Seed-Specific Overexpression of the Pyruvate Transporter BASS2 Increases Oil Content in Arabidopsis Seeds

Abstract

Seed oil is important not only for human and animal nutrition, but also for various industrial applications. Numerous genetic engineering strategies have been attempted to increase the oil content per seed, but few of these strategies have involved manipulating the transporters. Pyruvate is a major source of carbon for de novo fatty acid biosynthesis in plastids, and the embryo's demand for pyruvate is reported to increase during active oil accumulation. In this study, we tested our hypothesis that oil biosynthesis could be boosted by increasing pyruvate flux into plastids. We expressed the known plastid-localized pyruvate transporter BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2 (BASS2) under the control of a seed-specific soybean (Glycine max) glycinin-1 promoter in Arabidopsis thaliana. The resultant transgenic Arabidopsis plants (OEs), which expressed high levels of BASS2, produced seeds that were larger and heavier and contained 10-37% more oil than those of the wild type (WT), but were comparable to the WT seeds in terms of protein and carbohydrate contents. The total seed number did not differ significantly between the WT and OEs. Therefore, oil yield per plant was increased by 24-43% in the OE lines compared to WT. Taken together, our results demonstrate that seed-specific overexpression of the pyruvate transporter BASS2 promotes oil production in Arabidopsis seeds. Thus, manipulating the level of specific transporters is a feasible approach for increasing the seed oil content.

Keywords: BASS2; bioenergy; pyruvate transporter; seed oil yield; seed-specific promoter.

Figure 1

Our strategy to increase seed oil accumulation by overexpressing the plastidial pyruvate transporter BASS2 under the control of a seed-specific promoter in developing A. thaliana seeds. A simplified diagram depicting the oil metabolism pathway in developing A. thaliana seeds. During seed development, sucrose imported from maternal tissues is converted to G6P. Then, G6P is metabolized to PEP. In the cytosol, PEP is either converted to pyruvate or enters a plastid via PPT. In plastids, pyruvate is used as a substrate for fatty acid biosynthesis, and the resulting fatty acids are channeled into the TAG biosynthesis pathway in the ER. Cytosolic pyruvate is used for many metabolic pathways, such as protein biosynthesis and the TCA cycle. By increasing pyruvate flux into plastids by the seed-specific overexpression of the pyruvate transporter BASS2, we expected to increase fatty acid biosynthesis in the plastids and finally enhance TAG biosynthesis in developing Arabidopsis seeds. This figure was modified from van Erp et al. (2014) Plant Physiology. Ovals indicate transporters responsible for each transport process: GPT, Glucose-6-phosphate translocator (green); PPT, phosphoenolpyruvate/phosphate translocator (green); BASS2, bile-acid sodium symporter 2 (red); ABCA9; ATP-binding cassette A subfamily member 9 (blue). Other abbreviations are: Suc, sucrose; G3P, Glucose-3-phosphate; G6P, Glucose-6-phosphate; PEP, phosphoenolpyruvate; PKc, cytosolic pyruvate kinase; PKp, plastidic pyruvate kinase; Pyr, pyruvate; FAS, fatty acid synthase complex; MEP, methylerythritol phosphate; PC, phosphatidylcholine; DAG, diacylglycerol; TAG, triacylglycerol; Acyl-CoA, acyl-Coenzyme A; Acetyl-CoA, acetyl-Coenzyme A; TCA, tricarboxylic acid. Black lines indicate pathways active in the cell during seed maturation. Red indicates the flux of newly incorporated pyruvate by pyruvate transporter (BASS2) in transgenic lines.

Figure 2

Expression levels of the pyruvate transporter (BASS2) in the developing siliques of transgenic plants overexpressing BASS2. (A) Schematic representation of the T-DNA of the binary vector used to express BASS2 under the control of the seed-specific soybean glycinin-1 promoter. (B) Relative levels of BASS2 expression. RNA was extracted from T2 developing siliques (12–14 DAF) of four independent BASS2-overexpressing lines (OE1, OE2, OE3, and OE4) and the wild type (WT). BASS2 transcript levels were determined by real-time quantitative RT-PCR, normalized to transcript levels of the control gene UBQ11, and presented relative to values of the WT, which were set to 1. Error bars depict standard error (±SE; n = 3). Asterisks indicate significant difference from the WT (N = 3, 6 ≤ n ≤ 15, ^**P < 0.01, ^***P < 0.001), as determined using Student's t-test. Values above the columns indicate fold changes when compared with WT. pGly, soybean glycinin-1 promoter; BASS2, bile-acid sodium symporter 2; gly, glycinin-1 terminator; pNos, nopaline synthase promoter; BAR, BASTA resistance gene; nos, nopaline synthase terminator sequence; LB, left border; RB, right border; DAF, day after flowering.

Figure 3

Seed oil contents and fatty acid (FA) composition in seed-specific BASS2-overexpressing (OEs) seeds. (A) The oil contents of seeds produced by the T3 WT and OE lines. (B) FA composition (mol%) of seeds of WT and OE lines. Error bars depict standard error (±SE). Asterisks indicate significant difference from the WT (N = 3, 24 ≤ n ≤ 90, ^***P < 0.001), as determined using Student's t-test. Values above the columns indicate FA content as a percentage of that in WT seeds.

Figure 4

BASS2-overexpressing plants produce larger and heavier seeds than the WT. (A) Seed size of wild-type (WT) and BASS2-overexpressing lines (OE1, OE2, OE3, and OE4). The T3 seeds of BASS2-overexpressing lines were photographed and the mean values of the cross sectional area were compared to that of WT. Error bars depict standard error (±SE; n = 3). Asterisks indicate significant difference from the WT (1092 ≤ n ≤ 4308, ^***P < 0.001), as determined using Student's t-test. Values above the columns indicate seed size as a percentage of that of the WT. (B) Seed weight of WT and OEs. For each replicate, 300 seeds of the WT and BASS2-overexpressing lines (OEs) were collected and weighed. Seed weight was positively correlated with seed size. Error bars depict standard error (±SE). Asterisks indicate significant difference from the wild-type (N = 3, 8 ≤ n ≤ 30, ^***P < 0.001) as determined using Student's t-test. Values above the columns indicate seed weight as a percentage of that of the WT.

Figure 5

Protein and carbohydrate contents of WT and BASS2-overexpressing (OEs) seeds. (A,B) Protein and carbohydrate contents in T3 seeds of WT and BASS2-overexpressing lines. Carbohydrate contents were measured as the sum of sucrose and starch extracts. Values are the mean contents of each metabolite ±SE as a percentage of the corresponding WT value, which was set to 100%. Asterisks indicate significant difference from the wild-type (^*P < 0.05, ^**P < 0.01, ^***P < 0.001), as determined using Student's t-test. (A) N = 3, 42 ≤ n ≤90, for OE2, OE3, and OE4. N = 1, n = 3 for OE1. (B) N = 3, 6 ≤ n ≤ 29 for all samples.

Figure 6

Germination rate and early seedling growth rate of seed-specific BASS2-overexpressing lines (OEs). The time required for 50% of the seeds to germinate (A) and the time for seedlings to grow roots to 2 cm (B) were scored. Values are means ±SE. N = 2, 29 ≤ n ≤ 71, Student's t-test (^*P < 0.05).

Figure 7

Seed yield of WT and seed-specific BASS2-overexpressing (OEs) plants. (A) Seed yield of WT and OEs. Seed yield of plants was measured as the total seed weight from a plant (N = 2, n = 7, 8). (B) Silique number of the main stem of WT and OE plants. After inflorescence meristem growth of the main stem had ceased, the number of siliques on the main stem was counted (N = 6, 10 ≤ n ≤ 30). (C) Seed number per silique. Developing siliques were sampled, and the seed number in the siliques was counted under a dissecting microscope (N = 3,6 ≤ n ≤ 14). Error bars indicate standard error (SE). Asterisks indicate significant difference from the WT (^*P < 0.05), as determined using Student's t-test. Values above the columns indicate seed yield, the number of siliques per plant or the number of seeds per silique as a percentage of the corresponding WT value.