Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes

Abstract

Background: 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets.

Results: In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds.

Conclusion: Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.

Figure 1

The OPLS-DA score scatter plots of three 14-3-3 overexpressing and wild type samples for (A) shoots and (B) roots. Each point represents an independent plant sample in the score scatter plots. We used 55 shoots and 56 roots for the analysis. (A) The OPLS-DA model for shoot samples shows three significant components, with R²X, R²Y and Q²Y values of 0.37, 0.67 and 0.41, respectively. (B) The OPLS-DA model for root samples shows three significant components, with R²X, R²Y and Q²Y values of 0.30, 0.50 and 0.23, respectively. Black square, wild type; blue diamond, kappa-ox; yellow triangle, chi-ox; green circle, psi-ox.

Figure 2

The OPLS-DA score scatter plots (left) and loading scatter plots (right) of shoot samples of (A) kappa-ox and kappa-KO (B) chi-ox and chi-KO. Wild type samples were used as controls. Each point represents an independent plant in the score scatter plots and an individual metabolite peak in the loading plots. (A) The OPLS-DA model of kappa samples shows two significant components, with R²X, R²Y and Q²Y values of 0.53, 0.95 and 0.65, respectively. (B) The OPLS-DA model of chi samples shows three significant components, with R²X, R²Y and Q²Y values of 0.31, 0.67 and 0.41, respectively. These models were validated using analysis of variance of cross-validated predictive residuals (CV-ANOVA) (p_CV < 0.01). (Left) Black square, wild type; blue diamond, kappa-ox; pale-green star, kappa-KO; yellow triangle, chi-ox; green circle, psi-ox; pink-inverted triangle, chi-ox. (Right) Pale-green circle, amino acids; orange diamond, TCA intermediates; blue star, metabolites that consists of CHON-elemental composition; pink square, metabolites that consists of CHO-elemental composition; gray triangle, unclassified peaks. Number of biological replicates: wild type, n = 6; kappa-ox, n = 16; kappa-KO, n = 6; chi-ox, n = 16; chi-KO, n = 6; psi-ox, n = 17; psi-RNAi, n = 6. p_CV, p-value of the probability level of the F-test in each model.

Figure 3

Major metabolite changes in 14-3-3 overexpression plants during day and night. The levels of starch, sucrose, fructose, malate and amino acids were significantly decreased in 14-3-3 overexpression plants compared to wild type (WT). Plants were harvested 1 h before switching light conditions. T-tests were performed to determine significant difference compared to WT (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4

The activities of metabolic enzymes were altered in 14-3-3 overexpression plants. Enzyme activities were determined in 14-3-3 overexpression plants and wild type Col-0 plants (WT). Asterisks indicate significant differences compared to WT as determined by t-test with n > 7 (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 5

14-3-3 proteins interact with TCA cycle enzymes in yeast. Three 14-3-3 isoforms interact with isocitrate dehydrogenase (ICDH, At4G35650) and two malate dehydrogenases (MDH1, At1G04410; MDH2, At5G43330) that were previously isolated as possible targets of 14-3-3 proteins. AC indicates a pGADT7 and BD indicates a pGBKT7 vector. The positive control and negative control were described in methods section.

Figure 6

The schematic model of metabolic pathways that are regulated by 14-3-3 proteins. Grey box indicates unchanged metabolites; blue box indicates decreased metabolites in 14-3-3 overepxression plants compared to wild type plants; no colored box indicates that metabolites were not measured in this study; purple box (α-ketoglutarate) indicates that some overexpression lines showed higher level of metabolite compared to WT but others showed lower than WT. Black letters indicate that the activities of enzymes were unchanged in 14-3-3 overexpression plants compared to WT; Blue letters indicate that the enzyme's activity was decreased in 14-3-3 overexpression plants; Red letters indicate that the enzyme's activity was increased in 14-3-3 overexpression plants. AGPase, Glucose-1-phosphate adenylyltransferase; INV, β-fructofuranosidase; SPS, sucrose-phosphate synthase; UGP, UTP-glucose-1-phosphate uridylyltransferase; PGM, Phosphoglucomutase; PFK, 6-phosphofructokinase; cFBP, Fructose-bisphosphatase; TK, Transketolase; GAPDH-NADP, Glyceraldehyde-3-phosphate dehydrogenase (NADP⁺) (phosphorylating); TPI, Triose-phosphate isomerase; FBP Ald, Fructose-bisphosphate aldolase; PFK, 6-phosphofructokinase; PEPC, Phosphoenolpyruvate carboxylase; PK, Pyruvate kinase; Shikimate DH, Shikimate dehydrogenase; Ala AT, Alanine-glyoxylate transaminase; CS, Citrate synthase; NAD-ICDH, Isocitrate dehydrogenase (NAD⁺); NAD-MDH, Malate dehydrogenase; GLDH, Glutamate dehydrogenase; NR, Nitrate reductase; ASP AT, Aspartate transaminase.