extensive research, yet the underlying molecular mechanisms remain elusive. In this study, we identified a rice mutant of the OsSNDP4 gene that is impaired in pollen development. We demonstrated that OsSNDP4, a putative Sec14-nodulin domain protein, exhibits a preference for binding to phosphatidylinositol (3)-phosphate [PI(3)P], a lipid primarily found in endosomal and vacuolar membranes. The OsSNDP4 protein was detected in association with the endoplasmic reticulum (ER), vacuolar membranes, and the nucleus. OsSNDP4 expression was detected in all tested organs but was notably higher in anthers during exine development. Loss of OsSNDP4 function led to abnormal vacuole dynamics, inhibition in Ubisch body development, and premature degradation of cellular contents and organelles in the tapetal cells. Microspores from the ossndp4 mutant plant displayed abnormal exine formation, abnormal vacuole enlargement, and ultimately, pollen abortion. RNA-seq assay revealed that genes involved in the biosynthesis of fatty acid and secondary metabolites, the biosynthesis of lipid polymers, and exosome formation were enriched among the down-regulated genes in the mutant anthers, which correlated with the morphological defects observed in the mutant anthers. Base on these findings, we propose that OsSNDP4 regulates pollen development by binding to PI(3)P and influencing the dynamics of membrane systems. The involvement of membrane systems in the regulation of sporopollenin biosynthesis, Ubisch body formation, and exine formation provides a novel mechanism regulating pollen wall development.

Background Male fertility is crucial for rice yield, and the improvement of rice yield requires hybrid production that depends on male sterile lines. Although recent studies have revealed several important genes in male reproductive development, our understanding of the mechanisms of rice pollen development remains unclear. Results We identified a rice mutant oslap6 with complete male sterile phenotype caused by defects in pollen exine formation. By using the MutMap method, we found that a single nucleotide polymorphism (SNP) variation located in the second exon of OsLAP6/OsPKS1 was responsible for the mutant phenotype. OsLAP6/OsPKS1 is an orthologous gene of Arabidopsis PKSA/LAP6 , which functions in sporopollenin metabolism. Several other loss-of-function mutants of OsLAP6/OsPKS1 generated by the CRISPR/Cas9 genomic editing tool also exhibited the same phenotype of male sterility. Our cellular analysis suggested that OsLAP6/OsPKS1 might regulate pollen exine formation by affecting bacula elongation. Expression examination indicated that OsLAP6/OsPKS1 is specifically expressed in tapetum, and its product is localized to the endoplasmic reticulum (ER). Protein sequence analysis indicated that OsLAP6/OsPKS1 is conserved in land plants. Conclusions OsLAP6/OsPKS1 is a critical molecular switch for rice male fertility by participating in a conserved sporopollenin precursor biosynthetic pathway in land plants. Manipulation of OsLAP6/OsPKS1 has potential for application in hybrid rice breeding.