The impact of the GLOSSY2 and GLOSSY2-LIKE BAHD-proteins in affecting the product profile of the maize fatty acid elongase

The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic 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 haploid Saccharomyces cerevisiae strains 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

Effects of trans-acting Genetic Modifiers on Meiotic Recombination Across the a1–sh2 Interval of Maize

Meiotic recombination rates are potentially affected by cis- and trans-acting factors, i.e., genotype-specific modifiers that do or do not reside in the recombining interval, respectively. Effects of trans modifiers on recombination across the ∼140-kb maize a1–sh2 interval of chromosome 3L were studied in the absence of polymorphic cis factors in three genetically diverse backgrounds into which a sequence-identical a1–sh2 interval had been introgressed. Genetic distances across a1–sh2 varied twofold among genetic backgrounds. Although the existence of regions exhibiting high and low rates of recombination (hot and cold spots, respectively) was conserved across backgrounds, the absolute rates of recombination in these sequence-identical regions differed significantly among backgrounds. In addition, an intergenic hot spot had a higher rate of recombination as compared to the genome average rate of recombination in one background and not in another. Recombination rates across two genetic intervals on chromosome 1 did not exhibit the same relationships among backgrounds as was observed in a1–sh2. This suggests that at least some detected trans-acting factors do not equally affect recombination across the genome. This study establishes that trans modifier(s) polymorphic among genetic backgrounds can increase and decrease recombination in both genic and intergenic regions over relatively small genetic and physical intervals

Methods development parameters.

<p>Silk tissues were extracted with one of five different solvents for a defined period of time. Solvents are presented in order of increasing polarity, as denoted by the dielectric constants provided in brackets.</p

Evaluation of extracellular surface lipids from wildtype (<i>Gl</i>) and <i>glossy1</i> (<i>gl1</i>) seedlings.

<p>A. The water adherence phenotype in <i>gl1</i> seedlings impaired in surface lipid deposition. B. Extracellular surface lipids (fatty acids, aldehydes and alcohols) extracted from wildtype and <i>gl1</i> seedlings, using chloroform or hexanes:diethyl ether (90:10) solvent systems. C. Individual alcohol constituents of different carbon-chain lengths, extracted from wildtype seedlings with hexanes:diethyl ether (90:10) or chloroform. D. Individual alcohol constituents of different carbon-chain lengths, extracted from <i>gl1</i> seedlings with hexanes:diethyl ether (90:10) or chloroform. Asterisks denote statistically significant differences between different solvents in the recovery of specific constituents, at p<0.05, according to a Tukey’s HSD test. A minimum of two replicates were used per combination of genotype and solvent type, with an N = 13. Averages ± SE are reported.</p

Comparison of lipid recovery from lyophilized, powderized silks extracted with different solvents.

<p>A. Total extracted lipid recovery (including hydrocarbons, fatty acids, aldehydes) from lyophilized, powderized emerged silks of inbred B73 extracted for ten minutes with the specified solvents: hexanes, hexanes:diethyl ether (95:5 and 90:10), chloroform:hexanes (1:1) and chloroform. B. Relative abundances of the observed lipid classes, hydrocarbons and fatty acids. Aldehydes were not detected above the limit of quantitation in this experiment. C. Relative abundances (%) of saturated (alkanes) and unsaturated (alkenes) relative to total hydrocarbon recovery. D. Relative abundances (%) of unsaturated 18-carbon fatty acids relative to total fatty acid recovery. Different lowercase letters above or within data-bars for a specific lipid class or constituent denote statistically significant differences in recovery with different solvents, at p<0.05, according to a Tukey’s HSD test. In panel A, uppercase letters denote statistically significant differences among total extracellular surface lipid recoveries. A minimum of four replicates were used per solvent, with an N = 28. Averages ± SE are reported.</p

Optimization of extraction time for the recovery of extracellular surface lipids from silks.

<p>Recovery of total extracellular surface lipids (A), hydrocarbons (B), and fatty acids (C) from emerged maize silks of inbred B73 extracted for different periods of time with hexanes:diethyl ether (90:10), chloroform or hexanes. Aldehydes comprised <1% of observed metabolites, regardless of extraction period and therefore are not shown. The asterisks denote statistically significant differences in recoveries with different solvents, at p<0.05, according to a Tukey’s HSD test. In panels A and C, different letters above data-bars denote statistically significant differences in recoveries in response to time of extraction with a specific solvent, at p<0.05. No such differences were observed in the data presented in panel B. A minimum of three replicates were used per combination of extraction time and solvent type, with an N = 67. Averages ± SE are reported.</p

Optimization of extraction times for recovery of extracellular surface lipids from seedlings.

<p>Recovery of hydrocarbons (A), fatty acids (B), aldehydes (C), and alcohols (D) from seedling leaves of inbred B73 treated with hexanes:diethyl ether (90:10) or chloroform for different extraction times. Different letters above data-bars in panel B denote statistically significant differences in recoveries in response to time of extraction, at p<0.05, according to a Tukey’s HSD test. No such differences were observed for the data presented in panels A, C and D. Four replicates were used per combination of extraction period and solvent type, with an N = 48. Averages ± SE are reported.</p