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

Abstract

SummaryLipid 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.