The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

Abstract

Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.

The Plant Cuticle Is Required for Osmotic Stress Regulation of Abscisic Acid Biosynthesis and Osmotic Stress Tolerance inArabidopsis

“…As controls, we also included two wax mutants, mah1-1 and cer7. The mah1-1 is mutated in the cytochrome P450 midchain alkane hydroxylase (CYP96A15) required for producing the wax components secondary alcohols and ketones (Greer et al, 2007). The cer7 mutation is in a putative 39-59 exoribonuclease of core exosome that degrades the transcript of a negative regulator of the wax gene CER3/WAX2/YRE (Hooker et al, 2007).…”

The Formation and Function of Plant Cuticles

“…Aside from being a major component of the wax mixture, alkanes can undergo further modification to form secondary alcohols and ketones. In Arabidopsis, both of these oxidations are performed by the cytochrome P450 enzyme midchain alkane hydroxylase (MAH1; Greer et al, 2007).…”

Arabidopsis ABCG Transporters, Which Are Required for Export of Diverse Cuticular Lipids, Dimerize in Different Combinations

“…Alternatively, the inclusions could reflect the nature of lipid traffic during cuticular lipid secretion. All of the enzymes that have been characterized in wax synthesis have been localized to the ER membranes (Zheng et al, 2005;Greer et al, 2007;Bach et al, 2008;Li et al, 2008). If intermediate secretory compartments, such as the Golgi apparatus, are required for wax secretion to the cell surface, then wax accumulation in these compartments would be expected to occur when transport is blocked.…”