Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism

Abstract

Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions.

Fig. 1.

The gapcp1gapcp2 mutant shows ABA insensitivity to growth. (A) Fresh weight of the aerial part of 3-week-old gapcp1gapcp2 seedlings (g1.1g1.1 g2.1g2.1) as compared with the wild type (WT). (B) Fresh weight (%) of the aerial part of 3-week-old gapcp1gapcp2 seedlings (g1.1g1.1 g2.1g2.1) as compared with the WT under different ABA concentrations. Seedlings were treated for 13 d with the ABA concentrations indicated in the figure. Data are the mean ±SD, n ≥5 plates, each plate containing four (WT) and six (g1.1g1.1 g2.1g2.1) plants. *Significant at a P-value <0.05 as compared with the WT. For simplicity, g stands for gapcp.

Fig. 2.

The gapcp1gapcp2 mutant shows ABA insensitivity to stomatal closing. ABA-induced stomatal closing in the wild type (WT) and in two different alleles of gapcp1gapcp2 (g1.1g1.1 g2.1g2.1, g1.1g1.1 g2.3g2.3) plants. Data are the mean ±SD, n=30–40 stomata per experiment. *Significant at a P-value <0.05 as compared with non-ABA-treated plants. For simplicity, g stands for gapcp.

Fig. 3.

The gapcp1gapcp2 mutant shows ABA insensitivity during germination. (A) Germination of the wild type (WT) and a mixed population of GAPCp-deficient (double mutant, heterozygous and single mutant plants; G1g1.1 g2.1g2.1) seeds. Fresh seeds without a cold pre-treatment were plated in agar plates containing 0.2 g l⁻¹ MES. (B) Germination of 10-day-old seeds from the WT and single mutants of GAPCp1 (g1.1g1.1 G2G2) and GAPCp2 (G1G1 g2.1g2.1) with and without 1 μM ABA. (C) Germination of 10-day-old WT seeds, a mixed population of GAPCp-deficient seeds (double mutant, heterozygous, and single mutant; G1g1.1 g2.1g2.1), and GAPCp1-overexpressing (Oex-GAPCp1) seeds under different ABA concentrations. Data are the mean ±SD; each experiment consisted of at least four plates with 60 seeds each. Data from Oex-GAPCp1 are from a representative line, but similar results were obtained with two other T₃ overexpressing lines. The experiment was repeated several times with different pools of seeds. *Significant at a P-value <0.05 as compared with WT seeds. For simplicity, g stands for gapcp and G for GAPCp.

Fig. 4.

ABA gene expression is deregulated in gapcp1gapcp2. (A) Comparison of the wild type (WT) versus WT+ABA and gapcp1gapcp2 (g1.1g1.1 g2.1g2.1) versus g1.1g1.1 g2.1g2.1+ABA. (B) Comparison of the WT versus g1.1g1.1 g2.1g2.1 and WT+ABA versus g1.1g1.1 g2.1g2.1+ABA. (C) Quantification of transcript level changes in g1.1g1.1 g2.1g2.1 upon ABA treatment. RNA was extracted from 18-day-old gapcp1gapcp2 and WT seedlings grown in the presence or absence of 0.75 μM ABA for 10 d. The expression of a selection of up- and down-regulated genes in gapcp1gapcp2 was quantified using real-time PCR. Data are the mean ±SD; n=3. For simplicity, g stands for gapcp.

Fig. 5.

ABA reduces sugar content in gapcp1gapcp2. Starch and total soluble glucose in the aerial part and roots of 3-week-old wild-type (WT) and gapcp1gapcp2 (g1.1g1.1 g2.1g2.1) seedlings grown in the presence or absence of 0.75 μM ABA for 10 d. Values were normalized to the mean response of the WT in mg g fresh weight⁻¹ (starch, 1.75±0.33 in the aerial part of the WT, 0.19±0.05 in roots of the WT; soluble glucose, 0.31±0.10 in the aerial part of the WT, 0.87±0.15 in roots of the WT). Data are the mean ±SD, n ≥3;. *Significant at a P-value <0.05 as compared with non-ABA-treated plants. For simplicity, g stands for gapcp.

Fig. 6.

ABI4 expression is deregulated in gapcp1gapcp2. Quantification of changes in ABI4 expression in seeds from the wild type (WT) and from a mixed population deficient in GAPCp (double mutant, heterozygous, and single mutant; G1g1.1 g2.1g2.1). RNA was extracted from seeds which had been imbibed for 48 h and germinated in the presence or absence of 0.25 μM ABA. ABI4 expression was quantified using real-time PCR. Data are the mean ±SD; n=3. For simplicity, g stands for gapcp and G for GAPCp.

Fig. 7.

gapcp1gapcp2 ABA insensitivity to growth is abolished by serine supplementation. Fresh weight (%) of the aerial part of 18-day-old gapcp1gapcp2 seedlings (g1.1g1.1 g2.1g2.1) as compared with the wild type (WT) under different concentrations of ABA and serine. Seedlings were treated for 9 d with the concentrations of ABA and serine indicated in the figure. Data are the mean ±SD, n ≥5 plates; each plate contained 4 (WT) and six (g1.1g1.1 g2.1g2.1) plants. *Significant at a P-value <0.05 as compared with the WT. For simplicity, g stands for gapcp.

Fig. 8.

Serine supplementation confers hypersensitivity to ABA during germination. Germination of 10-day-old seeds from wild-type (WT) and heterozygous gapcp (G1g1.1 g2.1g2.1) plants under different concentrations of ABA and serine. Data are the mean ±SD; each experiment consisted of at least four plates with 60 seeds each. The experiment was repeated several times with different pools of seeds. •♦Significant at a P-value <0.05 as compared with the non-serine-treated WT seeds. *Significant at a P-value <0.05 as compared with the non-serine-treated seeds within each genotype. For simplicity, g stands for gapcp and G for GAPCp.