LPIAT, a lyso-Phosphatidylinositol Acyltransferase, Modulates Seed Germination in Arabidopsis thaliana through PIP Signalling Pathways and is Involved in Hyperosmotic Response

Abstract

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination.

Keywords: ABA; Acyltransferase; aleurone-like cells; hyperosmotic stress; phosphoinositides; seed germination.

Figure 1

Determination of the lyso-phospholipid specificity. Labelled-phospholipids were synthesized by in vitro acylation of lyso-phospholipids, catalysed with membranes from E. coli transformed by pET-15b::LPIAT plasmid (100 µg protein/assay) corrected by the amount of labelled phospholipids obtained with membranes transformed by empty pET15b. Assays were carried out with 3 nmol of [¹⁴C]-C18:1-CoA and 2 nmol of lyso-phospholipids. After 60min incubation, lipids were extracted and analysed by thin-layer chromatography (TLC) followed by radioimaging. Values represent mean ± SD of three biological replicates. LPC, lyso-phosphatidylcholine; LPS, lyso-phosphatidylserine; LPA, lyso-phosphatidic acid; LPI, lyso-phosphatidylinositol; LPE, lyso-phosphatidylethanolamine; LPG, lyso-phosphatidylglycerol.

Figure 2

Determination of the acyl-CoA specificity. In vitro acylation of lyso-phosphatidylinositol was catalysed with membranes from E. coli transformed by pET-15b::LPIAT plasmids (10 µg protein/assay) corrected by the amount of labelled phospholipids obtained with membranes transformed by empty pET15b. Assays were carried out with 2 nmol of [¹⁴C]-C18:1-CoA, 2 nmol unlabelled acyl-CoA and 4 nmol of LPI. After 10min incubation, lipids were extracted and analysed by TLC using chloroform/methanol/1-propanol/methyl acetate/0.25% aqueous KCl (10:4:10:10:3.6, v/v) as solvent followed by radioimaging. Values represent mean ± SD of 3 biological replicates with two technical replicates. The dashed line represents the theoretical level of labelled PI reached in presence of 2 nmol [¹⁴C]-C18:1-CoA and 2 nmol C18:1-CoA.

Figure 3

Determination of the regiospecificity of acyltransferase activity catalysed by LPIAT on lyso-phosphatidylinositol. Microsomal membrane proteins (20 or 40 µg) from E. coli transformed by empty pET15b or pET-15b::LPIAT plasmids were incubated with [¹⁴C]-palmitoyl-CoA (1 nmol) in the absence or in the presence of 1-lyso 2-acyl PI or 2-lyso 1-acyl PI (1 nmol). After 10 min of incubation, lipids were extracted and analysed by TLC followed by radioimaging. Results are from one experiment representative of three experiments performed with independent microsome preparations.

Figure 4

Spatial expression patterns of LPIAT in transgenic Arabidopsis harbouring the LPIAT promoter fused to the β-glucuronidase (GUS) gene. Promoter activity was visualized by histochemical GUS staining on (a) dry seed, (b) imbibed seed, (c) 2-day-old seedling, (d) 4-day-old seedling, (e) 9-day-old seedling, (f) cauline leaves and inflorescence, (g) flower. Scale bar: 100 µm. Arrow heads in (a) and (b) show cells from the aleurone-like layer.

Figure 5

Impact of LPIAT on lipid metabolism. (A), Relative quantification of LPIAT expression in wt, lpiat, LPIATOx (overexpression in wild-type background) and lpiat:LPIATOx (overexpression in knock-out background) in leaves from 1 week-old plants, normalized to two reference genes (actin and eIF4A-1). Relative expression quantities are represented related to wild type level at 1 week, which was set to one. Values represent mean ± SD of three technical replicates for two biological replicates (black and white boxes). (B), Phospholipid analysis of wt, lpiat or overexpressing leaves. Lipids from leaves from 3 week-old plants were quantified by GC-FID after transesterification. Values represent mean ± SD for four biological replicates. (C), Phosphatidylinositol (PI) fatty acid composition (in % of total FA). Values represent mean ± SD for four biological replicates. (D), Effect of LPIAT overexpression on the molecular fatty acid composition of PIP (top panel) and PIP2 (down panel) in leaves. Phosphoinositides were extracted from leaves from 3-week-old plants by acidic extraction and analysed by LC MS/MS after derivatization. Values represent mean ± SD for five biological replicates. Statistically significant differences from the wild type are indicated by * p < 0.05 or ** p < 0.01 as determined by Wilcoxon’s-test.

Figure 6

Germination and growth phenotype. (A), Seed germination in Col0, lpiat, LPIATOx and lpiat:LPIATOx lines. After 3 days stratification, seeds were germinated on 1/2 MS medium, 0.8% agar and 1% Sucrose. About 100 seeds were sowed by plates. % germination was estimated by plates as judged by radical extrusion. Values are means ± SD (n = 3). (B), Hypocotyl length from 3-day-old dark-grown seedlings from wild-type and the indicated LPIAT mutant lines. Seeds were germinated and grown in the dark for 3 days on 1/2 MS medium, 0.8% agar and 1% Sucrose. (C), Primary roots were measured after 3 days. Values are means ± SD (n = 60) from one of three independent experiments that gave similar results. Wilcoxon’s test comparisons between altered-LPIAT expression level mutants and the wild type were performed. ***, p-value was < 0.005; **, p-value was < 0.01.

Figure 7

ABA-related modifications in seed germination and drought stress resistance in LPIAT mutants. (A), Germination rate from wild-type and the indicated LPIAT mutant seeds. Seeds were germinated for 29 h on 1/2 MS medium, 0.8% agar and 1% Sucrose. The medium was supplemented with various ABA concentrations. Values are means ± SD of 4 independent experiments. (B), Effect of a drought stress on survival rate of the different LPIAT mutants grown on a medium supplemented with ABA before the stress. Seeds were sown on filter laying on ½ MS medium, 0.8% agar and 1% Sucrose. After 3 days stratification at 4 °C, filters were transferred on the same medium supplemented or not by 5 µM ABA for 10 days. Filters were removed from medium and dried at room temperature for 6 h. Then, they were deposited on ½ MS medium, 0.8% agar and 1% Sucrose; Survivals were counted after 4 days. All the seedlings that were not initially transferred on medium supplemented by 5µM ABA died after the drought stress (not shown). Values are means ± SD of 5 independent experiments.

Figure 8

Germination efficiency of LPIAT mutants under osmotic/salinity stress. (A) Germination rate of wild type, lpiat, LPIATOx and lpiat:LPIATOx lines on MS media. (B) Germination rate on MS media containing 0.2 M NaCl. (C) Germination rate on MS media containing 0.4 M mannitol. Values are means ± SD of three independent experiments.