Concerted transport and phosphorylation of diacylglycerol at ER-PM contact sites regulate phospholipid dynamics during stress

Abstract

A universal response of plants to environmental stresses is the activation of plasma membrane (PM) phospholipase C, which hydrolyzes phosphoinositides to produce soluble inositol phosphate and diacylglycerol (DAG). Because of their conical shape, DAG amounts have to be tightly regulated or they can destabilize membranes. We previously showed that upon stress, Synaptotagmin1 (SYT1) transports DAG from the PM to the endoplasmic reticulum (ER) at ER-PM Contact Sites (CS). Here, we addressed the fate of the incoming DAG in the ER. We show that diacylglycerol kinases (DGKs) DGK1 and DGK2 form a module with SYT1 functionally coupling DAG transport and phosphorylation at ER-PM CS. Although SYT1 and DGK1/DGK2 do not show exclusive ER-PM CS localization, their interaction occurs specifically at ER-PM CS and the removal of ER-PM CS abolishes the interaction. Lipidomic analysis of a dgk1dgk2 double mutant supports that DGK1 and DGK2 phosphorylate DAG at the ER and transcriptomic and phenotypic analyses indicate that SYT1 and DGK1/DGK2 are functionally related. Taken together, our results highlight a mechanism at ER-PM CS that coordinates the transfer of DAG from the PM to the ER by SYT1 upon stress and the concomitant phosphorylation of DAG by DGK1 and DGK2 at the ER. These findings underscore the critical role of spatial coordination in lipid metabolism during stress-induced membrane remodeling.

Keywords: DAG; PI cycle; abiotic stress; contact sites; signaling.

Fig. 1.

DGK1 and DGK2 interact with SYT1, share a similar structure, and show an ER localization. (A) SYT1 TurboID identifies DGK1 as one of the main putative interactors of SYT1, along with SYT5 and CLB1, whose interactions with SYT1 have already been described (37, 38). The bubble plot shows the enrichment score and Normalized Spectral Abundance Factor ratio of each protein. −log(P-value) is represented by the circle size. (B) A GFP pull-down assay shows that DGK1 and DGK2 coimmunoprecipitate SYT1. Proteins were transiently expressed in N. benthamiana, and tissue was harvested 2 d postinfiltration. Proteins tagged with GFP were IP using GFP Trap beads. Total (Input) and IP proteins were separated by SDS-PAGE. DGK2-GFP and DGK1-GFP were detected with a GFP antibody and SYT1-RFP was 1 detected using an anti-RFP antibody. Uniform sample loading was verified by Coomassie blue staining (CBB) on the input samples. (C) DGK1 coimmunoprecipitates DGK2. The GFP pull-down assay was performed as in (A) using DGK1-GFP and DGK2-RFP. (D) Schematic representation of SYT1, DGK1, and their truncated protein versions using in the FRET analysis. DGK1, DGK1ΔAcc, DGK1 C1s, SYT1, and SYT1ΔC2AB were tagged with both RFP and GFP at the C-terminus and all combinations were used in the FRET analysis. (E) FRET assays using the full-length and the truncated versions of SYT1, DGK1, and DGK2 (A). Protein pairs were coexpressed in N. benthamiana leaves and analyzed at 2 d postinfiltration. The protein pairs shown in the graph are labeled with the first protein fused to GFP and the second fused to RFP. RFP-tagged proteins were photobleached, and the intensity of the GFP-tagged proteins was quantified. The percentage increase in GFP intensity was calculated using the following formula: [(IAfter − IBefore)/IAfter] × 100, where IBefore and IAfter represent the means of the intensity from six measurements taken before and after photobleaching, respectively. The statistical analysis using one-way ANOVA and Tukey multiple comparisons is shown. (F) BiFC assay was performed in N. benthamiana by coexpressing pairs of proteins. DGK2-nYFP associates with SYT1-cYFP but not with SYT1ΔC2AB-cYFP, indicating a possible role of C2 domains in SYT1–DGK2 interaction. However, SYT1-nYFP is capable of associating with SYT1ΔC2AB-cYFP through the SMP domain. The scheme indicates that the images were taken at the cortical region of lower epidermis cells 2 d postinfiltration. (Scale bar, 10 µm.)

Fig. 2.

SYT1, DGK1, and DGK2 interact at ER–PM CS. (A) The ER–PM CS/ER ratio for DGK1 was quantified in plants coexpressing DGK2 with DGK1, SYT1, and MAPPER, and also for ER marker coexpressed with SYT1. ER–PM CS and ER segmentation was performed as described in (B). Error bars indicate SD. Letters indicate statistically significant differences using one-way ANOVA Tukey multiple comparisons, P < 0.05 (n > 20). DGK1 increased at the ER–PM CS when coexpressed with SYT1, but not when coexpressed with DGK2 or MAPPER. (B) MAPPER, SYT1, DGK2, and RTNLB6 tagged to GFP, and ER Marker-RFP (FaFAH) were infiltrated in N. benthamiana leaves. Images were taken 2 d post infiltration at the cortical region of lower epidermal cells. Two types of fluorescence patterns were distinguished: a more punctate pattern corresponding to the ER–PM CS and a more reticular pattern corresponding to the ER. MAPPER is characterized by the punctate pattern, ER marker, DGK2, and RTNLB6 by the reticular pattern, and SYT1 exhibits both of them. (C and D) The BiFC assay between SYT1-nYFP and DGK2-cYFP shows a punctate pattern, while the assay between SYT1-nYFP and RTNLB6-cYFP displays a reticular pattern. To distinguish between both, several images were segmented using the machine learning tool Ilastik (45) into: Background, ER–PM CS, ER, and overexposed regions where determining ERPM CS is not possible, and thus these regions are excluded from the ratio shown in (D). Area of each segmented ROI was measured with FIJI. Error bars indicate SD. Letters indicate statistically significant differences using one-way ANOVA Tukey multiple comparisons, P < 0.05 (n > 20). (E and F) SAC1 is a phosphatase that converts PI4P into PI at the PM. Since PI4P is crucial for the formation of ER–PM CS, SAC1 activity avoids the ER–PM CS formation. Coexpression of SYT1-nYFP/DGK2-cYFP and SYT1-nYFP/RTNLB6-cYFP results in BiFC fluorescence when coinfiltrated with an inactive version of SAC1 (SAC1 dead). However, when coexpressed with the active form of SAC1, SYT1-nYFP/DGK2-cYFP fluorescence disappears, indicating that the interaction of SYT1 and DGK2 depends on ER–PM CS formation.

Fig. 3.

dgk1dgk2 plants show higher DAG accumulation at the IM than WT plants after cold stress. (A) Lipid quantification of the molecular species of DAG in PM and IM using HR/AM. Fractions were isolated from 5-wk-old WT and dgk1dgk2 rosettes grown at control conditions followed by 3 d of cold treatment (4 °C). PM and IM samples were purified by two-phase partitioning protocol, and lipids were extracted as described in “Material and Methods.” Acyl chains are expressed as the number of acyl carbons: number of acyl double bonds. Distribution of the identified DAG molecular species in the PM and the IM of WT (black) and dgk1dgk2 (green) is represented. Column bars show the mean values of at least four biological replicates of MS signal % (pos/neg combined). To avoid polarity switching during a run, each sample replicate was run twice, once each in negative and positive mode. Lipids identified from the two runs were merged into a single, annotated dataset for that replicate. Error bars indicate the SEM. The Inset graph represents the sum of all molecular species of the specific lipid. The asterisks indicate statistically significant differences between dgk1dgk2 and WT as determined by a Dunnett's multiple comparisons test: **P < 0.0001; *P < 0.0002. (B and C) Cell viability quantification in 6-d-old Arabidopsis roots in control and after cold treatment. Seedlings were cultivated on a solidified 1/10 MS agar medium under long-day photoperiods at 23 °C. Then plants were subjected to a 30 min cold treatment (6 °C in prechilled 1/10 strength liquid MS). Cell viability was assessed using FDA staining, with fluorescence intensity quantified as the percentage of root area exceeding an automatic threshold set by the “Moments” algorithm in Fiji software. Each point represents a measurement from an individual ROI. Horizontal lines indicate the mean values. The experiment was repeated three times with consistent results. Letters indicate significant differences as determined by one-way ANOVA and Tukey's multiple comparisons test.

Fig. 4.

Global transcriptomic analyses indicate that SYT1 and DGK1/DGK2 regulate similar genes. RNAseq analysis was performed using the aerial parts of a pool of 2-wk-old plants (approximately 10 plants per biological replicate and at least three replicates per group) grown in soil under control conditions and subsequently cold-treated (4 °C) during 24 h. (A) Number of DEGs (q-value < 0.05) in dgk1dgk2 and syt1 compared to the WT. Up-regulated genes are represented in purple and down-regulated in green. (B) Venn diagram of DEGs in dgk1dgk2 and syt1 and their overlap. The percentage of shared genes are calculated relative to the number of total DEGs in the dgk1dgk2 mutant. The enrichment of the overlap is 55.2 and is calculated as the number of overlapping genes divided by the expected number of overlapping genes drawn from two independent groups. The expected genes are defined by the following formula: (no. of genes in dgk1dgk2 × no. of genes in syt1)/total genes analyzed in the whole genome. P-value of the enrichment is indicated. (C) Bubble plot of the GO terms from the 24 overlapping genes in panel (B). The fold enrichment of each category is shown on the X axis, while the FDR is represented by a color gradation from purple, lowest, to yellow, highest. The circle size corresponds to the number of genes involved in each GO term. Most of these GO terms are related abiotic and biotic stress responses. (D) WT, dgk1dgk2, syt1, and dgk1dgk2syt1 do not exhibit obvious phenotype differences when grown in standard conditions. Images show: 10-d-old seedlings grown on vertical plates; Rosettes of 3 and 5 wk grown in soil; and Internodes of 7-wk-old plants. Scale is indicated in the figure. (E and F) DGK1DGK2 and SYT1 are implicated in the same pathway related to freezing tolerance. Surviving plants of acclimated WT, dgk1dgk2, syt1, and dgk1dgk2syt1 after a freezing treatment 6 h at −9 °C and 1 wk of recovery under control conditions. Data represent mean ± SD (n > 20 plants per replicate) using one-way ANOVA and Tukey multiple comparisons.