Biosynthesis and secretion of cuticular wax

 

Previous work

1. Identification of wax biosynthetic enzymes



Previous work in my laboratory has established that the condensing enzyme (KCS) is the key activity of each ER-associated fatty acid elongation (FAE) system. Its expression determines whether VLCFAs are synthesized, it controls levels of VLCFA accumulation and the types of VLCFAs made in a particular cell. Isolation of CUT1/CER6 gene, which encodes a wax-specific condensing enzyme, allowed us to investigate the regulation of CER6 expression in response to developmental and environmental cues known to affect wax deposition, examine the potential for using CER6 gene to alter wax deposition in transgenic plants. My research group has also identified the CER10 enoyl reductase of the FAE, and using GFP-protein fusions and confocal microscopy demonstrated that the KCS and the ECR, and therefore likely the whold fatty acid elongation complex, reside in the membranes of the endoplasmic reticulum (ER).

 

We also identified and characterized the first wax biosynthetic enzyme downstream of fatty acid elongation, CER4, a fatty acyl-CoA reductase which catalyzes the formation of primary alcohols, and WSD1, a member of bifunctional wax ester synthase/ diacylglycerol acyltransferase (WS/DGAT) family, which plays a key role in wax ester synthesis in Arabidopsis. In collaboration with Reinhard Jetter and Lacey Samuels, we also discovered a cytochrome P450 enzyme (CYP96A15) which functions as a midchain alkane hydroxylase (MAH1) catalyzing the conversion of alkanes to secondary alcohols and possibly also the corresponding ketones. Using MAH1-GFP and YFP-WSD1 fusion proteins were localized these enzymes to the ER by confocal microscopy, providing evidence that both the alkane and the primary alcohol pathways of wax biosynthesis are associated with this organelle. Thus, the ER is the cellular compartment where all the cuticular wax components are generated.

 

2. Discovery of ABC transporters involved in wax export

our studies of the Arabidopsis wax-deficient cer5 mutant by transmission electron microscopy revealed striking sheet-like inclusions in the cytoplasm of wax-secreting epidermis. Gas chromatographic analysis showed that these inclusions represent abnormal deposits of cuticular wax that accumulate within the cells, while the wax load on the plant surface is substantially reduced. The isolation of the CER5 gene established that it encodes a plasma membrane localized ABC transporter, specific to the epidermal cells. The CER5 ABC transporter is the first component of the wax export pathway identified in plants and provides clues about the mechanism of wax transport to the plant cuticle. In collaboration with the Samuels-Jetter labs we have recently characterized the second ABC transporter of wax, WBC11, using a reverse genetics approach. Similar to mutations in the CER5 gene, lesions in WBC11 also cause reduced wax load and accumulation of intracellular lipidic inclusions, but in addition also result in dwarfism, sterility, post-genital organ fusions and reduced cutin load.

 

3. Discovery of a unique mechanism of regulation of wax biosynthesis by a core subunit of the exosome

Cloning of the CER7 gene identified by mutation in the wax-deficient cer7 mutant demonstrated that it encodes a functional homologue of the yeast RRP45p enzyme, a core subunit of the exosome, an evolutionarily conserved complex of 3’-5’exoribonucleases involved in RNA processing and degradation. In addition to performing general exosomal functions, as described for other eukaryotes, the CER7 ribonuclease has a unique role in epidermis-specific control of wax biosynthesis. The proposed target of the CER7 enzyme is an mRNA encoding a transcriptional repressor of the key wax biosynthetic gene CER3/WAX2/YRE. We hypothesize that in wild type plants the CER7 ribonuclease degrades the mRNA specifying the repressor, thereby allowing CER3 expression and wax production via the alkane (decarbonylation) pathway. Our work on the cer7 mutant provides the first experimental evidence for a mechanism of regulation of cuticular wax deposition by plants, but more significantly also demonstrates that in eukaryotes core ribonuclease subunits of the exosome can have dedicated roles in distinct cellular processes that require recognition of specific mRNA targets.