Fatty acid de novo biosynthesis in plastids: Key enzymes and their critical roles for male reproduction and other processes in plants.

Fatty acid de novo biosynthesis in plant plastids is initiated from acetyl-CoA and catalyzed by a series of enzymes, which is required for the vegetative growth, reproductive growth, seed development, stress response, chloroplast development and other biological processes. In this review, we systematically summarized the fatty acid de novo biosynthesis-related genes/enzymes and their critical roles in various plant developmental processes. Based on bioinformatic analysis, we identified fatty acid synthase encoding genes and predicted their potential functions in maize growth and development, especially in anther and pollen development. Finally, we highlighted the potential applications of these fatty acid synthases in male-sterility hybrid breeding, seed oil content improvement, herbicide and abiotic stress resistance, which provides new insights into future molecular crop breeding.

CRISPR/Cas9-based genome editing of 14 lipid metabolic genes 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.

A systematic analysis of the subtilase gene family and expression and subcellular localization investigation of anther-specific members in maize.

Subtilases (SBTs), also known as Subtilisin-like serine proteases, are extracellular alkaline protease proteins. SBTs function in all stages of plant growth, development and stress responses. Maize (Zea mays L.) is a crop widely used worldwide as food, feed, and industrial materials. However, information about the members and their functions of the SBT proteins in maize is lacking. In this study, we identified 58 ZmSBT genes from the maize genome and conducted a comprehensive investigation of ZmSBTs by phylogenetic, gene duplication event, gene structure, and protein conserved motif analyses. The ZmSBT proteins were phylogenetically classified into seven groups, and collinearity analysis indicated that many ZmSBTs originate from tandem or segmental duplications. Structural and homolog protein comparison revealed ZmSBTs have conserved protein structures with reported subtilase proteins, suggesting the conserved functions. Further analysis showed that ZmSBTs are expressed in different tissues, and many are responses to specific abiotic stress. Analysis of the anther-specific ZmSBT genes showed their expression peaked at different developmental stages of maize anthers. Subcellular localization analysis of selected maize ZmSBTs showed they are located in different cellular compartments. The information provided in this study is valuable for further functional study of ZmSBTs.

Structural and molecular basis of pollen germination.

Pollen germination is a prerequisite for double fertilization of flowering plants. A comprehensive understanding of the structural and molecular basis of pollen germination holds great potential for crop yield improvement. The pollen aperture serves as the foundation for most plant pollen germination and pollen aperture formation involves the establishment of cellular polarity, the formation of distinct membrane domains, and the precise deposition of extracellular substances. Successful pollen germination requires precise material exchange and signal transduction between the pollen grain and the stigma. Recent cytological and mutant analysis of pollen germination process in Arabidopsis and rice has expanded our understanding of this biological process. However, the overall changes in germination site structure and energy-related metabolites during pollen germination remain to be further explored. This review summarizes and compares the recent advances in the processes of pollen aperture formation, pollen adhesion, hydration, and germination between eudicot Arabidopsis and monocot rice, and provides insights into the structural basis and molecular mechanisms underlying pollen germination process.

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.

Promoting genotype-independent plant transformation by manipulating developmental regulatory genes and/or using nanoparticles.

KEY MESSAGE: This review summarizes the molecular basis and emerging applications of developmental regulatory genes and nanoparticles in plant transformation and discusses strategies to overcome the obstacles of genotype dependency in plant transformation. Plant transformation is an important tool for plant research and biotechnology-based crop breeding. However, Plant transformation and regeneration are highly dependent on species and genotype. Plant regeneration is a process of generating a complete individual plant from a single somatic cell, which involves somatic embryogenesis, root and shoot organogeneses. Over the past 40 years, significant advances have been made in understanding molecular mechanisms of embryogenesis and organogenesis, revealing many developmental regulatory genes critical for plant regeneration. Recent studies showed that manipulating some developmental regulatory genes promotes the genotype-independent transformation of several plant species. Besides, nanoparticles penetrate plant cell wall without external forces and protect cargoes from degradation, making them promising materials for exogenous biomolecule delivery. In addition, manipulation of developmental regulatory genes or application of nanoparticles could also bypass the tissue culture process, paving the way for efficient plant transformation. Applications of developmental regulatory genes and nanoparticles are emerging in the genetic transformation of different plant species. In this article, we review the molecular basis and applications of developmental regulatory genes and nanoparticles in plant transformation and discuss how to further promote genotype-independent plant transformation.

Bibliometric Analysis of Functional Crops and Nutritional Quality: Identification of Gene Resources to Improve Crop Nutritional Quality through Gene Editing Technology.

Food security and hidden hunger are two worldwide serious and complex challenges nowadays. As one of the newly emerged technologies, gene editing technology and its application to crop improvement offers the possibility to relieve the pressure of food security and nutrient needs. In this paper, we analyzed the research status of quality improvement based on gene editing using four major crops, including rice, soybean, maize, and wheat, through a bibliometric analysis. The research hotspots now focus on the regulatory network of related traits, quite different from the technical improvements to gene editing in the early stage, while the trends in deregulation in gene-edited crops have accelerated related research. Then, we mined quality-related genes that can be edited to develop functional crops, including 16 genes related to starch, 15 to lipids, 14 to proteins, and 15 to other functional components. These findings will provide useful reference information and gene resources for the improvement of functional crops and nutritional quality based on gene editing technology.

Triphasic regulation of ZmMs13 encoding an ABCG transporter is sequentially required for callose dissolution, pollen exine and anther cuticle formation in maize.

INTRODUCTION: ATP Binding Cassette G (ABCG) transporters are associated with plant male reproduction, while their regulatory mechanisms underlying anther and pollen development remain largely unknown. OBJECTIVES: Identify and characterize a male-sterility gene ZmMs13 encoding an ABCG transporter in modulating anther and pollen development in maize. METHODS: Phenotypic, cytological observations, and histochemistry staining were performed to characterize the ms13-6060 mutant. Map-based cloning and CRISPR/Cas9 gene editing were used to identify ZmMs13 gene. RNA-seq data and qPCR analyses, phylogenetic and microsynteny analyses, transient dual-luciferase reporter and EMSA assays, subcellular localization, and ATPase activity and lipidomic analyses were carried out to determine the regulatory mechanisms of ZmMs13 gene. RESULTS: Maize ms13-6060 mutant displays complete male sterility with delayed callose degradation, premature tapetal programmed cell death (PCD), and defective pollen exine and anther cuticle formation. ZmMs13 encodes a plasm membrane (PM)- and endoplasmic reticulum (ER)-localized half-size ABCG transporter (ZmABCG2a). The allele of ZmMs13 in ms13-6060 mutant has one amino acid (I311) deletion due to a 3-bp deletion in its fourth exon. The I311 and other conserved amino acid K99 are essential for the ATPase and lipid binding activities of ZmMS13. ZmMs13 is specifically expressed in anthers with three peaks at stages S5, S8b, and S10, which are successively regulated by transcription factors ZmbHLH122, ZmMYB84, and ZmMYB33-1/-2 at these three stages. The triphasic regulation of ZmMs13 is sequentially required for callose dissolution, tapetal PCD and pollen exine development, and anther cuticle formation, corresponding to transcription alterations of callose-, ROS-, PCD-, sporopollenin-, and anther cuticle-related genes in ms13-6060 anthers. CONCLUSION: ms13-6060 mutation with one key amino acid (I311) deletion greatly reduces ZmMS13 ATPase and lipid binding activities and displays multiple effects during maize male reproduction. Our findings provide new insights into molecular mechanisms of ABCG transporters controlling anther and pollen development and male fertility in plants.

The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide.

Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology.

Epigenetic Dynamics and Regulation of Plant Male Reproduction.

Flowering plant male germlines develop within anthers and undergo epigenetic reprogramming with dynamic changes in DNA methylation, chromatin modifications, and small RNAs. Profiling the epigenetic status using different technologies has substantially accumulated information on specific types of cells at different stages of male reproduction. Many epigenetically related genes involved in plant gametophyte development have been identified, and the mutation of these genes often leads to male sterility. Here, we review the recent progress on dynamic epigenetic changes during pollen mother cell differentiation, microsporogenesis, microgametogenesis, and tapetal cell development. The reported epigenetic variations between male fertile and sterile lines are summarized. We also summarize the epigenetic regulation-associated male sterility genes and discuss how epigenetic mechanisms in plant male reproduction can be further revealed.

Overexpression of HLH4 Inhibits Cell Elongation and Anthocyanin Biosynthesis in Arabidopsis thaliana.

In plants, many basic helix-loop-helix (bHLH) transcription factors are involved in controlling cell elongation. Three bHLH proteins, PACLOBTRAZOL RESISTANCE1 (PRE1), Cryptochrome Interacting Basic Helix-loop-helix 5 (CIB5), and Arabidopsis ILI1 binding bHLH1 (IBH1) form a triantagonistic system that antagonistically regulates cell elongation in a competitive manner. In this study, we identified a new player, HLH4, related to IBH1, that negatively regulates cell elongation in Arabidopsis thaliana. Overexpression of HLH4 causes dwarf and dark green phenotypes and results in the downregulation of many key regulatory and enzymatic genes that participate in the anthocyanin biosynthetic pathway. HLH4 interacts with CIB5 and PRE1. By interacting with CIB5, HLH4 interferes with the activity of CIB5, and thus inhibiting the transcription of cell elongation-related genes regulated by CIB5, including EXPANSINS8 and 11 (EXP8 and EXP11) and indole-3-acetic acid 7 and 17 (IAA7 and IAA17). The interference of HLH4 on CIB5 is counteracted by PRE1, in which these bHLH proteins form a new tri-antagonistic system.

The OsEIL1-OsERF115-target gene regulatory module controls grain size and weight in rice.

Grain size is one of the essential determinants of rice yield. Our previous studies revealed that ethylene plays an important role in grain-size control; however, the precise mechanism remains to be determined. Here, we report that the ethylene response factor OsERF115 functions as a key downstream regulator for ethylene-mediated grain development. OsERF115 encodes an AP2/ERF-type transcriptional factor that is specifically expressed in young spikelets and developing caryopses. Overexpression of OsERF115 significantly increases grain length, width, thickness and weight by promoting longitudinal elongation and transverse division of spikelet hull cells, as well as enhancing grain-filling activity, whereas its knockout mutations lead to the opposite effects, suggesting that OsERF115 positively regulates grain size and weight. OsERF115 transcription is strongly induced by ethylene, and OsEIL1 directly binds to the promoter to activate its expression. OsERF115 acts as a transcriptional repressor to directly or indirectly modulate a set of grain-size genes during spikelet growth and endosperm development. Importantly, haplotype analysis reveals that the SNP variations in the EIN3-binding sites of OsERF115 promoter are significantly associated with the OsERF115 expression levels and grain weight, suggesting that natural variations in the OsERF115 promoter contribute to grain-size diversity. In addition, the OsERF115 orthologues are identified only in grass species, implying a conserved and unique role in the grain development of cereal crops. Our results provide insights into the molecular mechanism of ethylene-mediated grain-size control and a potential strategy based on the OsEIL1-OsERF115-target gene regulatory module for genetic improvement of rice yield.

Mining of Potential Gene Resources for Breeding Nutritionally Improved Maize.

Maize is one of the leading food crops and its kernel is rich in starch, lipids, protein and other energy substances. In addition, maize kernels also contain many trace elements that are potentially beneficial to human health, such as vitamins, minerals and other secondary metabolites. However, gene resources that could be applied for nutrient improvement are limited in maize. In this review, we summarized 107 genes that are associated with nutrient content from different plant species and identified 246 orthologs from the maize genome. In addition, we constructed physical maps and performed a detailed expression pattern analysis for the 246 maize potential gene resources. Combining expression profiles and their potential roles in maize nutrient improvement, genetic engineering by editing or ectopic expression of these genes in maize are expected to improve resistant starch, oil, essential amino acids, vitamins, iron, zinc and anthocyanin levels of maize grains. Thus, this review provides valuable gene resources for maize nutrient improvement.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize.

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

Genetic background and cis-organization regulate ALDH7B4 gene expression in Eutrema salsugineum: a promoter analysis case study.

MAIN CONCLUSION: ALDH7B4 promoter analysis in A. thaliana and E. salsugineum reveals that both genetic background and promoter architecture contribute to gene expression in response to stress in different species. Many genes are differentially regulated in a comparison of salinity-sensitive and salinity-tolerant plant species. The aldehyde dehydrogenase 7B4 (ALDH7B4) gene is turgor-responsive in A. thaliana and encodes a highly conserved detoxification enzyme in plants. This study compared the ALDH7B4 gene in A. thaliana (salinity-sensitive) and in the salinity-tolerant close relative Eutrema salsugineum. EsALDH7B4 in E. salsugineum is the ortholog of AtALDH7B4 and the expression is also salinity, drought, and wound responsive. However, E. salsugineum requires higher salinity stress to induce the EsALDH7B4 transcriptional response. The GUS expression driven either by the promoter AtALDH7B4 or EsALDH7B4 was induced under 300 mM NaCl treatment in A. thaliana while 600 mM NaCl treatment was required in E. salsugineum, suggesting that the genetic background plays a crucial role in regulation of gene expression. Promoter sequences of ALDH7B4 are less conserved than the protein coding region. A series of EsALDH7B4 promoter deletion fragments were fused to the GUS reporter gene and promoter activity was determined in A. thaliana. The promoter region that contains two conserved ACGT-containing motifs was identified to be essential for stress induction. Furthermore, a 38 bp "TC" rich motif in the EsALDH7B4 promoter, absent from the AtALDH7B4 promoter, negatively affects EsALDH7B4 expression. A MYB-like transcription factor was identified to bind the "TC" motif and to repress the EsALDH7B4 promoter activity. This study reveals that genetic background and cis-acting elements coordinately regulate gene expression.

Epigenome and Epitranscriptome: Potential Resources for Crop Improvement.

Crop breeding faces the challenge of increasing food demand, especially under climatic changes. Conventional breeding has relied on genetic diversity by combining alleles to obtain desired traits. In recent years, research on epigenetics and epitranscriptomics has shown that epigenetic and epitranscriptomic diversity provides additional sources for crop breeding and harnessing epigenetic and epitranscriptomic regulation through biotechnologies has great potential for crop improvement. Here, we review epigenome and epitranscriptome variations during plant development and in response to environmental stress as well as the available sources for epiallele formation. We also discuss the possible strategies for applying epialleles and epitranscriptome engineering in crop breeding.

Genome-Wide Identification and Characterization of Polygalacturonase Gene Family in Maize (Zea mays L.).

Polygalacturonase (PG, EC 3.2.1.15) is a crucial enzyme for pectin degradation and is involved in various developmental processes such as fruit ripening, pollen development, cell expansion, and organ abscission. However, information on the PG gene family in the maize (Zea mays L.) genome and the specific members involved in maize anther development are still lacking. In this study, we identified 55 PG family genes from the maize genome and further characterized their evolutionary relationship and expression patterns. Phylogenetic analysis revealed that ZmPGs are grouped into six Clades, and gene structures of the same Clade are highly conserved, suggesting their functional conservation. The ZmPGs are randomly distributed across maize chromosomes, and collinearity analysis showed that many ZmPGs might be derived from tandem duplications and segmental duplications, and these genes are under purifying selection. Furthermore, gene expression analysis provided insights into possible functional divergence among ZmPGs. Based on the RNA-seq data analysis, we found that many ZmPGs are expressed in various tissues while 18 ZmPGs are highly expressed in maize anther, and their detailed expression profiles in different anther developmental stages were further investigated by using RT-qPCR analysis. These results provide valuable information for further functional characterization and application of the ZmPGs in maize.

ZmFAR1 and ZmABCG26 Regulated by microRNA Are Essential for Lipid Metabolism in Maize Anther.

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.

RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts.

Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.

ZmMs25 encoding a plastid-localized fatty acyl reductase is critical for anther and pollen development in maize.

Fatty acyl reductases (FARs) catalyse 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 by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, 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, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 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, indicating its application potential in maize and other crops.