Two Isoforms of NADPH:Cytochrome P450 Reductase in Arabidopsis thaliana
Gene Structure, Heterologous Expression in Insect Cells, and Differential Regulation

Masaharu Mizutani and Daisaku Ohta^*

Takarazuka Research Institute, Novartis Pharma K.K., 10-66 Miyuki-cho, Takarazuka 665, Japan (M.M.); and Research Institute for Biological Sciences, Okayama, 7459-1 Yoshikawa, Kayo-cho, Okayama 716-12, Japan (D.O.)

We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (AR1 and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar K_m values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min¹ nmol¹ P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PAL1) gene. On the contrary, the AR1 expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.

Plant Physiol. (1998) 116: 357-367
Copyright Clearance Center:   0032-0889/98/116/0357/11
© 1998 American Society of Plant Physiologists

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