Exine Formation Research Articles

Identification of the new allele ptc1-2 and analysis of the regulatory role of PTC1 gene in rice anther development.

Anther development involves a series of important biological events that are precisely regulated by many genes. Although several important genes involved in rice anther development have been identified, the regulatory network involved in tapetal development and pollen wall formation is still largely unclear. PERSISTENT TAPETAL CELL 1 (PTC1) encodes a PHD-Finger protein, which plays a critical role in the regulation of tapetal cell death and pollen development in rice. Here, we report the isolation and characterization of a new allele ptc1-2 with 2-base deletion in the third exon, causing the absent of the PHD domain due to the sequence change. Cytological analysis revealed delayed tapetal PCD, defective pollen exine formation and abnormal ubisch bodies development. Transcriptome analysis revealed that genes related to pollen wall formation (secondary metabolism, phenylalanine synthesis, and cutin and wax biosynthesis pathways), cell death (cysteine and methionine metabolism and DNA repair pathways), and carbohydrate synthesis (starch and sucrose metabolism pathways) were significantly altered in ptc1-2 mutant. A total of 13 reported anther development genes exhibited significant expression changes in the ptc1-2 mutant. Yeast two-hybrid and BiFC analyses showed that PTC1 could interact with API5, an inhibitor of apoptosis, and the citrin-binding enzyme EDT1. This work is helpful in deepening the understanding of the regulatory network of male reproductive development in rice.

Analysis of Rfo-Mediated Network in Regulating Fertility Restoration in Brassica oleracea

Ogura cytoplasmic male sterility (CMS) lines play a crucial role in the utilization of heterosis. However, valuable traits, such as disease resistance genes from Ogura CMS hybrids, are challenging to incorporate for germplasm innovation, particularly in cabbage and broccoli. To date, the Rfo-mediated network regulating fertility restoration remains largely unexplored. In this study, we conducted a transcriptomic analysis of broccoli flower buds from Ogura CMS SFB45 and its Rfo-transgenic fertility restoration line, pRfo, at different stages of pollen development. Gene Ontology (GO) terms such as “pollen exine formation”, “flavonoid metabolic and biosynthetic processes”, and “pollen wall assembly”, along with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including “flavonoid biosynthesis”, “MAPK signaling pathway-plant”, and “ABC transporters”, were significantly enriched. We identified five differentially expressed genes (DEGs) involved in tapetum-mediated callose metabolism, thirty-four DEGs related to tapetum-mediated pollen wall formation, three DEGs regulating tapetum programmed cell death (PCD), five MPKs encoding DEGs, and twelve DEGs associated with oxidative phosphorylation. Additionally, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that RFO directly interacts with ORF138 at the protein level. These findings provide valuable insights into the fertility recovery mechanisms regulated by Rfo in broccoli and offer important clues for breeders aiming to enhance Ogura CMS hybrids in Brassica oleracea.

Arabidopsis KNS3 and its two homologs mediate endoplasmic reticulum-to-plasma membrane traffic of boric acid channels.

Membrane proteins targeted to the plasma membrane are first transported from the endoplasmic reticulum (ER) to the Golgi apparatus. This study explored the mechanisms controlling plasma membrane trafficking of the boric acid channel AtNIP5;1 from the ER. Imaging-based screening using transgenic Arabidopsis identified six mutants in which GFP-NIP5;1 was localized in the ER in addition to the plasma membrane. Genetic mapping and whole-genome resequencing identified the responsible gene in four among the six mutants as KAONASHI3 (KNS3)/SPOTTY1/IMPERFECTIVE EXINE FORMATION. Among the plasma membrane-localized proteins tested, NIP5;1 and its homolog NIP6;1 were retained in the ER of the kns3 mutants. Our genetic analysis further discovered that two homologs of KNS3, KNSTH1 and KNSTH2, were also involved in the ER exit of NIP5;1. In Arabidopsis protoplasts and tobacco leaves, mCherry-fused KNS3 localized to the ER and Golgi, whereas KNSTH2 localized to the ER. The cytosolic C-terminal tail of KNS3 contains amino acids important for Golgi-to-ER trafficking. Furthermore, the ER-to-Golgi trafficking of KNS3 depended on KNSTH1 and KNSTH2, and the accumulation of these three proteins in Arabidopsis roots depended on each other. We propose that KNS3, KNSTH1, and KNSTH2 function as a cargo-receptor complex mediating the ER exit of NIP5;1.

RNA-seq analyses on gametogenic tissues of alfalfa (Medicago sativa) revealed plant reproduction- and ploidy-related genes

BackgroundIn alfalfa (Medicago sativa), the coexistence of interfertile subspecies (i.e. sativa, falcata and coerulea) characterized by different ploidy levels (diploidy and tetraploidy) and the occurrence of meiotic mutants capable of producing unreduced (2n) gametes, have been efficiently combined for the establishment of new polyploids. The wealth of agronomic data concerning forage quality and yield provides a thorough insight into the practical benefits of polyploidization. However, many of the underlying molecular mechanisms regarding gene expression and regulation remained completely unexplored. In this study, we aimed to address this gap by examining the transcriptome profiles of leaves and reproductive tissues, corresponding to anthers and pistils, sampled at different time points from diploid and tetraploid Medicago sativa individuals belonging to progenies produced by bilateral sexual polyploidization (dBSP and tBSP, respectively) and tetraploid individuals stemmed from unilateral sexual polyploidization (tUSP).ResultsConsidering the crucial role played by anthers and pistils in the reduced and unreduced gametes formation, we firstly analyzed the transcriptional profiles of the reproductive tissues at different stages, regardless of the ploidy level and the origin of the samples. By using and combining three different analytical methodologies, namely weighted-gene co-expression network analysis (WGCNA), tau (τ) analysis, and differentially expressed genes (DEGs) analysis, we identified a robust set of genes and transcription factors potentially involved in both male sporogenesis and gametogenesis processes, particularly in crossing-over, callose synthesis, and exine formation. Subsequently, we assessed at the same floral stage, the differences attributable to the ploidy level (tBSP vs. dBSP) or the origin (tBSP vs. tUSP) of the samples, leading to the identification of ploidy and parent-specific genes. In this way, we identified, for example, genes that are specifically upregulated and downregulated in flower buds in the comparison between tBSP and dBSP, which could explain the reduced fertility of the former compared to the latter materials.ConclusionsWhile this study primarily functions as an extensive investigation at the transcriptomic level, the data provided could represent not only a valuable original asset for the scientific community but also a fully exploitable genomic resource for functional analyses in alfalfa.

OsSNDP4, a Sec14-nodulin Domain Protein, is Required for Pollen Development 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.

Integrated transcriptomic and metabolomic analysis provides insight into the pollen development of CMS-D1 rice

BackgroundCytoplasmic male sterility (CMS) has greatly improved the utilization of heterosis in crops due to the absence of functional male gametophyte. The newly developed sporophytic D1 type CMS (CMS-D1) rice exhibits unique characteristics compared to the well-known sporophytic CMS-WA line, making it a valuable resource for rice breeding.ResultsIn this research, a novel CMS-D1 line named Xingye A (XYA) was established, characterized by small, transparent, and shriveled anthers. Histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays conducted on anthers from XYA and its maintainer line XYB revealed that male sterility in XYA is a result of delayed degradation of tapetal cells and abnormal programmed cell death (PCD) of microspores. Transcriptome analysis of young panicles revealed that differentially expressed genes (DEGs) in XYA, compared to XYB, were significantly enriched in processes related to chromatin structure and nucleosomes during the microspore mother cell (MMC) stage. Conversely, processes associated with sporopollenin biosynthesis, pollen exine formation, chitinase activity, and pollen wall assembly were enriched during the meiosis stage. Metabolome analysis identified 176 specific differentially accumulated metabolites (DAMs) during the meiosis stage, enriched in pathways such as α-linoleic acid metabolism, flavone and flavonol biosynthesis, and linolenic acid metabolism. Integration of transcriptomic and metabolomic data underscored the jasmonic acid (JA) biosynthesis pathway was significant enriched in XYA during the meiosis stage compared to XYB. Furthermore, levels of JA, MeJA, OPC4, OPDA, and JA-Ile were all higher in XYA than in XYB at the meiosis stage.ConclusionsThese findings emphasize the involvement of the JA biosynthetic pathway in pollen development in the CMS-D1 line, providing a foundation for further exploration of the molecular mechanisms involved in CMS-D1 sterility.

Global gene expression profile and functional analysis reveal the conservation of reproduction-associated gene networks in Gossypium hirsutum.

Lastly, the bZIP gene family encompasses genes that have been reported to play a role in flower development, such as bZIP14 (FD). Notably, bZIP14 is essential for Flowering Locus T (FT) initiation of floral development in Arabidopsis (Abe et al. 2005). Cotton (Gossypium hirsutum L.) is the world's most extensively cultivated fiber crop. However, its reproductive development is poorly characterized at the molecular level. Thus, this study presents a detailed transcriptomic analysis of G. hirsutum at three different reproductive stages. We provide evidence that more than 64,000 genes are active in G. hirsutum during flower development, among which 94.33% have been assigned to functional terms and specific pathways. Gene set enrichment analysis (GSEA) revealed that the biological process categories of floral organ development, pollen exine formation, and stamen development were enriched among the genes expressed during the floral development of G. hirsutum. Furthermore, we identified putative Arabidopsis homologs involved in the G. hirsutum gene regulatory network (GRN) of pollen and flower development, including transcription factors such as WUSCHEL (WUS), INNER NO OUTER (INO), AGAMOUS-LIKE 66 (AGL66), SPOROCYTELESS/NOZZLE (SPL/NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), ABORTED MICROSPORES (AMS), and ASH1-RELATED 3 (ASHR3), which are known crucial genes for plant reproductive success. The cotton MADS-box protein-protein interaction pattern resembles the previously described patterns for AGAMOUS (AG), SEEDSTICK (STK), SHATTERPROOF (SHP), and SEPALLATA3 (SEP3) homolog proteins from Arabidopsis. In addition to serving as a resource for comparative flower development studies, this work highlights the changes in gene expression profiles and molecular networks underlying stages that are valuable for cotton breeding improvement.

CRISPR/Cas9-based genome editing of 14 lipid metabolicgenes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.

Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.

The Role of Physical Processes in Pollen Wall Morphogenesis: Hypothesis and Experimental Confirmation

The review is devoted to the analysis and generalization of modern knowledge about the mechanisms underlying the ontogeny of the male gametophyte envelope. New and earlier data on exine development аre discussed, and recurrent phases in the development of exine of phylogenetically distant plant species are emphasized. Though exine formation has been shown to be dependent on plenty of genes, the reiteration of exine patterns in different plant species (e. g. columellate, granular, “white-lined” lamellae) suggests that these patterns are based on some non-biological principles of space-filling operations. However, mechanisms involved remained obscure until it became clear that the sequence of structures observed during exine development coincided with the sequence of self-assembling micellar mesophases. It was discovered later that another physical-chemical process – phase separation – participated in exine formation. To confirm that exine-like patterns are capable of generating in vitro by simple physical processes, and their formation does not require regulation at the genome level, some our and other authors’ in vitro experiments were undertaken; the data obtained are discussed. Several series of our new experiments on modeling exine development with mixtures of urface-active substances resulted in some patterns simulating the main types of natural exine. Transmission electron microscopy analysis of the samples has shown that patterns simulating the full range of exine types were obtained by joint action of phase separation and micellar self-assembly. The reconsideration and analysis of our and other authors’ morphogenetic and modeling data revealed that molecular-genetic mechanisms and physical forces work in tandem, with considerable input of physical processes.

Characterization and expression analysis of chalcone synthase gene family members suggested their roles in the male sterility of a wheat temperature-sensitive genic male sterile (TGMS) line

Chalcone synthase (CHS), also known as the plants-specific type III polyketide synthases (PKSs), catalyzes the first key step in the biosynthesis of plant flavonoids. Flavonoids are one of the most important secondary metabolites which participate in flower pigmentation and pollen fertility. Recent reports have demonstrated the role of the CHS family in plant pollen exine formation. This study focused on the potential roles of CHS in the pollen exine formation of wheat. In the present study, a genome-wide investigation of the CHS family was carried out, and 87 CHS genes were identified in wheat. TaCHS3, TaCHS10, and TaCHS13 are wheat orthologs of Arabidopsis LESS ADHESIVE POLLEN (LAP5); TaCHS58, TaCHS64, and TaCHS67 are wheat orthologs of AtLAP6. TaCHS3, TaCHS10, and TaCHS67 showed anther-specific patterns. The expression of TaCHS3, TaCHS10, and TaCHS67 was positively co-expressed with sporopollenin biosynthetic genes, including TaCYP703A2, TaCYP704B1, TaDRL1, TaTKPR2, and TaMS2. Coincidently, the expression of TaCHS3, TaCHS10, and TaCHS67, together with those sporopollenin biosynthetic genes, were repressed at the tetrads and uninucleate stages in the temperature-sensitive genic male-sterile (TGMS) line BS366 under sterile conditions. Wheat anther-specific CHS genes might participate in the exine formation of BS366 through co-expressing with sporopollenin biosynthetic genes, which will undoubtedly provide knowledge of the roles of CHS in wheat pollen development.

Putting the brakes on pollen wall development: A conserved negative feedback loop regulates pollen exine formation in flowering plants

Pollen grains are covered by a complex multi-layered structure, the pollen wall. Exine, the outermost layer of this wall, is made from the biopolymer sporopollenin that exhibits remarkable physical and chemical stability. In different plants, exine, which shields pollen from harsh environmental stresses, has very diverse morphologies and forms different patterns (Wang and Dobritsa, 2018). It also contributes to pollination, pollen-stigma interaction, pollen hydration, and eventual release of the pollen tube. Formation of exine begins after male meiosis when four microspores are arranged in a tetrad configuration and enclosed by the callose wall (Ariizumi and Toriyama, 2011). At this stage, microspores secrete primexine, the precursor of exine that accumulates between the microspore plasma membrane and the callose wall, providing a scaffold for exine patterning (Wang et al., 2021). As the callose wall gradually gets degraded, sporopollenin is secreted by the nearby tapetal cells and deposited onto the primexine template to form mature exine. This process requires coordination of activities between microspores and the surrounding tapetum.

ZmMS1/ZmLBD30-orchestrated transcriptional regulatory networks precisely control pollen exine development

Because of its significance for plant male fertility and, hence, direct impact on crop yield, pollen exine development has inspired decades of scientific inquiry. However, the molecular mechanism underlying exine formation and thickness remains elusive. In this study, we identified that a previously unrecognized repressor, ZmMS1/ZmLBD30, controls proper pollen exine development in maize. Using an ms1 mutant with aberrantly thickened exine, we cloned a male-sterility gene, ZmMs1, which encodes a tapetum-specific lateral organ boundary domain transcription factor, ZmLBD30. We showed that ZmMs1/ZmLBD30 is initially turned on by a transcriptional activation cascade of ZmbHLH51-ZmMYB84-ZmMS7, and then it serves as a repressor to shut down this cascade via feedback repression to ensure timely tapetal degeneration and proper level of exine. This activation-feedback repression loop regulating male fertility is conserved in maize and sorghum, and similar regulatory mechanism may also exist in other flowering plants such as rice and Arabidopsis. Collectively, these findings reveal a novel regulatory mechanism of pollen exine development by which a long-sought master repressor of upstream activators prevents excessive exine formation.

OsTKPR1 proteins with a single amino acid substitution fail the synthesis of a specific sporopollenin precursor and cause abnormal exine and pollen development in rice

Fatty acid derivatives are key components of rice pollen exine. The synthesis of aliphatic sporopollenin precursors are initiated in the plastids of the tapetal cells, followed by multiple-step reactions conducted in the endoplasmic reticulum (ER). However, the relative contribution of different precursors to the precise structure of sporopollenin remains largely elusive, let alone the underlying mechanism. Here, we report that two complete male sterile mutants ostkpr1–3 (Tetraketide α-pyrone reductase 1–3, with OsTKPR1P124S substitution) and ostkpr1–4 (with truncated OsTKPR1stop) are defective in pollen exine, Ubisch body and anther cuticle development where ostkpr1–4 display severer phenotypes. Remarkably, OsTKPR1 could produce reduced hydroxylated tetraketide α-pyrone and reduced tetraketide α-pyrone, whereas OsTKPR1P124S fails to produce the latter. Pairwise interaction assays show that mutated OsTKPR1P124S is able to integrate into a recently characterized metabolon, thus its altered catalytic activity is not due to dis-integrity of the metabolon. In short, we find that reduced tetraketide α-pyrone is a key sporopollenin precursor required for normal exine formation, and the conserved 124th proline of OsTKPR1 is essential for the reduction activity. Therefore, this study provided new insights into the sporopollenin precursor constitution critical for exine formation.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/β Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1–1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

OsTKPR2 is part of a sporopollenin-producing metabolon required for exine formation in rice.

The sporopollenin polymer is a major component of the pollen exine. Fatty acid derivatives synthesized in the tapetum are among the precursors of sporopollenin. Progress has been made to understand sporopollenin metabolism in rice; however, the underlying molecular mechanisms remain elusive. We found that OsTKPR2 and OsTKPR1 share a similar expression pattern, and their coding proteins have a similar subcellular localization and enzyme activities towards reduced tetraketide α-pyrone and hydroxylated tetraketide α-pyrone. Unexpectedly, OsTKPR1pro:OsTKPR2-eGFP could not rescue the phenotype of ostkpr1-4. Three independent ostkpr2 mutant lines generated by CRISPR/Cas9 displayed reduced male fertility to various extents which were correlated with the severity of gene disruptions. Notably, the anther cuticle, Ubisch bodies, and pollen development were affected in the ostkpr2-1 mutant, where a thinner pollen exine was noticed. OsTKPR1 and OsTKPR2 were integrated into a metabolon including OsACOS12 and OsPKS2, which resulted in a significant increased enzymatic efficiency when both OsTKPR1 and OsTKPR2 were present, indicating the mutual dependence of OsTKPR2 and OsTKPR1 for their full biochemical activities. Thus, our results demonstrated that OsTKPR2 is required for anther and pollen development where an OsTKPR2-containing metabolon is functional during rice sporopollenin synthesis. Furthermore, the cooperation and possible functional divergence between OsTKPR2 and OsTKPR1 is also discussed.

Genome-Wide Identification of the Cytochrome P450 Superfamily Genes and Targeted Editing of BnCYP704B1 Confers Male Sterility in Rapeseed.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

Duplicate mutations of GhCYP450 lead to the production of ms5m6 male sterile line in cotton.

The duplicated male sterile genes ms5m6 in cotton were map-based cloned and validated by the virus-induced gene silencing assays. Duplicate mutations of the GhCYP450 gene encoding a cytochrome P450 protein are responsible for the male sterility in cotton. The utilization of male sterility in cotton plays a vital role in improving yield and fiber quality. A complete male sterile line (ms5ms6) has been extensively used to develop hybrid cotton worldwide. Using Zhongkang-A (ZK-A) developed by transferring Bt and ms5ms6 genes into the commercial cultivar Zhongmiansuo 12, the duplicate genes were map-based cloned and confirmed via the virus-induced gene silencing (VIGS) assays. The duplicate mutations of GhCYP450 genes encoding a cytochrome P450 protein were responsible for producing male sterility in ms5ms6 in cotton. Sequence alignment showed that GhCYP450-Dt in ZK-A differed in two critical aspects from the fertile wild-type TM-1: GhCYP450-Dt has three amino acid (D98E, E168K, G198R) changes in the coding region and a 7-bp (GGAAAAA) insertion in the promoter domain; GhCYP450-At appears to be premature termination of GhCYP450 translation. Further morphological observation and cytological examination of GhCYP450-silenced plants induced by VIGS exhibited shorter filaments and no mature pollen grains. These results indicate that GhCYP450 is essential for pollen exine formation and pollen development for male fertility. Investigating the mechanisms of ms5ms6 male sterility will deepen our understanding of the development and utilization of heterosis.

Comparative transcriptome analysis provides insight into the important pathways and key genes related to the pollen abortion in the thermo-sensitive genic male sterile line 373S in Brassica napus L.

The thermo-sensitive genic male sterility (TGMS) system plays a key role in the production of two-line hybrids in rapeseed (Brassica napus). To uncover key cellular events and genetic regulation associated with TGMS, a combined study using cytological methods and RNA-sequencing analysis was conducted for the rapeseed TGMS line 373S. Cytological studies showed that microspore cytoplasm of 373S plants was condensed, the microspore nucleus was degraded at an early stage, the exine was irregular, and the tapetum developed abnormally, eventually leading to male sterility. RNA-sequencing analysis identified 430 differentially expressed genes (298 upregulated and 132 downregulated) between the fertile and sterile samples. Gene ontology analysis demonstrated that the most highly represented biological processes included sporopollenin biosynthetic process, pollen exine formation, and extracellular matrix assembly. Kyoto encyclopedia of genes and genomes analysis indicated that the enriched pathways included amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Moreover, 26 transcript factors were identified, which may be associated with abnormal tapetum degeneration and exine formation. Subsequently, 19 key genes were selected, which are considered to regulate pollen development and even participate in pollen exine formation. Our results will provide important insight into the molecular mechanisms underlying TGMS in rapeseed.

Rice anther tapetum: a vital reproductive cell layer for sporopollenin biosynthesis and pollen exine patterning.

The tapetum is the innermost layer of the four layers of the rice anther that provides protection and essential nutrients to pollen grain development and delivers precursors for pollen exine formation. The tapetum has a key role in the normal development of pollen grains and tapetal programmed cell death (PCD) that is linked with sporopollenin biosynthesis and transport. Recently, many genes have been identified that are involved in tapetum formation in rice and Arabidopsis. Genetic mutation in PCD-associated genes could affect normal tapetal PCD, which finally leads to aborted pollen grains and male sterility in rice. In this review, we discuss the most recent research on rice tapetum development, including genomic, transcriptomic and proteomic studies. Furthermore, tapetal PCD, sporopollenin biosynthesis, ROS activity for tapetum function and its role in male reproductive development are discussed in detail. This will improve our understanding of the role of the tapetum in male fertility using rice as a model system, and provide information that can be applied in rice hybridization and that of other major crops.