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.

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 ZmMYB84-ZmPKSB regulatory module controls male fertility through modulating anther cuticle-pollen exine trade-off in maize anthers.

Anther cuticle and pollen exine are two crucial lipid layers that ensure normal pollen development and pollen-stigma interaction for successful fertilization and seed production in plants. Their formation processes share certain common pathways of lipid biosynthesis and transport across four anther wall layers. However, molecular mechanism underlying a trade-off of lipid-metabolic products to promote the proper formation of the two lipid layers remains elusive. Here, we identified and characterized a maize male-sterility mutant pksb, which displayed denser anther cuticle but thinner pollen exine as well as delayed tapetal degeneration compared with its wild type. Based on map-based cloning and CRISPR/Cas9 mutagenesis, we found that the causal gene (ZmPKSB) of pksb mutant encoded an endoplasmic reticulum (ER)-localized polyketide synthase (PKS) with catalytic activities to malonyl-CoA and midchain-fatty acyl-CoA to generate triketide and tetraketide α-pyrone. A conserved catalytic triad (C171, H320 and N353) was essential for its enzymatic activity. ZmPKSB was specifically expressed in maize anthers from stages S8b to S9-10 with its peak at S9 and was directly activated by a transcription factor ZmMYB84. Moreover, loss function of ZmMYB84 resulted in denser anther cuticle but thinner pollen exine similar to the pksb mutant. The ZmMYB84-ZmPKSB regulatory module controlled a trade-off between anther cuticle and pollen exine formation by altering expression of a series of genes related to biosynthesis and transport of sporopollenin, cutin and wax. These findings provide new insights into the fine-tuning regulation of lipid-metabolic balance to precisely promote anther cuticle and pollen exine formation in plants.

Genetic Structure and Molecular Mechanisms Underlying the Formation of Tassel, Anther, and Pollen in the Male Inflorescence of Maize (Zea mays L.).

Maize tassel is the male reproductive organ which is located at the plant's apex; both its morphological structure and fertility have a profound impact on maize grain yield. More than 40 functional genes regulating the complex tassel traits have been cloned up to now. However, the detailed molecular mechanisms underlying the whole process, from male inflorescence meristem initiation to tassel morphogenesis, are seldom discussed. Here, we summarize the male inflorescence developmental genes and construct a molecular regulatory network to further reveal the molecular mechanisms underlying tassel-trait formation in maize. Meanwhile, as one of the most frequently studied quantitative traits, hundreds of quantitative trait loci (QTLs) and thousands of quantitative trait nucleotides (QTNs) related to tassel morphology have been identified so far. To reveal the genetic structure of tassel traits, we constructed a consensus physical map for tassel traits by summarizing the genetic studies conducted over the past 20 years, and identified 97 hotspot intervals (HSIs) that can be repeatedly mapped in different labs, which will be helpful for marker-assisted selection (MAS) in improving maize yield as well as for providing theoretical guidance in the subsequent identification of the functional genes modulating tassel morphology. In addition, maize is one of the most successful crops in utilizing heterosis; mining of the genic male sterility (GMS) genes is crucial in developing biotechnology-based male-sterility (BMS) systems for seed production and hybrid breeding. In maize, more than 30 GMS genes have been isolated and characterized, and at least 15 GMS genes have been promptly validated by CRISPR/Cas9 mutagenesis within the past two years. We thus summarize the maize GMS genes and further update the molecular regulatory networks underlying male fertility in maize. Taken together, the identified HSIs, genes and molecular mechanisms underlying tassel morphological structure and male fertility are useful for guiding the subsequent cloning of functional genes and for molecular design breeding in maize. Finally, the strategies concerning efficient and rapid isolation of genes controlling tassel morphological structure and male fertility and their application in maize molecular breeding are also discussed.

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.