Functional analysis of the tandem-duplicated P450 genes SPS/BUS/CYP79F1 and CYP79F2 in glucosinolate biosynthesis and plant development by Ds transposition-generated double mutants.

A significant fraction (approximately 17%) of Arabidopsis genes are members of tandemly repeated families and pose a particular challenge for functional studies. We have used the Ac-Ds transposition system to generate single- and double-knockout mutants of two tandemly duplicated cytochrome P450 genes, SPS/BUS/CYP79F1 and CYP79F2. We have previously described the Arabidopsis supershoot mutants in CYP79F1 that exhibit massive overproliferation of shoots. Here we use a cytokinin-responsive reporter ARR5::uidA and an auxin-responsive reporter DR5::uidA in the sps/cyp79F1 mutant to show that increased levels of cytokinin, but not auxin, correlate well with the expression pattern of the SPS/CYP79F1 gene, supporting the involvement of this gene in cytokinin homeostasis. Further, we isolated Ds gene trap insertions in the CYP79F2 gene, and find these mutants to be defective mainly in the root system, consistent with a root-specific expression pattern. Finally, we generated double mutants in CYP79F1 and CYP79F2 using secondary transpositions, and demonstrate that the phenotypes are additive. Previous biochemical studies have suggested partially redundant functions for SPS/CYP79F1 and CYP79F2 in aliphatic glucosinolate synthesis. Our analysis shows that aliphatic glucosinolate biosynthesis is completely abolished in the double-knockout plants, providing genetic proof for the proposed biochemical functions of these genes. This study also provides further demonstration of how gluconisolate biosynthesis, regarded as secondary metabolism, is intricately linked with hormone homeostatis and hence with plant growth and development.