Genic male sterility (GMS) is very useful for hybrid vigor utilization and hybrid seed production. Although a large number of GMS genes have been identified in plants, little is known about the roles of GDSL lipase members in anther and pollen development. Here, we report a maize GMS gene, ZmMs30, which encodes a novel type of GDSL lipase with diverged catalytic residues. Enzyme kinetics and activity assays show that ZmMs30 has lipase activity and prefers to substrates with a short carbon chain. ZmMs30 is specifically expressed in maize anthers during stages 7-9. Loss of ZmMs30 function resulted in defective anther cuticle, irregular foot layer of pollen exine, and complete male sterility. Cytological and lipidomics analyses demonstrate that ZmMs30 is crucial for the aliphatic metabolic pathway required for pollen exine formation and anther cuticle development. Furthermore, we found that male sterility caused by loss of ZmMs30 function was stable in various inbred lines with different genetic background, and that it didn't show any negative effect on maize heterosis and production, suggesting that ZmMs30 is valuable for crossbreeding and hybrid seed production. We then developed a new multi-control sterility system using ZmMs30 and its mutant line, and demonstrated it is feasible for generating desirable GMS lines and valuable for hybrid maize seed production. Taken together, our study sheds new light on the mechanisms of anther and pollen development, and provides a valuable male-sterility system for hybrid breeding maize.
Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and female fertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops.
Summary Identifying genic male‐sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with Arabidopsis and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9‐mediated gene mutagenesis, and then, 12 genes in eight types, including ZmbHLH51, ZmbHLH122, ZmTGA9‐1/‐2/‐3, ZmTGA10, ZmMYB84, ZmMYB33‐1/‐2, ZmPHD11 and ZmLBD10/27, were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably, ZmTGA9‐1/‐2/‐3 triple‐gene mutants and ZmMYB33‐1/‐2 double‐gene mutants displayed complete male sterility, but their double‐ or single‐gene mutants showed male fertility. Similarly, ZmLBD10/27 double‐gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition, ZmbHLH51 was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co‐segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14‐type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and Arabidopsis. These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male‐sterility lines in maize.
Genic male sterility (GMS) is critical for heterosis utilization and hybrid seed production. Although GMS mutants and genes have been studied extensively in plants, it has remained unclear whether chloroplast-associated photosynthetic and metabolic activities are involved in the regulation of anther development. In this study, we characterized the function of ZmMs33/ZmGPAT6, which encodes a member of the glycerol-3-phosphate acyltransferase (GPAT) family that catalyzes the first step of the glycerolipid synthetic pathway. We found that normal structure and function of endothecium (En) chloroplasts maintained by ZmMs33-mediated lipid biosynthesis in tapetal cells are crucial for maize anther development. ZmMs33 is expressed mainly in the tapetum at early anther developmental stages and critical for cell proliferation and expansion at late stages. Chloroplasts in En cells of wild-type anthers function as starch storage sites before stage 10 but as photosynthetic factories since stage 10 to enable starch metabolism and carbohydrate supply. Loss of ZmMs33 function inhibits the biosynthesis of glycolipids and phospholipids, which are major components of En chloroplast membranes, and disrupts the development and function of En chloroplasts, resulting in the formation of abnormal En chloroplasts containing numerous starch granules. Further analyses reveal that starch synthesis during the day and starch degradation at night are greatly suppressed in the mutant anthers, leading to carbon starvation and low energy status, as evidenced by low trehalose-6-phosphate content and a reduced ATP/AMP ratio. The energy sensor and inducer of autophagy, SnRK1, was activated to induce early and excessive autophagy, premature PCD, and metabolic reprogramming in tapetal cells, finally arresting the elongation and development of mutant anthers. Taken together, our results not only show that ZmMs33 is required for normal structure and function of En chloroplasts but also reveal that starch metabolism and photosynthetic activities of En chloroplasts at different developmental stages are essential for normal anther development. These findings provide novel insights for understanding how lipid biosynthesis in the tapetum, the structure and function of En chloroplasts, and energy and substance metabolism are coordinated to maintain maize anther development.
The “competing endogenous RNA (ceRNA) hypothesis” has recently been proposed for a new type of gene regulatory model in many organisms. Anther development is a crucial biological process in plant reproduction, and its gene regulatory network (GRN) has been gradually revealed during the past two decades. However, it is still unknown whether ceRNAs contribute to anther development and sexual reproduction in plants. We performed RNA and small RNA sequencing of anther tissues sampled at three developmental stages in two maize lines. A total of 28,233 stably transcribed loci, 61 known and 51 potentially novel microRNAs (miRNAs) were identified from the transcriptomes. Predicted ceRNAs and target genes were found to conserve in sequences of recognition sites where their corresponding miRNAs bound. We then reconstructed 79 ceRNA-miRNA-target gene regulatory networks consisting of 51 known miRNAs, 28 potentially novel miRNAs, 619 ceRNA-miRNA pairs, and 869 miRNA-target gene pairs. More than half of the regulation pairs showed significant negative correlations at transcriptional levels. Several well-studied miRNA-target gene pairs associated with plant flower development were located in some networks, including miR156-SPL, miR159-MYB, miR160-ARF, miR164-NAC, miR172-AP2, and miR319-TCP pairs. Six target genes in the networks were found to be orthologs of functionally confirmed genes participating in anther development in plants. Our results provide an insight that the ceRNA-miRNA-target gene regulatory networks likely contribute to anther development in maize. Further functional studies on a number of ceRNAs, miRNAs, and target genes will facilitate our deep understanding on mechanisms of anther development and sexual plants reproduction.
The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.
Fatty acyl reductases (FARs) catalyze the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism of FARs involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticle, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25 gene, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro, and four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther at stage 9, and was directly regulated by transcriptional factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing most of cutin monomers and internal lipids, and altering composition of cuticular wax in anthers. Moreover, loss of ZmMs25 function significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, implying its application potential in maize and other crops.
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