Dual Fatty Acid Elongase Complex Interactions in Arabidopsis

Abstract

Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting.

Fig 1. A. thaliana PTPLA complements inducible yeast phs1 mutant.

(A) PTPLA expression restores growth of Tet-PHS1 in presence of DOX. Tet-PHS1 was transformed with yeast expression vector pFL61 alone or with yeast PHS1, Arabidopsis PAS2 or PTPLA. R1158 is the wild type control strain. (B) Growth kinetic of Tet-PHS1 strain expressing PHS1, PAS2 and PTPLA in presence of DOX. Three independent PTPLA expressing clones were analyzed and the mean (+/- sd) is shown. (C-D) Fatty acid content of PTPLA expressing yeasts. PTPLA expression induces fatty acid elongation in yeast Tet-PHS1 in presence of DOX (C) and in wild type strain (D). The graph shows FAMES analysis from n = 5–12 and n = 9–15 independent clones for respectively (C) and (D). Data shows means (+/- se). Significant differences between Tet-PHS1 (C) or the wild-type (D) and overexpressing strains were determined using the Wilcoxon-test: *p<0,05, **p<0,01, ***p < 0.001.

Fig 2. PTPLA expression enhances VLCFA contents in A. thaliana.

Relative fatty acid content of pas2-1 mutant (A and B) and Col0 (C and D) lines expressing 3 pPAS2:PTPLA independent constructs or the control pPAS2:PAS2. FAMES analysis were performed on roots of 14 days-old seedling (n = 3). (B and D) VLC/LCFA ratio shows means (+/- sd) of the ratio between very long chain (C20 to C26) and the long fatty acids (C16 and C18). Significant differences between pas2-1 (A) or Col0 (B) and overexpressing lines were determined using the student’s t-test: *p<0,05, **p<0,01, ***p < 0.001.

Fig 3. PTPLA is expressed during root development.

(A-T) Expression of GUS constructs under the control of pPTPLA (A-E), pPAS2 (F-J), pKCR1 (K-O) or pKCR2 promoters (P-T) in 14 days-old Arabidopsis seedlings. GUS staining was observed in different organs: seedling apical part (A, F, K, P), hypocotyl/root transition (B, G, L,Q), root hair transition zone of the primary root (C, H, M, R), secondary root (D, I, N, S) and primary root tip (E, J, O, T). Scale: 50 μm (except pictures A, F, K, P: 2mm). n = 18. (U). Coexpression of pPTPLA:mRFP1 and pPAS2:GFP in A. thaliana primary root. Images were taken from the tip (R1), the root hair initiation zone (R2), the lateral root initiation zone and lateral root emergence zone (R4). Scale: 50μm. (V) Effect of ptpla mutation of primary root length. Vertically grown 10 days-old seedling (top) and the corresponding primary root length (bottom). n = 43–57. Scale: 500μm. Significant differences were determined using the student’s t-test: *p<0,05, **p<0,01, ***p < 0.001.

Fig 4. PTPLA interacts with the elongase complex subunits in the ER.

(A-C) Subcellular distribution in N.benthamiana epidermal cells of 35S:mcherry-PTPLA (A) and pPAS2:GFP-PAS2 (B). Merged channels showed colocalization (C). Chloroplast autofluorescence is shown in blue. Scale: 50μm. (D-I). BiFC assays between PTPLA and subunits of the elongase complex in the ER. Scale: 25μm. (J) Results of BiFC assays between PTPLA and several enzymes of the elongase complex. The plus sign (+) indicates an interaction, the minus sign (-) no interaction. Root Exp.: Root expression of the proteins or genes when is known [1,4,5,31] is indicated by “Yes”. The question mark indicates unclear information about the KCS expression. “No” indicates no or unknown expression of the protein in the root.

Fig 5. PTPLA is involved in very long chain fatty acids elongation.

(A) 3-hydroxy-acyl-CoA profile of pas2-1 and ptpla mutant roots compared to wild type. n = 4. Significant differences were determined using the Wilcoxon-test: *p<0,05, **p<0,01, ***p < 0.001. (B) Three independent experiments showing the VLC/LCFA ratio in pas2-1 and ptpla mutant roots compared to wild type. Three independent ptpla mutant lines expressing pPTPLA:PTPLA were used for comparison in the second and third experiments. n = 3. (C) VLC/LCFA ratio in pas2-1 and pas2-1/ptpla mutants. n = 3. (D) VLCFA levels in three independent kcr2 mutant liness compared to wild type. n = 3. Significant differences were determined using the student’s t-test: *p<0,05, **p<0,01, ***p < 0.001.

Fig 6. Model of PTPLA mode of action.

PTPLA encodes a new dehydratase of the elongase complex specifically localized in the pericycle and vascular tissues that impact the elongase activity in the root endodermis. EC1: PAS2-associated elongase complex, EC2: PTPLA-associated elongase complex. KCS: 3-ketoacyl-CoA synthase. KCR: β-ketoacyl-CoA reductase. ECR: enoyl-CoA reductase. Green thickening: suberin. Blue thickening: vascular tissues. Red band: Caspary band. Red dotted line: non-cell autonomous signal.