Arabidopsis 3-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development

The biosynthesis of C(27-2)9 sterols from their C30 precursor squalene involves C24-alkylation and the removal of three methyl groups, including two at the C4 position. The two C4 demethylation reactions require a bifunctional enzyme known as 3 beta-hydroxysteroid dehydrogenase/C4-decarboxylase (3 beta HSD/D), which removes an oxidized methyl (carboxylic) group at C4 while simultaneously catalyzing the 3 beta-hydroxyl -> 3-keto oxidation. Its loss-of-function mutations cause ergosterol-dependent growth in yeast and congenital hemidysplasia with ichthyosiform erythroderma and limb defect (CHILD) syndrome in humans. Although plant 3 beta HSD/D enzymes were well studied enzymatically, their developmental functions remain unknown. Here we employed a CRISPR/Cas9-based genome-editing approach to generate knockout mutants for two Arabidopsis 3 beta HSD/D genes, HSD1 and HSD2, and discovered the male gametophytic lethality for the hsd1 hsd2 double mutation. Pollen-specific expression of HSD2 in the heterozygous hsd1 hsd2/+ mutant not only rescued the pollen lethality but also revealed the critical roles of the two HSD genes in embryogenesis. Our study thus demonstrated the essential functions of the two Arabidopsis 3 beta HSD/D genes in male gametogenesis and embryogenesis.