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The maizeglossy2andglossy2-likegenes are homologs, which encode proteins that belong to the BAHD family of acyltransferases.In plantagenetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploidSaccharomyces cerevisiaestrains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.

 

Abstract Eukaryotes express a multi-component fatty acid elongase to produce very long chain fatty acids (VLCFAs), which are building blocks of diverse lipids. Elongation is achieved by cyclical iteration of four reactions, the first of which generates a new carbon–carbon bond, elongating the acyl-chain. This reaction is catalyzed by either ELONGATION DEFECTIVE LIKE (ELO) or 3-ketoacyl-CoA synthase (KCS) enzymes. Whereas plants express both ELO and KCS enzymes, other eukaryotes express only ELOs. We explored the Zea mays KCS enzymatic redundancies by expressing each of the 26 isozymes in yeast strains that lacked endogenous ELO isozymes. Expression of the 26 maize KCS isozymes in wild-type, scelo2 or scelo3 single mutants did not affect VLCFA profiles. However, a complementation screen of each of the 26 KCS isozymes revealed five that were capable of complementing the synthetically lethal scelo2; scelo3 double mutant. These rescued strains express novel VLCFA profiles reflecting the different catalytic capabilities of the KCS isozymes. These novel strains offer a platform to explore the relationship between VLCFA profiles and cellular physiology. 

We demonstrate two synthetic single-cell systems that can be used to better understand how the acquisition of an orphan gene can affect complex phenotypes. The Arabidopsis orphan gene,Qua-Quine Starch(QQS) has been identified as a regulator of carbon (C) and nitrogen (N) partitioning across multiple plant species.QQSmodulates this important biotechnological trait by replacing NF-YB (Nuclear Factor Y, subunit B) in its interaction with NF-YC. In this study, we expand on these prior findings by developingChlamydomonas reinhardtiiandSaccharomyces cerevisiaestrains, to refactor the functional interactions between QQS and NF-Y subunits to affect modulations in C and N allocation. Expression ofQQSinC. reinhardtiimodulates C (i.e., starch) and N (i.e., protein) allocation by affecting interactions between NF-YC and NF-YB subunits. Studies inS. cerevisiaerevealed similar functional interactions between QQS and the NF-YC homolog (HAP5), modulating C (i.e., glycogen) and N (i.e., protein) allocation. However, inS. cerevisiaeboth the NF-YA (HAP2) and NF-YB (HAP3) homologs appear to have redundant functions to enable QQS and HAP5 to affect C and N allocation. The genetically tractable systems that developed herein exhibit the plasticity to modulate highly complex phenotypes.