Molecular regulation of ZmMs7 required for maize male fertility and development of a dominant male-sterility system in multiple species.

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.

IRREGULAR POLLEN EXINE2 Encodes a GDSL Lipase Essential for Male Fertility in Maize.

Anther cuticle and pollen exine are two physical barriers protecting plant reproductive cells against environmental stresses; defects in either often cause male sterility. Here, we report the characterization of a male-sterile mutant irregular pollen exine2 (ipe2) of maize (Zea mays), which displays shrunken anthers and no starch accumulation in mature pollen grains. We cloned the causal gene IPE2 and confirmed its role in male fertility in maize with a set of complementary experiments. IPE2 is specifically expressed in maize developing anthers during stages 8 to 9 and encodes an endoplasmic-reticulum-localized GDSL lipase. Dysfunction of IPE2 resulted in delayed degeneration of tapetum and middle layer, leading to defective formation of anther cuticle and pollen exine, and complete male sterility. Aliphatic metabolism was greatly altered, with the contents of lipid constituents, especially C16/C18 fatty acids and their derivatives, significantly reduced in ipe2 developing anthers. Our study elucidates GDSL function in anther and pollen development and provides a promising genetic resource for breeding hybrid maize.

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.

Genome-wide analysis of maize GPAT gene family and cytological characterization and breeding application of ZmMs33/ZmGPAT6 gene.

KEY MESSAGE: Genome-wide analysis of maize GPAT gene family, cytological characterization of ZmMs33/ZmGPAT6 gene encoding an ER-localized protein with four conserved motifs, and its molecular breeding application in maize. Glycerol-3-phosphate acyltransferase (GPAT) mediates the initial step of glycerolipid biosynthesis and plays pivotal roles in plant growth and development. Compared with GPAT genes in Arabidopsis, our understanding to maize GPAT gene family is very limited. Recently, ZmMs33 gene has been identified to encode a sn-2 GPAT protein and control maize male fertility in our laboratory (Xie et al. in Theor Appl Genet 131:1363-1378, 2018). However, the functional mechanism of ZmMs33 remains elusive. Here, we reported the genome-wide analysis of maize GPAT gene family and found that 20 maize GPAT genes (ZmGPAT1-20) could be classified into three distinct clades similar to those of ten GPAT genes in Arabidopsis. Expression analyses of these ZmGPAT genes in six tissues and in anther during six developmental stages suggested that some of ZmGPATs may play crucial roles in maize growth and anther development. Among them, ZmGPAT6 corresponds to the ZmMs33 gene. Systemic cytological observations indicated that loss function of ZmMs33/ZmGPAT6 led to defective anther cuticle, arrested degeneration of anther wall layers, abnormal formation of Ubisch bodies and exine and ultimately complete male sterility in maize. The endoplasmic reticulum-localized ZmMs33/ZmGPAT6 possessed four conserved amino acid motifs essential for acyltransferase activity, while ZmMs33/ZmGPAT6 locus and its surrounding genomic region have greatly diversified during evolution of gramineous species. Finally, a multi-control sterility system was developed to produce ms33 male-sterile lines by using a combination strategy of transgene and marker-assisted selection. This work will provide useful information for further deciphering functional mechanism of ZmGPAT genes and facilitate molecular breeding application of ZmMs33/ZmGPAT6 gene in maize.

Discovering and Constructing ceRNA-miRNA-Target Gene Regulatory Networks during Anther Development in Maize.

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.

Map-Based Cloning, Phylogenetic, and Microsynteny Analyses of ZmMs20 Gene Regulating Male Fertility in Maize.

Genic male sterility (GMS) mutant is a useful germplasm resource for both theory research and production practice. The identification and characterization of GMS genes, and assessment of male-sterility stability of GMS mutant under different genetic backgrounds in Zea may (maize) have (1) deepened our understanding of the molecular mechanisms controlling anther and pollen development, and (2) enabled the development and efficient use of many biotechnology-based male-sterility (BMS) systems for hybrid breeding. Here, we reported a complete GMS mutant (ms20), which displays abnormal anther cuticle and pollen development. Its fertility restorer gene ZmMs20 was found to be a new allele of IPE1 encoding a glucose methanol choline (GMC) oxidoreductase involved in lipid metabolism in anther. Phylogenetic and microsynteny analyses showed that ZmMs20 was conserved among gramineous species, which provide clues for creating GMS materials in other crops. Additionally, among the 17 maize cloned GMS genes, ZmMs20 was found to be similar to the expression patterns of Ms7, Ms26, Ms6021, APV1, and IG1 genes, which will give some clues for deciphering their functional relationships in regulating male fertility. Finally, two functional markers of ZmMs20/ms20 were developed and tested for creating maize ms20 male-sterility lines in 353 genetic backgrounds, and then an artificial maintainer line of ms20 GMS mutation was created by using ZmMs20 gene, ms20 mutant, and BMS system. This work will promote our understanding of functional mechanisms of male fertility and facilitate molecular breeding of ms20 male-sterility lines for hybrid seed production in maize.

ABNORMAL POLLEN VACUOLATION1 (APV1) is required for male fertility by contributing to anther cuticle and pollen exine formation in maize.

Anther cuticle and pollen exine are the major protective barriers against various stresses. The proper functioning of genes expressed in the tapetum is vital for the development of pollen exine and anther cuticle. In this study, we report a tapetum-specific gene, Abnormal Pollen Vacuolation1 (APV1), in maize that affects anther cuticle and pollen exine formation. The apv1 mutant was completely male sterile. Its microspores were swollen, less vacuolated, with a flat and empty anther locule. In the mutant, the anther epidermal surface was smooth, shiny, and plate-shaped compared with the three-dimensional crowded ridges and randomly formed wax crystals on the epidermal surface of the wild-type. The wild-type mature pollen had elaborate exine patterning, whereas the apv1 pollen surface was smooth. Only a few unevenly distributed Ubisch bodies were formed on the apv1 mutant, leading to a more apparent inner surface. A significant reduction in the cutin monomers was observed in the mutant. APV1 encodes a member of the P450 subfamily, CYP703A2-Zm, which contains 530 amino acids. APV1 appeared to be widely expressed in the tapetum at the vacuolation stage, and its protein signal co-localized with the endoplasmic reticulum (ER) signal. RNA-Seq data revealed that most of the genes in the fatty acid metabolism pathway were differentially expressed in the apv1 mutant. Altogether, we suggest that APV1 functions in the fatty acid hydroxylation pathway which is involved in forming sporopollenin precursors and cutin monomers that are essential for the development of pollen exine and anther cuticle in maize.

Case Report: A PD-L1-Positive Patient With Pleomorphic Rhabdomyosarcoma Achieving an Impressive Response to Immunotherapy.

There is currently a lack of effective systemic treatment for patients with advanced pleomorphic rhabdomyosarcoma (PRMS). Although programmed death protein 1 (PD-1) inhibitors have shown efficacy in various solid tumors, their effects on PRMS have not been well established. Here, we present a case of a 12-year-old Chinese male adolescent with metastatic PRMS who benefited from the PD-1 inhibitor nivolumab. The patient initially underwent primary tumor resection but failed to respond to subsequent first-line chemotherapy and second-line pazopanib treatment. Pathological examination showed positive PD-L1 expression and tumor-infiltrating lymphocytes in the tumor tissue, and the patient was administered nivolumab as a posterior-line treatment. After attaining a clinically partial response (PR), surgical resection was performed, which was followed by adjuvant nivolumab. At the time of the submission of this manuscript, the patient achieved recurrence-free survival (RFS) lasting 45 months and counting. This is the first clinical evidence that a patient with refractory PRMS was controlled by anti-PD-1 antibody, with an RFS lasting more than 3 years. This case suggests that PD-L1 expression and T-cell infiltration could be used as potential biomarkers for PRMS immunotherapy.

Lipid Metabolism: Critical Roles in Male Fertility and Other Aspects of Reproductive Development in Plants.

Fatty acids and their derivatives are essential building blocks for anther cuticle and pollen wall formation. Disruption of lipid metabolism during anther and pollen development often leads to genic male sterility (GMS). To date, many lipid metabolism-related GMS genes that are involved in the formation of anther cuticle, pollen wall, and subcellular organelle membranes in anther wall layers have been identified and characterized. In this review, we summarize recent progress on characterizing lipid metabolism-related genes and their roles in male fertility and other aspects of reproductive development in plants. On the basis of cloned GMS genes controlling biosynthesis and transport of anther cutin, wax, sporopollenin, and tryphine in Arabidopsis, rice, and maize as well as other plant species, updated lipid metabolic networks underlying anther cuticle development and pollen wall formation were proposed. Through bioinformatics analysis of anther RNA-sequencing datasets from three maize inbred lines (Oh43, W23, and B73), a total of 125 novel lipid metabolism-related genes putatively involved in male fertility in maize were deduced. More, we discuss the pathways regulating lipid metabolism-related GMS genes at the transcriptional and post-transcriptional levels. Finally, we highlight recent findings on lipid metabolism-related genes and their roles in other aspects of plant reproductive development. A comprehensive understanding of lipid metabolism, genes involved, and their roles in plant reproductive development will facilitate the application of lipid metabolism-related genes in gene editing, haploid and callus induction, molecular breeding and hybrid seed production in crops.

Maize Genic Male-Sterility Genes and Their Applications in Hybrid Breeding: Progress and Perspectives.

As one of the most important crops, maize not only has been a source of the food, feed, and industrial feedstock for biofuel and bioproducts, but also became a model plant system for addressing fundamental questions in genetics. Male sterility is a very useful trait for hybrid vigor utilization and hybrid seed production. The identification and characterization of genic male-sterility (GMS) genes in maize and other plants have deepened our understanding of the molecular mechanisms controlling anther and pollen development, and enabled the development and efficient use of many biotechnology-based male-sterility (BMS) systems for crop hybrid breeding. In this review, we summarize main advances on the identification and characterization of GMS genes in maize, and construct a putative regulatory network controlling maize anther and pollen development by comparative genomic analysis of GMS genes in maize, Arabidopsis, and rice. Furthermore, we discuss and appraise the features of more than a dozen BMS systems for propagating male-sterile lines and producing hybrid seeds in maize and other plants. Finally, we provide our perspectives on the studies of GMS genes and the development of novel BMS systems in maize and other plants. The continuous exploration of GMS genes and BMS systems will enhance our understanding of molecular regulatory networks controlling male fertility and greatly facilitate hybrid vigor utilization in breeding and field production of maize and other crops.

ZmMs30 Encoding a Novel GDSL Lipase Is Essential for Male Fertility and Valuable for Hybrid Breeding in Maize.

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 cross-breeding 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.

MALE STERILE6021 (MS6021) is required for the development of anther cuticle and pollen exine in maize.

The anther cuticle and pollen wall function as physical barriers that protect genetic material from various environmental stresses. The anther cuticle is composed of wax and cutin, the pollen wall includes exine and intine, and the components of the outer exine are collectively called sporopollenin. Other than cuticle wax, cutin and sporopollenin are biopolymers compounds. The precise constituents and developmental mechanism of these biopolymeric are poorly understood. Here, we reported a complete male sterile mutant, male sterile6021, in maize. The mutant displayed a smooth anther surface and irregular pollen wall formation before anthesis, and its tapetum was degraded immaturely. Gas chromatography-mass spectrometry analysis revealed a severe reduction of lipid derivatives in the mutant anther. We cloned the gene by map based cloning. It encoded a fatty acyl carrier protein reductase that was localized in plastids. Expression analysis indicated that MS6021 was mainly expressed in the tapetum and microspore after the microspore was released from the tetrad. Functional complementation of the orthologous Arabidopsis mutant demonstrated that MS6021 is conserved between monocots and dicots and potentially even in flowering plants. MS6021 plays a conserved, essential role in the successful development of anther cuticle and pollen exine in maize.