Biosynthesis and secretion of cuticular wax
- a genetic screen for suppressors of the cer7 glossy phenotype, and
- transcriptional profiling using microarrays.
Current projects
1. Isolation and characterization of alkane biosynthetic genes
Genetic studies indicate that a large number of proteins
participates in cuticular wax production. To date, over 30 recessive
wax-related eceriferum (cer = without wax) mutant
loci have been described in Arabidopsis. However, based on cer
mutation frequency, it is conceivable that there are ~65 loci involved
in this process. Lines carrying mutations in the CER genes
are readily discernable by naked eye due to their bright green appearance
as a result of reduced or dramatically altered composition of surface
wax. Thus, a genetic approach is an excellent strategy for the discovery
of wax-related genes that we have successfully employed in my laboratory
over the past decade. 
Several Arabidopsis cer mutants show stem wax phenotypes with reduced alkane levels, suggesting defects in genes coding for enzymes required for the formation of alkanes, key components of cuticular wax. The sequence of biosynthetic steps leading from very long chain fatty acid precursors to alkanes is currently unknown and represents the last fundamental element of wax biosynthesis that remains to be elucidated (red box).
We are working on several of these mutants in order to fill this important gap in our understanding of wax biosynthesis. In addition, alkane biosynthetic genes identified in these mutant lines are valuable tools for metabolic engineering of alkane production in plants and microorganisms. Alkanes are important constituents of high-energy fuels, gasoline and diesel, currently provided by crude fossil oil. The discovery of alkane biosynthetic genes is a prerequisite for a large-scale production of renewable high-efficiency transportation biofuels that can replace fossil fuels.
2. Analysis of CER7-mediated regulation of wax biosynthesis
This research builds on our previous work on the wax-deficient cer7 mutant which resulted in a recent discovery of the CER7 ribonuclease involved in the regulation of cuticular wax biosynthesis in epidermal cells. 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.
To identify the putative repressor, as well as additional protein components downstream of CER7, we are pursuing two different approaches:
Genetic screen for suppressors of the cer7 glossy phenotype
The suppressor screen in the cer7 mutant background generated a number of wax restorer (war) mutants with mutations in genes distinct from CER7 that fall into two general classes: class 1 which includes waxy plants with wild type CER3 transcript levels, and class 2 which includes waxy plants with reduced, cer7-like levels of the CER3 transcript. Characterization of these mutants is currently in progress.
Transcriptional profiling using microarrays
Genes whose expression is directly or indirectly controlled by the CER7 ribonuclease are expected to have altered transcript levels in the epidermis of cer7 mutants in comparison with the wild type. We have used the Affymetrix gene chip microarrays to detect such genes, and are using reverse genetic approaches (T-DNA insertional mutants, RNAi) to confirm their involvement in wax formation.