Isolation of mutants deficient in expression of a particular gene (T-DNA or transposon knockouts, or by RNAi) is a powerful tool to understand gene function but may initially be quite uninformative. For example, the lacs2 mutant was at first puzzling, but ultimately provided the phenotypes that allowed us to recognize that LACS2 is essential for cuticle development. For LACS6 and LACS7, the single mutants were indistinguishable from wild type under a range of conditions, so knowledge of their overlapping substrate specificities (and their colocalization to the peroxisome) was key to our decision to make the double mutant. Overlapping functions are also an issue with the 4CL genes and it may be a common problem with the Coenzyme A (CoA) ligases and also with the CoA thioesterases. It would be anticipated that very closely related genes are more likely to encode enzymes with overlapping functions, and our experience with the lacs6/lacs7 single and double mutants supports this. There are 7-10 closely paired genes among the CoA ligases, and four pairs among the 15 CoA thioesterases. In some cases, the pairs are tightly linked indicating relatively recent gene duplications. This genomic information will guide us in the choice of which multiple mutants to generate in this project. Overexpression of our genes may also perturb metabolism and generate informative phenotypes, and we will explore this possibility also, as circumstances indicate.
An alternative to producing double mutants is to construct hairpin antisense constructs against both genes and to transform these as a single construct into wild-type plants to generate RNAi lines. RNAi becomes an attractive approach when we contemplate the possibility of reducing the expression of three or more genes in a single line. RNAi is also valuable in situations where knockout (null) alleles are lethal.