Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism

Abstract

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.

Keywords: Arabidopsis thaliana; acyl-CoA-binding proteins; hypoxic tolerance; sphingolipids; very-long-chain fatty acids.

Figure 1

Expression profiles of Arabidopsis ACBPs under submergence.Total RNA was extracted from 4-week-old soil-grown seedlings upon dark and DS (a), or LS (b) treatment. The samples were harvested at 0, 3, 6, 9, 12 or 24 h after treatment, and the relative expression levels of Arabidopsis ACBPs (ACBP1 to ACBP6) were determined by qRT-PCR. Expression levels relative to 0 h for each time point were normalized to that of ACTIN2. Data are means ± SD of three independent replicates. Asterisks indicate significant difference from untreated control (0 h); *P < 0.05; **P < 0.01 by Student's t-test. The experiment was repeated with similar results.

Figure 2

Phenotypes of ACBP3-OEs and ACBP3-KOs to hypoxic stress. (a) Phenotypes of 4-week-old wild type (WT), ACBP3-OEs (OE-1 and OE-4), ACBP3-KOs (acbp3 and ACBP3-RNAi) plants before treatment (day 0) and after 2-day (for ACBP3-OEs) or 3-day (for ACBP3-KOs) DS treatment, followed by 3 days' recovery. (b, c) Survival rates (b) and dry weights (c) of WT, ACBP3-OEs and ACBP3-KOs after DS treatment followed by 3 days' recovery. (d) Phenotypes of 4-week-old WT, ACBP3-OEs, ACBP3-KOs before treatment (day 0) and after 5 days (for ACBP3-OEs) or 7 days (for ACBP3-KOs) of LS treatment, followed by 3 days' recovery. (e, f) Survival rates (e) and dry weights (f) of the WT, ACBP3-OEs and ACBP3-KOs after LS treatment followed by 3 days' recovery. Bars represent means ± SD (n = 3) of three independent experiments (for one experiment, >15 plants were scored for each genotype). Asterisks indicate significant differences from WT; **P < 0.01 by Student's t-test.

Figure 3

Expression of hypoxia marker genes in OE-1 and acbp3 after DS and LS treatments. Total RNA was isolated from 4-week-old WT, OE-1 and acbp3 at 0, 1, 3 and 6 h after DS (a) treatment or 6, 12 and 24 h after LS (b) treatment. Relative expression levels of ADH1, PDC1 and SUS1 were analyzed by normalizing to a WT sample at 0 h. Data are means ± SD of three independent replicates. Asterisks indicate significant differences from WT; *P < 0.05; **P < 0.01 by Student's t-test. The experiment was repeated with similar results.

Figure 4

H₂O₂ accumulation in ACBP3-OEs and ACBP3-KOs upon hypoxic stress. Rosettes of 4-week-old WT, ACBP3-OEs (OE-1 and OE-4), ACBP3-KOs (acbp3 and ACBP3-RNAi) before treatment (CK) and after LS (LS day 1, LS day 2 and LS day 3) or DS (DS day 1) treatments were collected and stained by DAB solution.

Figure 5

Effects of exogenous ethanol application on ACBP3-OEs and ACBP3-KOs. (a) Phenotypes of 4-week-old WT, ACBP3-OEs (OE-1 and OE-4) and ACBP3-KOs (acbp3 and ACBP3-RNAi) before treatment and at 5 days after spraying with either 0.5% ethanol or water as a control. (b) Relative chlorophyll contents of plants in (a) treated with ethanol or water control after 5 days. The chlorophyll contents of plants following ethanol treatment were expressed relative to the values of water treatment. Data are average of three samples from three independent plants. Asterisks indicate significant differences to WT; **P < 0.01 by Student's t-test. (c) Seeds of WT, ACBP3-OEs and ACBP3-KOs germinated on MS medium supplemented with 0 (MS), 50 or 75 mm ethanol. Images were taken 2 weeks after germination. (d) Statistical frequencies of seedlings in (c). The values in the columns correspond to seedlings with true leaves (1 and 2), seedlings with green (3) or brown (4) cotyledons, etiolated seedlings (5), and ungerminated seeds (6).

Figure 6

The hypoxia-sensitive phenotype of OE-1 is dependent on NPR1 and CTR1. (a) Phenotypes of 4-week-old WT, OE-1, OE-1 coi1, coi1-2, OE-1 npr1 and npr1-5 plants before treatment (day 0) and after 2-day DS or 8-day LS treatment followed by 3 days recovery. (b, c) Phenotypes of 4-week-old WT, OE-1, ein3-1, OE-1 ein3 (b), and ctr1-1 and OE-1 ctr1 (c) plants before (day 0) and after 2-day DS or 8-day LS treatment followed by 3 days recovery.

Figure 7

Lipid profiles in the rosettes of WT, ACBP3-OEs and ACBP3-KOs after LS treatment. (a) Content of lipid species of 4-week-old WT, ACBP3-OEs (OE-1 and OE-4) and ACBP3-KOs (acbp3 and ACBP3-RNAi) before treatment (day 0) and after LS treatment for 4 days (LS day 4; for ACBP3-OEs) or 6 days (LS day 6; for ACBP3-KOs). MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid. (b) Lipid compositions of PS in WT and ACBP3-OEs before treatment (day 0) or after 4-day LS treatment (upper two graphs), and WT and ACBP3-KOs before treatment (day 0) or after 6-day LS treatment (lower two graphs). Asterisks indicate significant differences from WT; *P < 0.05; **P < 0.01 by Student's t-test. Values represent means ± SD (n = 4) of four independent samples and each sample was pooled from the rosettes of three plants.

Figure 8

Sphingolipid contents in rosettes of WT, ACBP3-OEs and ACBP3-KOs after LS treatment. For GIPC and GlcCer profiling, 4-week-old WT, ACBP3-OEs (OE-1 and OE-4) and ACBP3-KOs (acbp3 and ACBP3-RNAi) were untreated (CK), LS-treated for 4 days (a; for ACBP3-OEs), or LS-treated for 6 days (b; for ACBP3-KOs). For ceramide profiling, 4-week-old WT, OE-1 and acbp3 plants were untreated (CK) or LS-treated for 2 days (LS). The rosette samples were harvested at the indicated times. The amounts of GIPC (left panels in a, b), GlcCer (right panels in a, b) and Cer (c) were calculated by normalizing to the dry weights of tissues. Asterisks indicate significant differences from WT; *P < 0.05; **P < 0.01 by Student's t-test. Values represent means ± SD (n = 4) of four independent samples and each sample was pooled from the rosettes of three plants.

Figure 9

rACBP3 protein binds VLC acyl-CoA esters in vitro.The interactions between rACBP3 and 18:2-, 20:0-, 22:0- and 24:0-acyl-CoA esters were determined by MST analyses. The dissociation constant (K_d) calculated for each binding assay is shown.

Figure 10

Regulation of ACBP3-mediated hypoxia response by MYB30.(a) Phenotypes of 4-week-old WT, ACBP3-RNAi, RNAi myb30, and myb30-1 plants before treatment (day 0) and after 5-day LS treatment followed by 3 days' recovery. (b) Seeds of WT, ACBP3-RNAi, RNAi myb30, and myb30-1 were germinated on MS medium supplemented with 0 (MS) or 75 mm ethanol. Images were taken 2 weeks after germination.