DIACYLGLYCEROL ACYLTRANSFERASE1 Contributes to Freezing Tolerance

Abstract

Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Figure 1.

Freezing survival of seedlings of B. stricta SAD12 (from Colorado) and LTM (from Idaho) ecotypes. Non-acclimated (A) and cold acclimated plants (B) were subjected to a range of subzero temperatures, and survival was scored after thawing. Fitted response curves were used for calculation of LT50, the temperature at which 50% of the plants died. Significant differences (P < 0.01, Student’s t test) are indicated by asterisks. Data represent means (±sd) of five agar plates containing 30 seedlings each.

Figure 2.

Mapping of the SKI QTL for freezing tolerance of B. stricta seedlings. A, QTL analysis results of freezing tolerance across the seven LGs using the SAD12 × LTM RIL population. The red line shows the LOD significance threshold of 2.8. B, A single significant QTL above the LOD threshold for seedling survival assay was identified on LG5 (top) and its estimated additive effect (bottom). The negative value of the additive effect indicates that the LTM ecotype allele confers enhanced freezing tolerance. C, The precise two-LOD interval and marker names of the SKI major QTL on B. stricta LG5.

Figure 3.

Time course of relative BstDGAT1 expression in B. stricta SAD12 and LTM seedlings during cold acclimation at 4°C. Values are relative to the control (no cold treatment). Significant ecotypic differences are indicated by asterisks (*P < 0.05 and **P < 0.01; Student’s t test ); data represent means (±se) of three biological replicates.

Figure 4.

Polar glycerolipids of B. stricta SAD12 and LTM seedlings subjected to control conditions, cold acclimation, or cold acclimation followed by freezing. Immediately after treatment, total lipids were extracted from the aerial parts of plants for quantitative lipidomics analysis. The minor phospholipids PA and LPC are shown in the inset. Bars marked with different letters indicate statistical differences within a lipid class (P < 0.05, ANOVA); differences between genotypes under the same conditions are at P < 0.01 unless marked with a prime (P < 0.05); data represent means (±sd, n = 5–6).

Figure 5.

Molecular species of selected polar glycerolipid classes of B. stricta SAD12 and LTM seedlings subjected to control conditions, cold acclimation, or cold acclimation followed by freezing. Bar colors indicate ecotype and treatment, and letters indicate statistical differences as in Figure 4. For molecular species that do not display genotype-by-treatment interactions, significant differences from control (Ct) or cold-acclimated (CA) values within the same genotype are indicated by >/<Ct or >/<CA, respectively. Data represent means (±sd, n = 5–6).

Figure 6.

Accumulation of TAG, TGDG, and TeGDG in B. stricta SAD12 and LTM seedlings subjected to control conditions, cold acclimation, or cold acclimation followed by freezing. Significant ecotypic differences are indicated by asterisks (*P < 0.05 and **P < 0.01; ANOVA); data represent means (±sd, n = 5–6).

Figure 7.

Molecular species of TAG accumulated in B. stricta SAD12 and LTM seedlings subjected to control conditions, cold acclimation, or cold acclimation followed by freezing. Bar colors indicate ecotype and treatment, and letters indicate statistical differences as in Figure 4; data represent means (±sd, n = 5–6).

Figure 8.

Relative molecular species compositions of TAG in B. stricta SAD12 and LTM seedlings subjected to control conditions, cold acclimation, or cold acclimation followed by freezing. TAG classes indicated by different colors were characterized by distinctive acyl chain compositions. Chloroplastic species contained 16:3, indicative of a galactolipid precursor. Based on a χ² test, overall relative TAG molecular species compositions in control conditions and in response to freezing were similar, but TAGs formed during cold acclimation differed (P < 0.01). Three groups of TAG molecular species formed during acclimation showed ecotypic differences, as indicated by asterisks (**P < 0.01, n = 5–6; ANOVA).

Figure 9.

Freezing tolerance of Arabidopsis AtDGAT1 overexpressor plants. A, Two-week-old seedlings of Arabidopsis wild type (WT) and 35S-DGAT1 lines DGAT1-OE1 and DGAT1-OE2 were cold-acclimated for four 4 d (CA) and subsequently subjected to freezing temperatures as indicated. Pictures were taken 3 d after thawing of plates. B, Survival rates were scored, and significant differences compared to the wild type are indicated by asterisks (* P < 0.01, Student’s t test); data represent means (±sd) of three to four agar plates containing 40 to 50 seedlings each.

Figure 10.

Model of DGAT1 function in B. stricta seedlings in response to cold acclimation and subsequent freezing. During cold acclimation (top panel), BstDGAT1 is transcriptionally upregulated. As low temperature reduces the requirement for eukaryotic galactolipid biosynthesis (blue arrow), excess PC is converted to DAG and subsequently acylated to 18:1-, 18:2-, and 18:3-rich molecular species of TAG. Simultaneously, turnover of MGDG in the chloroplast results in accumulation of low amounts of 16:3-containing, chloroplastic TAG. In contrast, in response to freezing (bottom panel), high amounts of 16:3-TAG accumulate, together with oligogalactolipids DGDG, TGDG, and TeGDG, suggesting concomitant activation of SFR2. DGAT1, which is most likely coresident with SFR2 in the chloroplast outer envelope membrane (Chl OeM), is suggested to use SFR2-generated DAG as acylation substrate and promote chloroplast membrane resilience during freezing by removing membrane destabilizing DAG molecules, while allowing SFR2-mediated synthesis of membrane-stabilizing DGDG, TGDG, and TeGDG. Our study of natural variation in B. stricta ecotypes has identified BstDGAT1 as a candidate QTL locus for freezing survival and found firm positive associations of freezing tolerance with acclimation-induced BstDGAT1 expression and accumulation of TAGs and oligogalactolipids during freezing. The associated differential activities of DGAT1 and SFR2 support their proposed functional link underlying an adaptive mechanism of freezing tolerance. Freezing also induces PC hydrolysis to PA, presumably by PLD activity, which correlates negatively with freezing tolerance in B. stricta ecotypes. DAG acylation reactions indicated outside the chloroplast are localized to the ER or to the chloroplast outer envelope membrane. TAGs are contained in cytoplasmic oil bodies (o.b.). Lipids with significant genotype-by-treatment interactions that positively associate with freezing tolerance are indicated in red and by asterisks. (L)PC, (lyso)phosphatidylcholine; ACP, acyl carrier protein.