Two putative fatty acid synthetic genes of BgFas3 and BgElo1 are responsible for respiratory waterproofing in Blattella germanica

Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investigated, we know little about other mechanisms for preventing water loss in insects. Here, we report two fatty acid synthetic genes that are independent of hydrocarbon production but crucial for water retention in the German cockroach Blattella germanica (L.). First, an integument enriched fatty acid elongase gene (BgElo1) was identified as a critical gene for desiccation resistance in B. germanica;however, knockdown of BgElo1 surprisingly failed to cause a decline in cuticular lipids. In addition, RNA interference (RNAi)-knockdown of an upstream fatty acid synthase gene (BgFas3) showed a similar phenotype, and transmission electron microscopy analysis revealed that BgFas3- or BgElo1-RNAi did not affect cuticle architecture. Bodyweight loss test showed that repression of BgFas3 and BgElo1 significantly increased the weight loss rate, but the difference disappeared when the respiration was closed by freeze killing the cockroaches. A water immersion test was performed, and we found that BgFas3- and BgElo1-RNAi made it difficult for cockroaches to recover from drowning, which was supported by the upregulation of hypoxia-related genes after a 10-h recovery from drowning. Moreover, a dyeing assay with water-soluble Eosin Y showed that this was caused by the entry of water into the respiratory system. Our research suggests that BgFas3 and BgElo1 are required for both inward and outward waterproofing of the respiratory system. This study benefits the understanding of water retention mechanisms in insects.

Identification of KCS gene family and functional analysis of FAE-like genes from Malania oleifera

KCS(3-ketoacyl-CoA synthase)is the key enzyme catalyzing the first step of very long chain fatty acid(VLCFA)biosynthesis.Studies showed that different KCSs possessed different substrate preference.Malania oleifera are abundance of VLCFAs in its mature seeds,especially the nervonic acid,which is essential for human health.In this study,we identified and characterized 18 KCS genes in M.oleifera genome.Phylogenetic analysis showed that these KCS genes were classified into four subfamilies,including two FAE-like,six KCS-like,eight FDH-like and two CER6.We concentrated on the functional role of two FAE-like genes,Maole003085.T1 and Maole004215.T1 which encoded predicted amino acid residues of 516 and 518 in protein,respectively.Multiple sequence alignment showed that their two proteins contained the known and conserved active sites among FAE-like subfamily.Upon heterologous expression in wild type yeast(Saccharomyces cerevisiae)INVSc1,we found that Maole004215.T1 could produce four new fatty acids including C22:0/C22:1 and C24:0/C24:1,but Maole003085.T1 only produced C22:1.Besides,upon heterologous expression in mutant yeast BY4741-△elo3,we found the Maole003085.T1 could produce C24:0 and C26:0,while the Maole004215.T1 could catalyze the formation of fatty acids C24:0,C26:0 and C28:0.These results showed Maole003085.T1 and Maole004215.T1 had fatty acid elongation activity in yeast,and possessed different substrate preference in the production of different VLCFAs.Interestingly,we found Maole004215.T1 could produce nervonic acid in yeast,which provides molecular basis on the genetic improvement and genic engineering for producing nervonic acid resources by using biotechnological methods.

HOS3, an ELO-Like Gene, Inhibits Effects of ABA and Implicates a S-1-P/Ceramide Control System for Abiotic Stress Responses in Arabidopsis thaliana

A hyper-osmotically sensitive mutant of Arabidopsis thaliana, designated hos3-1 （high expression of osmotically responsive genes）, was identified based on its hyper-luminescence of RD29A：LUC promoter fusion plants upon treatment with NaCI and ABA. These responses implicate the disrupted gene as a direct or indirect negative regulator of the RD29A stress-responsive pathway. By sequencing the flanking regions of the T-DNA borders, it was determined that the disrupted gene is at locus At4g36830, annotated as encoding a putative protein with high homology to CIG30 （ELO2/FEN1）. CIG30 has been implicated in synthesis of very long chain fatty acids （VLCFA）, which are essential precursors for sphingolipids and ceramides. Altered stress responses characteristic of ABA-hypersensitivity, including reduced root growth inhibition and reduced germination with ABA treatment and reduced water loss from leaves, were exhibited by allelic hos3-1 and hos3-2 mutants. The hos3-2 mutant is partially suppressed in its transcript abundance and is inherited as a recessive trait. Further, the HOS30RF under the control of the 35SCaMV promoter restored wild-type NaCI- and ABA-root growth sensitivity as well as RD29A：LUC luminescence in mutant plants. We also show here that the HOS3 wild-type gene functionally complements the sensitivity of elo2 and elo3 yeast mutants to monensin. Furthermore, both hos3-1 and hos3-2 alleles shared increased sensitivity to the herbicide Metolachlor, which inhibits acyl chain elongation in synthesis of VLCFA, and HOS3 functionally complemented both elo2 and elo3 and restored levels of VLCFA. Together, these data establish that HOS3 inhibits ABA-mediated stress responses and implicate the VLCFA pathway and products as control points for several aspects of abiotic stress signaling and responses. The results also provide support for a role of ceramide in the control of stomatal behavior.

Genome-scale analysis of the cotton KCS gene family revealed a binary mode of action for gibberellin A regulated fiber growth

Production of b-ketoacyl-Co A, which is catalyzed by 3-ketoacyl-CoA synthase（KCS）, is the first step in very long chain fatty acid（VLCFA） biosynthesis. Here we identified 58 KCS genes from Gossypium hirsutum, 31 from G. arboreum and 33 from G. raimondii by searching the assembled cotton genomes. The gene family was divided into the plant-specific FAE1-type and the more general ELO-type. KCS transcripts were widely expressed and 32 of them showed distinct subgenome-specific expressions in one or more cotton tissues/organs studied. Six Gh KCS genes rescued the lethality of elo2Δelo3Δ yeast double mutant,indicating that this gene family possesses diversified functions.Most KCS genes with GA-responsive elements（GAREs） in the promoters were significantly upregulated by gibberellin A_3（GA）.Exogenous GA_3 not only promoted fiber length, but also increased the thickness of cell walls significantly. GAREs present also in the promoters of several cellulose synthase（CesA） genes required for cell wall biosynthesis and they were all induced significantly by GA_3. Because GA treatment resulted in longer cotton fibers with thicker cell walls and higher dry weight per unit cell length, we suggest that it may regulate fiber elongation upstream of the VLCFA-ethylene pathway and also in the downstream steps towards cell wall synthesis.