Cytosolic phosphorylating glyceraldehyde-3-phosphate dehydrogenases affect Arabidopsis cellular metabolism and promote seed oil accumulation

Abstract

The cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPC) catalyzes a key reaction in glycolysis, but its contribution to plant metabolism and growth are not well defined. Here, we show that two cytosolic GAPCs play important roles in cellular metabolism and seed oil accumulation. Knockout or overexpression of GAPCs caused significant changes in the level of intermediates in the glycolytic pathway and the ratios of ATP/ADP and NAD(P)H/NAD(P). Two double knockout seeds had ∼3% of dry weight decrease in oil content compared with that of the wild type. In transgenic seeds under the constitutive 35S promoter, oil content was increased up to 42% of dry weight compared with 36% in the wild type and the fatty acid composition was altered; however, these transgenic lines exhibited decreased fertility. Seed-specific overexpression lines had >3% increase in seed oil without compromised seed yield or fecundity. The results demonstrate that GAPC levels play important roles in the overall cellular production of reductants, energy, and carbohydrate metabolites and that GAPC levels are directly correlated with seed oil accumulation. Changes in cellular metabolites and cofactor levels highlight the complexity and tolerance of Arabidopsis thaliana cells to the metabolic perturbation. Further implications for metabolic engineering of seed oil production are discussed.

Figure 1.

KO of GAPCs Decreases Arabidopsis Seed Oil Accumulation. (A) NAD-dependent GAPDH activity in developing seeds. Total protein was extracted from seeds in two developing stages (8 to 10 DAF and 12 to 14 DAF). Values are means ± sd (n = 3). (B) Seed weight of wild-type and GAPC double KOs. Seed weight was average weight based on calculation of the total weight (∼3 mg) and total number of seeds. Values are means ± sd (n = 3). (C) Seed oil content of wild-type and GAPC mutants. Seed oil content was analyzed after seeds were harvested and dried under room temperature for at least 2 weeks. Seed oil content was calculated as the percentage of oil over the seed weight. Values are means ± sd (n = 3). (D) Oil content per seed. Values are means ± sd (n = 3). “L” in (A) to (D) indicates significantly lower than the wild type (Student’s t test, P < 0.05).

Figure 2.

Increased Seed Oil Content in GAPC OE Seeds. (A) Immunoblotting of overexpressed GAPC1 and GAPC2 in Arabidopsis seeds (>12 DAF). GAPCs were either fused with a FLAG tag (GAPC-OE) or with both YFP and FLAG tags (YFP-GAPC-OE) under the control of the cauliflower mosaic virus 35S promoter. Total protein was extracted from the seeds of T3 homozygous lines and the wild type was used as negative control. Total protein (10 μg) was separated on a 10% SDS-PAGE gel, transferred to a membrane, and blotted with anti-FLAG antibody. Two independent lines were examined for each transformation. (B) GAPDH activity assay using total proteins extracted from developing seeds (12 to 14 DAF) of T3 lines. Values are means ± sd (n = 3). (C) Seed oil analyses of GAPC OE seeds (T3). Seed oil content was calculated as the percentage of oil over the seed weight. Values are means ± sd (n = 3). (D) Fatty acid composition was altered in GAPC OE seeds. Fatty acid composition was calculated as mol %. Values are means ± sd (n = 3). (E) Fatty acid double bond index was increased in OE seeds. Fatty acid double bond index was calculated as DBI = mol % of unsaturated fatty acids × number of double bonds of each unsaturated fatty acid. Values are means ± sd (n = 3). “H” and “L” in (B) to (E) indicate significantly higher and lower than the wild type, respectively (Student’s t test, P < 0.05). [See online article for color version of this figure.]

Figure 3.

OE of GAPCs under the Control of the 35S Promoter Reduced Fertility. (A) P35S-driven OE of GAPCs reduced fertility and silique size in transgenic plants. (B) Seed yield per plant for eight independent T3 lines compared with the wild type. Values are means ± se (n = 8). “L” indicates significantly lower than the wild type (Student’s t test, P < 0.05). [See online article for color version of this figure.]

Figure 4.

Seed-Specific OE of GAPC1 and GAPC2. (A) Immunoblotting of overexpressed GAPC1 and GAPC2. GAPCs were fused with a FLAG tag under the control of a seed-specific soybean β-conglycinin promoter (ProCON:GAPC). Total proteins were extracted from the developing seed of T1 plants and the wild type was used as a negative control. Total protein (10 μg) was separated on a 10% SDS-PAGE gel, transferred to a membrane, and blotted with an anti-FLAG antibody. Ten lines were examined for each transformation. (B) Transcript level of GAPCs in developing seeds. YFP-GAPC1-OE6 and YFP-GAPC2-OE3 lines driven by the 35S promoter were transformed into the wild type. ProCON:GAPC1-OE2 and ProCON:GAPC2-OE1 lines driven by a seed-specific soybean β-conglycinin promoter were also transformed into the wild type. gapc1-1 gapc2-1, GAPC double knockout. Values are means ± sd (n = 3). (C) GAPDH activity assay using total proteins extracted from developing seeds (8 to 10 DAF and 12 to 14 DAF) of T1 lines. Values are means ± sd (n = 3). H indicates significantly higher than the wild type (Student’s t test, P < 0.05).

Figure 5.

Seed Oil Content Is Increased in GAPC Seed-Specific OE Seeds. (A) Seed oil content of WT and T1 seed-specific OE seeds. Values are means ±sd (n = 3). Data of three independent lines are presented. (B) Seed oil content of the wild type and T2 seed-specific OE seeds. Heterozygous (OE2-1, OE3-1, and OE4-1 for ProCON:GAPC1; OE1-1, OE3-2, and OE4-1 for ProCON:GAPC2) and homozygous (OE2-9, OE3-6, and OE4-5 for ProCON:GAPC1; OE1-5, OE3-5, and OE4-9 for ProCON:GAPC2) seeds for each independent line were analyzed. Values are means ± sd (n = 3). “H” in (A) and (B) indicates significantly higher than the wild type (Student’s t test, P < 0.05).

Figure 6.

Manipulation of GAPCs Alters the Expression Level of Genes Involved in Oil Biosynthesis in Developing Seeds. KO of GAPCs decreases while OE of GAPCs increases the expression level of genes involved in oil biosynthesis in developing seeds. RNA was extracted from T2 developing seeds (12 to 14 DAF), and the transcript level was determined by real-time PCR normalized to UBQ10. Values are means ± sd (n = 3). “H” and “L” indicate significantly higher and lower than the wild type (Student’s t test, P < 0.05).

Figure 7.

Altered Metabolite Level in the Glycolysis Pathway in Developing Seeds of gapc1-1 gapc2-1 and Seed-Specific OEs. Metabolites were extracted from T2 developing seeds (12 to 14 DAF) and determined and quantified by comparison with authentic standards. The y axis is metabolite content in nmol/g fresh weight (FW). 1,3-BPG was below the detection level and thus not measured. Values are means ± se (n = 4). One-way ANOVA was performed, and different lowercase letters mark significant differences among the wild type, gapc1-1 gapc2-1, and OEs (P < 0.05). G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-diphosphate; DHAP, dihydroxy-acetone-phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 3PGA, glycerate-3-phosphate; 2PGA, glycerate-2-phosphate.

Figure 8.

Metabolic Impact of GAPCs on ATP/ADP, NADH/NAD⁺, and NADPH/NADP⁺ in Developing Seeds. (A) ATP content. (B) ADP content. (C) ATP/ADP ratio. (D) NADH content. (E) NAD⁺ content. (F) NADH/NAD⁺ ratio. (G) NADPH content. (H) NADP⁺ content. (I) NADPH/NADP⁺ ratio. Compounds were extracted from T2 developing seeds (12 to 14 DAF) and quantified by comparison with authentic standards. Values are means ± se (n = 4). “H” and “L” indicate significantly higher and lower than the wild type (Student’s t test, P < 0.05).