| Fatty acids and their derivatives are important components in the formation of anther cuticle and pollen wall.Lipid metabolism mainly occurs in tapetum during anther and pollen development,and the destruction of this process often leads to genic male sterility(GMS).The lipid metabolism includes lipid biosynthesis and lipid transport.In plants,although many lipid metabolism genes related to anther and pollen development have been identified,but the function of some lipid metabolism genes remain to be unexplored.In this study,the two homologous lipid metabolism-related genes ZmACOS5-1 and ZmACOS5-2,which encoded fatty acyl CoA synthetase,were identified by using CRISPR/Cas9 gene editing method in maize.In addition,ZmMs13,a male-sterility gene encoding ABCG transporter ABCG2a,was identified by using map-based cloning strategy.ZmMs13 displayed an anther-specific expression with three peaks at stages S5,S8b,and S10 and had different biological functions at stages of anther development.Here,the molecular mechanisms of ZmACOS5-1/-2 and ZmMS13 controling anther and pollen development were investigated by using combinational strategy of genetics,molecular biology,biochemistry,cytology,bioinformatics and lipidomics,and the function of ABCG subfamily genes was identified and predicted based on the whole-genome analysis.The main results of this study were as follows:1.The maize fatty acyl CoA synthetase genes ZmACOS5-1/-2,which controling anther and pollen development,were identified for the first time by reverse genetic strategy.The two genes were highly expressed in stage S9 of anther development of maize,and the single mutant of acos5-1 or zmacos5-2 showed normal male fertility,while the microspores cannot vacuolize in their double mutant anthers at stage S10,and showed complete male sterility.2.ZmACOS5-1/-2 were essential for the formation of pollen exine and anther cuticle.Compared with the WT,the double mutant of acos5-1/-2 showed abnormal in sporopolysin synthesis and failed to form a normal three-dimensional network of cuticle on the outer surface of anther.3.ZmACOS5-1/-2 interacted with ZmPKSB,a polyketide synthase,and existed cascaded reaction of enzyme activity.ZmACOS5-1/-2 catalyzed C16:0 and C18:0 to produce C16-CoA and C18-CoA,and further generated triketones and tetraketones alpha-pyranone under the catalysis of allylene CoA and ZmPKSB.4.ZmACOS5-1/-2 were directly regulated by transcription factor ZmMYB84 during anther and pollen development.It also affected the expression of sporopollenine synthesis-related genes(ZmPKSA1,ZmPKSA2,ZmIPE1,ZmMs45,etc.),wax-related genes(ZmHCT12,ZmMs13,etc.)and cutin-related genes(ZmCER8,ZmGL8,ZmKCAS4,etc.)and thus affected the formation of pollen extine and anther wall.5.The male sterile gene ZmMs13(Zm00001d013960),which encodes a new ABCG transporter ZmABCG2a in maize was identified by mapping cloning method.ZmMs13 was specifically expressed in anthers and had 3 expression peaks in stage S5,S8b and S10 of anther development.6.ZmMS13 had ATPase activity,which was affected by fatty acids and fatty alcohols.The absence of a single amino acid(1311)of ZmMS13 in the ms13-6060 mutant resulted in decreased ATP hydrolyzing activity,which may be related to resulting in the male sterility phenotype.7.It was confirmed that a ABCG transporter gene was regulated by different transcription factors at different anther developmental stages,participating in different biological processes.We found that ZmMs13/ABCG2a was regulated by transcription factors ZmbHLH122,ZmMYB84 and ZmMYB33-1/-2 at early(S5),middle(S8b)and late(S10)stages,respectively.And the different regulation modules participated in callose degradation,tapetal PCD(Programmed cell death)and pollen exine development,anther cuticle formation in maize,consecutively.8.We established the link between ZmACOS5-1/-2 and ZmMs13 during anther development.In other words,precursors of sporopollenin and anther cuticle were synthesized under the action of lipid synthetases,such as ZmACOS5-1 and ZmACOS5-2,then are transported to destination by the action of lipid transporters,such as ZmMS13/ABCG2a,leading to the form ation of sporopollenin and anther cuticle.9.Based on the whole genome analysis,51 ABCG transporters were identified in maize,and these maize ABCG genes were named systematically and characterized for the first time.The functions of maize ABCG subfamily genes were predicted by phylogenetic tree and bioinformatics analysis in this study.In conclusion,this study revealed the mechanism of ZmACOS5-1 and ZmACOS5-2 as well as the ABCG transporter ZmMS13 in anther and pollen development of maize.ZmACOS5-1 and ZmACOS5-2,the key enzymes in lipid synthesis,are functionally conserved in different plants and play important roles in the synthesis of pollen exine and anther cuticle.As a transporter,ZmMS13 has multiple functions at different stages during anther development.ZmACOS5-1/-2 were associated with the transporter gene ZmMs13 during anther development,which will deepen our understanding of the anther and pollen development and their lipid metabolism process.These results provide a reference for investigating the mechanism of lipid metabolism in plant reproductive development. |
