DataSheet_1_Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.pdf

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. </p

Table_2_Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.xlsx

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. </p

Table_1_Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.xlsx

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. </p

Table_3_Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.xlsx

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. </p

DataSheet_2_Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.pdf

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. </p

Overall lipid compositions according to GC analyses.

<p>Class of neutral lipids (A) and types of fatty acyl-chains (B) were quantified and compared between the 8 samples according to GC analyses. Amounts are given in % of total. IBA+2MBA, relative amount of isobutyric and 2-methylbutyric acids; IVA, relative amount of isovaleric acid; ω3 FA, relative amount of omega3-fatty acids; Iso FA, relative amount of isobranched fatty acyl-chains; Linear FA, relative amount of linear fatty acyl-chains.</p

Assignment of the 24 major peaks detected by HR-MAS NMR in the blubber and melon of the harbour porpoise and long-finned pilot whale, and calculations used to determine peak area.

<p>Assignment of the 24 major peaks detected by HR-MAS NMR in the blubber and melon of the harbour porpoise and long-finned pilot whale, and calculations used to determine peak area.</p

Representative ¹H HR-MAS spectra of the samples.

<p>Intact tissues were placed in a zirconium oxide MAS rotor, D₂O was added for ²H field locking and ¹H HR-MAS NMR spectra were acquired at room temperature (spinning speed = 5000 Hz and ns = 64). The assignment of peaks <i>a</i> to <i>x</i> is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180597#pone.0180597.t001" target="_blank">Table 1</a>.</p

Overall lipid compositions according to HR-MAS NMR analyses.

<p>Class of neutral lipids (A) and types of fatty acyl-chains (B) were quantified and compared between the 8 samples according to HR-MAS NMR analyses. Amounts are given in % of total. IBA+2MBA, relative amount of isobutyric and 2-methylbutyric acids; IVA, relative amount of isovaleric acid; ω3 FA, relative amount of omega3-fatty acids; Iso FA, relative amount of isobranched fatty acyl-chains; Linear FA, relative amount of linear fatty acyl-chains.</p

WE profiling of the central melon and outer blubber from the long-finned pilot whale.

<p>WE from the central melon (A) and outer blubber (B) were purified by TLC before analysis by GC-MS. Note the different retention time windows between panel A and B. Identification of the major WE molecular species is provided in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180597#pone.0180597.g003" target="_blank">Fig 3</a>.</p