Molecular Mechanism Of Polyketide Synthases Gene ZmPKSB And Tetraketide α-pyrone Reductase Gene ZmTKPR1 In Controlling Maize Anther Development

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,the molecular mechanism underlying a trade-off of lipid metabolic products to promote the proper formation of the two lipid layers remains elusive.In this study,a male-sterile mutant pksb was screened from the maize EMS mutant library,and the ZmPKSB gene was identified by map-based cloning.The ZmPKSB gene encodes a type Ⅲ Polyketide synthase(PKS),which can produce tetraketide α-pyrone by continuous condense fatty acyl-CoA and malonyl-CoA in vitro.PKS and TKPR(Tetraketide α-pyrone reductase)are commonly involved in the biosynthesis of sporopollenin precursors.In this study,single-and double-gene homozygous mutants of ZmTKPR1-1 and ZmTKPR1-2 were generated in maize by CRISPR/Cas9.The functions of ZmPKSB,ZmTKPR1-1 and ZmTKPR1-2 in maize anther and pollen development were investigated by using cytological,molecular biology,biochemistry,and cutin/wax determination.The major results and conclusions of this study are as follows:1.Systemically cytological observations indicated that maize male-sterile mutant pksb,which exhibited delayed tapetum degradation,larger Ubisch bodys,dense anther cuticle,and thinner pollen exine resulted in male sterility.Based on map-based cloning and gene function verification,the causal gene(ZmPKSB)of the pksb mutant was identified as Zm00001d019478,located on chromosome 7 of maize.2.Phylogenetic analysis and microsynteny analysis showed that the structure and function of ZmPKSB and its orthologs were relatively conserved in a variety of plants.ZmPKSB worked as a polyketide synthase,and its activity was dependent on the catalytic triad.The fatty acyl-CoA was used as the starting substrate of ZmPKSB to produce triketide and tetraketide α-pyrone.3.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 ZmMYB84ZmPKSB 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.4.The maize ZmTKPR1-1 and ZmTKPR1-2 are homologous to Arabidopsis AtTKPRl and rice OsTKPR1.Mutations of ZmTKPR1-1 and ZmTKPR1-2 by CRISPR/Cas9 suggested redundant roles in controlling maize anther and pollen development and male fertility and made complete male sterility.Cytological observations indicated that the zmtkprl-1/zmtkprl-2 mutant showed delayed tapetum degradation,smaller Ubischs,thinner anther cuticle and pollen exine,and microspore degradation that eventually led to the appearance of a male sterile phenotype.5.ZmTKPR1-1 and ZmTKPR1-2 are two GMS genes that are specifically expressed in the anthers.ZmTKPR1-1 presented two expression peaks at stages 7 and 9,while ZmTKPR1-2 exhibited only one expression peak at stage 9.ZmMYB84 was able to bind to the ZmTKPR1-1 and ZmTKPR1-2 promoters and activate their expression.6.As tetraketide α-pyrone reductases,the activity of ZmTKPR1-1 and ZmTKPR1-2 was not only dependent on three amino acid residues Ser,Tyr,and Lys in the catalytic center,but also on amino acid residue Trp at the NADPH binding site.ZmTKPR1-1 was significantly more active than ZmTKPR1-2 in vitro.7.ZmPKSB,ZmTKPR1-1 and ZmTKPR1-2 function by forming protein complexes in the endoplasmic reticulum.In summary,this study revealed light on the molecular mechanisms of ZmPKSB,ZmTKPR1-1 and ZmTKPR1-2 in the phenylpropanoid metabolic pathway,which regulated anther and pollen development in maize.These findings provide new insights into the lipid metabolism trade-off during the formation of the anther cuticle and pollen exine,and shed new light on further study of the process of plant fertility.