Metabolic pathways involved in cold acclimation identified by integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A

Abstract

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.

Figure 1.

Venn diagrams of identified metabolites that are increased relative to the controls. The diagrams illustrate the number of identified metabolites in six kinds of plants: cold exposed (1 and 4 d), dehydration exposed (2 and 3 d), 35S:DREB1A, and 35S:DREB2A-CA.

Figure 2.

Statistical analyses of metabolite profiles. We analyzed two independent lines of 35S:DREB1A (α and β) and 35S:DREB2A-CA (α and β) plants. The levels of metabolites for both DREB1A and DREB2A in each β line were higher than those in each α line. A, PCA of metabolites. The y and x axes represent PC1 and PC2, respectively. The circles indicate untreated, cold-exposed, and dehydration-exposed plants. The diamonds represent control, 35S:DREB1A, and 35S:DREB2A-CA plants. B, Representative metabolites for which the eigenvector values were the first and second highest or lowest. In each case, the maximum level of the metabolite was set to 100. Error bars indicate sd for three experiments. A star indicates that the metabolite was not detected. Metabolites in the α and β lines of each transgenic plant are shown by the left and right bars, respectively. C, Selected metabolites that were increased in both cold-exposed and 35S:DREB1A plants but minimal in 35S:DREB2A-CA plants.

Figure 3.

Functional categorization of DREB1A and DREB2A-CA downstream genes. Shown are 20 functional categories of DREB1A and DREB2A-CA downstream genes.

Figure 4.

Map of starch degradation and Suc metabolism pathways. Each small square indicates the expression level of the gene that shows the highest expression in each gene family. Red squares show more than 4-fold increase relative to the control. Orange squares show between 2- and 4-fold increase. Octagons indicate metabolite accumulations. Each pink section indicates increased metabolites in each plant. We could not measure starch, maltooligosaccharides, Glc-1-P, Fru-6-P, Suc-6-P, UDP-Glc, and UDP-Gal. C, D, 1A, and 2A indicate cold-exposed, dehydration-exposed, 35S:DREB1A, and 35S:DREB2A-CA plants, respectively. We analyzed two independent lines of 35S:DREB1A (α and β) and 35S:DREB2A-CA (α and β) plants. GWD, Glucan-water dikinase.

Figure 5.

Expression of genes for starch degradation- and Suc metabolism-related enzymes. A, Each small square indicates the level of gene expression. Red squares show more than 4-fold increase relative to the control. Orange squares show between 2- and 4-fold increase. Light blue squares show between ½- and ¼-fold increase. Dark blue squares show less than ¼-fold increase. C1, C4, D2, D3, 1A, and 2A indicate cold-exposed (1 d), cold-exposed (4 d), dehydration-exposed (2 d), dehydration-exposed (3 d), 35S:DREB1A, and 35S:DREB2A-CA plants, respectively. We analyzed two independent lines of 35S:DREB1A (α and β) and 35S:DREB2A-CA (α and β) plants. The levels of BAM3, BAM1, AtGolS3, AtGolS2, DIN10, and SIP transcripts were detected by qRT-PCR. B, The levels of transcripts for genes encoding β-amylase, galactinol, and raffinose synthase determined by qRT-PCR. Panels 1 and 2 show the levels of transcripts for BAM3 and BAM1 encoding β-amylase, respectively. Panels 3 and 4 show the levels of transcripts for AtGolS3 and AtGolS2 encoding galactinol synthase, respectively. Panels 5 and 6 show the levels of transcripts for DIN10 and SIP1 encoding raffinose synthase, respectively. Error bars indicate sd for three experiments. A star indicates that the transcript was not detected.