ZmMs33 promotes anther elongation via modulating cell elongation regulators,metabolic homeostasis,and cell wall remodeling in maize

Plant cell elongation depends on well-defined gene regulations,adequate nutrients,and timely cell wall modifications.Anther size is positively correlated with the number and viability of pollen grains,while little is known about molecular mechanisms underlying anther cell elongation.Here,we found that properly activated cell elongation regulators at transcriptional levels in loss-of-function ZmMs33 mutant(ms33-6038)anthers failed to promote maize anther elongation.ZmMs33 deficiency disrupted metabolic homeostasis mainly by inhibiting both photosynthesis in anther endothecium and lipid accumulation in anther tapetum.Importantly,ms33-6038 anthers displayed ectopic,premature and excessive secondary cell wall thickening in anther middle layer,which constrained cell elongation structurally and blocked nutrient flows across different anther wall layers.The metabolic disorder was only found in ms33-6038 mutant rather than several representative male-sterility lines at transcriptional and post-translational levels.Collectively,the disordered metabolisms and blocked nutrient flows defeated the activated cell elongation regulators,and finally inhibited anther elongation and growth with a unique‘‘idling effect”in ms33-6038 mutant.

Jasmonic acid-mediated stress responses share the molecular mechanism underlying male sterility induced by deficiency of ZmMs33 in maize

Plant male reproduction is a fine-tuned developmental process that is susceptible to stressful environments and influences crop grain yields.Phytohormone signaling functions in control of plant normal growth and development as well as in response to external stresses,but the interaction or crosstalk among phytohormone signaling,stress response,and male reproduction in plants remains poorly understood.Cross-species comparison among 514 stress-response transcriptomic libraries revealed that ms33-6038,a genic male sterile mutant deficient in the Zm Ms33/Zm GPAT6 gene,displayed an excessive drought stress-like transcriptional reprogramming in anthers triggered mainly by disturbed jasmonic acid(JA)homeostasis.An increased level of JA appeared in Zm Ms33-deficient anthers at both meiotic and postmeiotic stages and activated genes involved in JA biosynthesis and signaling as well as genes functioning in JA-mediated drought response.Excessive accumulation of JA elevated expression level of a gene encoding a WRKY transcription factor that activated the Zm Ms33 promoter.These findings reveal a feedback loop of Zm Ms33-JA-WRKY-Zm Ms33 in controlling male sterility and JA-mediated stress response in maize,shedding light on the crosstalk of stress response and male sterility mediated by phytohormone homeostasis and signaling.

Genome-wide analyses on transcription factors and their potential microRNA regulators involved in maize male fertility

Anther development is a programmed biological process crucial to plant male reproduction. Genomewide analyses on the functions of transcriptional factor(TF) genes and their microRNA(miRNA) regulators contributing to anther development have not been comprehensively performed in maize. Here, using published RNA-Seq and small RNA-Seq(sRNA-Seq) data from maize anthers at ten developmental stages in three genic male-sterility(GMS) mutants(ocl4, mac1, and ms23) and wild type W23, as well as newly sequenced maize anther transcriptomes of ms7-6007 and lob30 GMS mutants and their WT lines, we analyzed and found 1079 stage-differentially expressed(stage-DE) TF genes that can be grouped into six(premeiotic, meiotic, postmeiotic, premeiotic-meiotic, premeiotic-postmeiotic, and meiotic-postmeiotic clusters) expression clusters. Functional enrichment combined with cytological and physiological analyses revealed specific functions of genes in each expression cluster. In addition, 118 stage-DE miRNAs and99 miRNA-TF gene pairs were identified in maize anthers. Further analyses revealed the regulatory roles of zma-miR319 and zma-miR159 as well as ZmMs7 and ZmLOB30 on ZmGAMYB expression. Moreover,ZmGAMYB and its paralog ZmGAMYB-2 were demonstrated as novel maize GMS genes by CRISPR/Cas9 knockout analysis. These results extend our understanding on the functions of miRNA-TF gene regulatory pairs and GMS TF genes contributing to male fertility in plants.

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 molecularmechanismunderlying 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.Weshowed thatZmMs1/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.

Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana

Abiotic stresses cause serious crop losses. Knowledge on genes functioning in plant responses to adverse growth conditions is essential for developing stress tolerant crops. Here we report that transgenic expression of MYB15, encoding a R2R3 MYB transcription factor in Arabidopsis thaliana, conferred hypersensitivity to exogenous abscisic acid （ABA） and improved tolerance to drought and salt stresses. The promoter of MYB15 was active in not only vegetative and reproductive organs but also the guard cells of stomata. Its transcript level was substantially upregulated by ABA, drought or salt treatments. Compared with wild type （WT） control, MYB15 overexpression lines were hypersensitive to ABA in germination assays, more susceptible to ABA-elicited inhibition of root elongation, and more sensitive to ABA-induced stomatal closure. In line with the above findings, the transcript levels of ABA biosynthesis （ABA1, ABA2）, signaling （AB13） and responsive genes （AtADH1, RD22, RD29B, AtEM6） were generally higher in MYB15 overexpression seedlings than in WT controls after treatment with ABA. MYB15 overexpression lines displayed improved survival and reduced water loss rates than WT control under water deficiency conditions. These overexpression lines also displayed higher tolerance to NaCI stress. Collectively, our data suggest that overexpression of MYB15 improves drought and salt tolerance in Arabidopsis possibly by enhancing the expression levels of the genes involved in ABA biosynthesis and signaling, and those encoding the stress-protective proteins.

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 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 valu. able 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.

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 con struct 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.

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\r\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.

Normal Structure and Function of Endothecium Chloroplasts Maintained by ZmMs33-Mediated Lipid Biosynthesis in Tapetal Cells Are Critical for Anther Development 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.