Long-Chain acyl-CoA Synthetase LACS2 Contributes to Submergence Tolerance by Modulating Cuticle Permeability in Arabidopsis

Abstract

In Arabidopsis thaliana, LONG-CHAIN ACYL-COA SYNTHETASEs (LACSs) catalyze the synthesis of long-chain acyl-CoAs and function in diverse biological processes. We have recently revealed that LACS2 is primarily involved in the production of polyunsaturated linolenoyl-CoA, essential for the activation of ethylene response transcription factors-mediated hypoxia signaling. Here, we further reported the dual role of LACS2 in the regulation of submergence tolerance by modulating cuticle permeability in Arabidopsis cells. LACS2-overexpressors (LACS2-OEs) showed improved tolerance to submergence, with higher accumulation of cuticular wax and cutin in their rosettes. In contrast, knockout of LACS2 in the lacs2-3 mutant resulted in hypersensitivity to submergence with reduced wax crystals and thinner cutin layer. By analyses of plant surface permeability, we observed that the hypoxic sensitivities in the LACS2-OEs and lacs2-3 mutant were physiologically correlated with chlorophyll leaching, water loss rates, ionic leakage, and gas exchange. Thus, our findings suggest the role of LACS2 in plant response to submergence by modulating cuticle permeability in plant cells.

Keywords: Arabidopsis thaliana; LONG-CHAIN ACYL-COA SYNTHETASE; cuticle; permeability; submergence.

Figure 1

Wild type (WT), lacs2-3, and LACS2-OEs leaves showed changed hydrophobicities upon submergence. The hydrophobic cuticular wax of WT, lacs2-3, and LACS2-OE lines were detected by water droplets under normal growth (Air) and submergence (Submergence) conditions. Hydrophobicity of the leaf cuticular wax was assessed using the contact angle of small droplets of water, and contact angles <90° indicate a hydrophilic surface, whereas those of >90° indicate a hydrophobic surface. The experiments have been biologically repeated three times and similar results were obtained. For each experiment, at least six leaves were used per genotype. Data are average values ± SD (n = 3) calculated from three independent experiments.

Figure 2

Alteration of cuticular wax and cutin loads on the stems of wild type (WT), lacs2-3, and LACS2-OE (OE-1) in response to submergence. (A) Scanning electron micrographs showing epicuticular wax crystals on the stems of six-week-old WT, lacs2-3, and OE-1 under normal growth conditions (air) or submergence for three days (submergence). Scale bar, 10 μm. (B) Transmission electron microscopy images showing cuticle layers in the stems of six-week-old WT, lacs2-3, and OE-1 under normal growth conditions (air) or submergence for three days (submergence). Scale bar, 500 nm. For each experiment, at least six leaves were used per genotype. Data are average values ± SD (n = 3) calculated from three independent experiments. The experiments have been biologically repeated three times and similar results were obtained.

Figure 3

Cuticular wax and cutin Polyester monomer profiles of wild type (WT), lacs2-3, and LACS2-OEs leaves upon submergence exposure. (A) The levels of total amounts of wax and cutin loads of WT, lacs2-3, and LACS2-OE (OE-1) leaves under normal growth (air) and submergence (submergence) conditions. (B) and (C) The levels of different molecular species of wax (B) and cutin loads (C) of WT, lacs2-3, and LACS2-OE (OE-1) leaves under normal growth (Air) and submergence (Submergence) conditions. Leaves of four-week-old WT, lacs2-3, and OE-1 were collected before submergence (air) and after three-day submergence treatment (submergence). Wax (B) and cutin (C) coverage is expressed as μg/cm² leaf surface area. Contents of different molecular species of fatty acids and ω-hydroxy fatty acids are shown at the top left (C). For each experiment, at least six plants (technical replicates) were analyzed per genotype. Asterisks with “H” or “L” indicate significantly higher or lower level than that of WT (* P < 0.05, ** P < 0.01 by Student’s t-test).

Figure 4

LACS2 regulated cuticle permeability in response to submergence treatment. (A) Cuticle permeability analysis in the leaves of four-week-old WT, lacs2-3, and LACS-OEs under normal growth (air) and submergence (submergence) conditions. The leaves were collected before and three days after treatment and stained with 0.05% toluidine blue. (B, C and D) Relative rates of chlorophyll leaching (B), water loss (C), and ion leakage (D) showing the cuticle permeability of four-week-old wild type (WT), lacs2-3, and LACS2-OEs leaves after submergence treatment for the indicated times. All of the experiments were performed for three times with similar results. The data are means ± SD (n = 6 technical replicates). Asterisks indicate significant differences from WT (** P < 0.01 by Student’s t-test). “H” and “L” indicate values that are significantly higher or lower, respectively, compared to that of WT.

Figure 5

Altered expression of LACS2 leads to apoplastic air availability in plant cells. Apoplastic water (A) and air volumes (B) of WT, lacs2-3, and LACS2-OEs before treatment and after submergence treatment for 1, 2, and 3 days. All of the experiments were performed for three times with similar results. The data are means ± SD (n = 6 technical replicates). Asterisks indicate significant differences from WT (** P < 0.01 by Student’s t-test). “H” and “L” indicate values that are significantly higher or lower, respectively, compared to that of WT.

Figure 6

Model of the role of LACS2 in plant responses to hypoxia stress. Lipid remodeling is vitally necessary for hypoxic tolerance in plant [7,8,18,35]. By inactivation of fatty acid synthetase (FAS) and accelerating the fatty acid degradation, submergence-induced hypoxia decreases the fatty acid levels. LACS2 is essential for plant hypoxic tolerance and acyl-CoA metabolism during hypoxia. Specifically, the hypoxia-induced 18:3-CoA catalyzed by LACS2 and FAD3, interacts with ACBP1 or ACBP2, leads to the dissociation of the ACBPs–ERF-VII complex and subsequently activates the signaling cascades of ACBPs–ERF-VII. On the other hand, VLCACoAs produced by LACS2 are shuttled by ACBP1 or ACBP2 to facilitate the biosynthesis of cuticular lipids, which may contribute to plant surface permeability, cellular hyperhydrivity, and gas exchange under submergence conditions. ACBP; acyl-CoA-binding protein; ACP, acyl carrier protein; ERF, ethylene response factor; FAS, fatty acid synthase; VLCACoA, very-long-chain acyl-CoA.