Two Triacylglycerol Lipases Are Negative Regulators of Chilling Stress Tolerance in Arabidopsis

Abstract

Cold stress is one of the abiotic stress conditions that severely limit plant growth and development and productivity. Triacylglycerol lipases are important metabolic enzymes for the catabolism of triacylglycerols and, therefore, play important roles in cellular activities including seed germination and early seedling establishment. However, whether they play a role in cold stress responses remains unknown. In this study, we characterized two Arabidopsis triacylglycerol lipases, MPL1 and LIP1 and defined their role in cold stress. The expression of MPL1 and LIP1 is reduced by cold stress, suggesting that they may be negative factors related to cold stress. Indeed, we found that loss-of-function of MPL1 and LIP1 resulted in increased cold tolerance and that the mpl1lip1 double mutant displayed an additive effect on cold tolerance. We performed RNA-seq analysis to reveal the global effect of the mpl1 and lip1 mutations on gene expression under cold stress. The mpl1 mutation had a small effect on gene expression under both under control and cold stress conditions whereas the lip1 mutation caused a much stronger effect on gene expression under control and cold stress conditions. The mpl1lip1 double mutant had a moderate effect on gene expression under control and cold stress conditions. Together, our results indicate that MPL1 and LIP1 triacylglycerol lipases are negative regulators of cold tolerance without any side effects on growth in Arabidopsis and that they might be ideal candidates for breeding cold-tolerant crops through genome editing technology.

Keywords: Arabidopsis; chilling stress tolerance; cold stress; triacylglycerol lipases.

Figure 1

The evolutionary relationships of LIP1, MPL1, and their close homologs. The evolutionary tree was generated with MEGA11 using the neighbor-joining method with the JTT-matrix-based model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Identities of the proteins shown in this tree are provided in the Materials and Methods section.

Figure 2

LIP1 and MPL1 display differential expression patterns in organs and developmental stages and under cold stress. (A,B) Expression profiles of LIP1 (At2g15230) and MPL1 (At5g14180) in different organs and developmental stages of wild-type plants as determined by microarrays [16]. (C,D) Expression profiles of LIP1 and MPL1 under cold stress as determined by microarrays with 13-day-old wild-type seedlings subjected to cold treatments [17]. (E,F) Expression profiles of LIP1 and MPL1 under salt stress as determined by microarrays with 13-day-old wild-type seedlings subjected to cold treatments [17]. (G,H) Expression profiles of LIP1 and MPL1 under salt stress as determined by microarrays with 16-day-old wild-type seedlings subjected to heat stress at 38 °C for 0, 0.25 (+0 h recovery at 25 °C), 0.5 (+0 h recovery at 25 °C), 1 (+0 h recovery at 25 °C), or 3 h (+3 h recovery at 25 °C = 6 h; +9 h recovery at 25 °C = 12 h; +21 h recovery at 25 °C = 24 h) [17]. Data are means ± sd (n = 3).

Figure 3

MPL1 and LIP1 expression level in Col-0, mpl1 (SALK_101919), lip1 (SALK_114605), and mpl1lip1 plants under different conditions. (A) Gene structures of MPL1 and LIP1 with T-DNA insertions. mpl1: SALK_101919, lip1: SALK_114605. (B,C) Expression levels of MPL1 and LIP1 in two-week-old Col-0, mpl1, lip1, and mpl1lip1 seedlings under control conditions. (D,E) Expression profiles of MPL1 and LIP1 in two-week-old Col-0 seedlings subjected to cold stress (4 °C). Data are means ± sd (n = 4). One-way ANOVA (Tukey test) was performed, and significant difference is indicated by different lowercase letters (p < 0.05).

Figure 4

Chilling phenotype of mpl1, lip1, and mpl1lip1 plants. (A–D) Seeds of Col-0, mpl1, lip1, and mpl1lip1 were sown on MS medium plates and immediately incubated in growth chambers at 22 °C for 7 days (Control) or at 4 °C (Chilling) for 39 days under a long-day photoperiod (16 h day and 8 h night; light intensity ~80 μmol/m²/s). There were 50–80 seedlings for each genotype per MS plate. (A) Morphology of Col-0, mpl1, lip1, and mpl1lip1 plants under control and chilling conditions. Bar = 1 cm. (B) Chlorophyll content of the plants shown in (A) under control conditions. (C) Chlorophyll content of the plants shown in (A) under chilling stress for 39 days. (D) Percentage of green seedlings of Col-0, mpl1, lip1, and mpl1lip1 under chilling stress for 39 days. Data are means ± sd (n = 3 in (B), ≥6 in (C,D) (n indicates the number of MS medium plates)). One-way ANOVA (Tukey test) was performed, and significant difference is indicated by different lowercase letters (0.007 < p < 0.01).

Figure 5

Hypocotyl elongation of the mpl1, lip1, and mpl1lip1 seedlings under chilling stress. (A–C) Seeds of Col-0, mpl1, lip1, and mpl1lip1 were sown in two rows on vertical MS medium plates and immediately incubated in growth chambers while kept in darkness at 22 °C for 7 days (Control) or at 4 °C (Chilling) for 42 days. There are 14–20 seeds for each genotype per MS medium plate. (A) Morphology of Col-0, mpl1, lip1, and mpl1lip1 under control and chilling conditions. (B) Hypocotyl elongation of Col-0, mpl1, lip1, and mpl1lip1 under control conditions for 7 days. (C) Hypocotyl elongation of Col-0, mpl1, lip1, and mpl1lip1 under chilling stress for 42 days. Data are means ± sd (n = 4 in (B) 12 in (C) (n indicates the number of MS medium plates)). One-way ANOVA (Tukey test) was performed, and significant difference is indicated by different lowercase letters (p < 0.03).

Figure 6

Responses of the mpl1, lip1, and mpl1lip1 plants to freezing and heat stresses. (A) Electrolyte leakage indictive of damage of biological membranes caused by freezing temperatures. NC, non-cold acclimated; AC, cold acclimated. (B) Hypocotyl elongation of mpl1, lip1, and mpl1lip1 in response to heat stress. Seeds were sown in two rows on vertical MS medium plates and incubated at 22 °C for 36 h in darkness to ensure uniform seed germination and initial hypocotyl elongation. There were 14–20 seeds for each genotype per MS medium plate. The germinated seedlings were treated at 45 °C for 0 or 1 h and allowed to grow in darkness in a growth room at 22 °C for an additional 3 days. Data are means ± sd (n = 18 in (A) (n = number of detached leaves), 4 (Control), 9 (Heat) in (B) (n = number of MS medium plates)). One-way ANOVA (Tukey test) was performed, and significant difference was indicated by different lowercase letters (p < 0.05).

Figure 7

Deferentially expressed genes in the mpl1, lip1, and mpl1lip1 plants determined by RNA-seq analysis. The RNA-seq and qRT-PCR experiments were performed with two-week-old seedlings subjected to cold stress at 4 °C for 0, 6, or 48 h. (A) Differentially expressed genes in the mpl1, lip1, and mpl1lip1 plants determined by RNA-seq analysis. (B–D) Validation of the RNA-seq results by qRT-PCR analysis. Values represent means ± se (n = 4 (n indicates number of experiments and there were 4 biological replicates in each experiment)). Significant differences in mean values are indicted by asterisk(s) determined by Student’s t-tests (* p < 0.05; ** p < 0.01; *** p < 0.001).