Tackling functional redundancy of Arabidopsis fatty acid elongase complexes

Very-long-chain fatty acids (VLCFA) are precursors for various lipids playing important physiological and structural roles in plants. Throughout plant tissues, VLCFA are present in multiple lipid classes essential for membrane homeostasis, and also stored in triacylglycerols. VLCFA and their derivatives are also highly abundant in lipid barriers, such as cuticular waxes in aerial epidermal cells and suberin monomers in roots. VLCFA are produced by the fatty acid elongase (FAE), which is an integral endoplasmic reticulum membrane multi-enzymatic complex consisting of four core enzymes. The 3-ketoacyl-CoA synthase (KCS) catalyzes the first reaction of the elongation and determines the chain-length substrate specificity of each elongation cycle, whereas the other three enzymes have broad substrate specificities and are shared by all FAE complexes. Consistent with the co-existence of multiple FAE complexes, performing sequential and/or parallel reactions to produce the broad chain-length-range of VLCFA found in plants, twenty-one KCS genes have been identified in the genome of Arabidopsis thaliana. Using CRISPR-Cas9 technology, we established an expression platform to reconstitute the different Arabidopsis FAE complexes in yeast. The VLCFA produced in these yeast strains were analyzed in detail to characterize the substrate specificity of all KCS candidates. Additionally, Arabidopsis candidate proteins were transiently expressed in Nicotiana benthamiana leaves to explore their activity and localization in planta. This work sheds light on the genetic and biochemical redundancy of fatty acid elongation in plants

The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual TDNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity

Suspensions cellulaires embryogènes de bananiers et bananiers plantain

This guideline presents two protocols to produce embryogenic cell suspensions by using scalps or immature male flowers. The protocols have been developed by the Laboratory of Tropical Crop Improvement of Katholieke Universiteit Leuven (KULeuven) and the cellular biology laboratory of the Centre de coopération internationale en recherche agronomique pour le développement (Cirad)

Banana and plantain embryogenic cell suspensions

This guideline presents two protocols to produce embryogenic cell suspensions by using scalps or immature male flowers. The protocols have been developed by the Laboratory of Tropical Crop Improvement of Katholieke Universiteit Leuven (KULeuven) and the cellular biology laboratory of the Centre de coopération internationale en recherche agronomique pour le développement (Cirad)

Suspensiones de células embriogénicas de banano y plátano

This guideline presents two protocols to produce embryogenic cell suspensions by using scalps or immature male flowers. The protocols have been developed by the Laboratory of Tropical Crop Improvement of Katholieke Universiteit Leuven (KULeuven) and the cellular biology laboratory of the Centre de coopération internationale en recherche agronomique pour le développement (Cirad)

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1 upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance