MATE-Type Proteins Are Responsible for Isoflavone Transportation and Accumulation in Soybean Seeds

Abstract

Soybeans are nutritionally important as human food and animal feed. Apart from the macronutrients such as proteins and oils, soybeans are also high in health-beneficial secondary metabolites and are uniquely enriched in isoflavones among food crops. Isoflavone biosynthesis has been relatively well characterized, but the mechanism of their transportation in soybean cells is largely unknown. Using the yeast model, we showed that GmMATE1 and GmMATE2 promoted the accumulation of isoflavones, mainly in the aglycone forms. Using the tobacco BrightYellow-2 (BY-2) cell model, GmMATE1 and GmMATE2 were found to be localized in the vacuolar membrane. Such subcellular localization supports the notion that GmMATE1 and GmMATE2 function by compartmentalizing isoflavones in the vacuole. Expression analyses showed that GmMATE1 was mainly expressed in the developing soybean pod. Soybean mutants defective in GmMATE1 had significantly reduced total seed isoflavone contents, whereas the overexpression of GmMATE1 in transgenic soybean promoted the accumulation of seed isoflavones. Our results showed that GmMATE1, and possibly also GmMATE2, are bona fide isoflavone transporters that promote the accumulation of isoflavones in soybean seeds.

Keywords: daidzein; genistein; glycitein; isoflavone; multidrug and toxic compound extrusion (MATE) transporter; seed; soybean.

Figure 1

Yeast uptake assay using a mixture of genistein, daidzein, and glycitein. The level of (A) genistein, (B) daidzein, (C) glycitein, or (D) total aglycone (genistein, daidzein and glycitein) was determined. Metabolites were extracted from the yeast after feeding for 24 h and analyzed using UPLC against the respective standards. Values obtained from the yeast expressing GmMATE1 or GmMATE2 were normalized to those from the empty vector control (EV) in the same experiment. Values shown were the mean of six independent experiments ± SEM. Significant differences compared to EV were determined by Mann-Whitney test. *, p < 0.05; **, p < 0.01.

Figure 2

Yeast uptake assay with a mixture of genistin, daidzin, and glycitin. Metabolites were extracted from the yeast after feeding for 24 h and analyzed using UPLC against the respective standards. It appeared some of the glycosides were converted to aglycones in the yeast by endogenous enzymes. Values obtained from the yeast expressing GmMATEs were normalized to those from the empty vector control (EV) in the same experiment. The relative uptake levels of individual isoflavone species (A–C,E–G) and different isoflavone combinations (D,H,I–L) were determined. Total aglycones (D) refers to the combination of genistein, daidzein, and glyciteins. Total glycosides (H) refers to the combination of genistin, daidzin, and glycitin. The relative uptake level of total aglycones and glycosides is shown in (L). Values shown are the mean of six independent experiments ± SEM. Significant differences compared to EV were determined by Mann-Whitney test. *, p < 0.05; **, p < 0.01; ns, not significant.

Figure 3

Subcellular localizations of GmMATE1-YFP and GmMATE2-YFP were determined using confocal microscopy. The cells were incubated with FM^TM4-64 in the dark at room temperature for 24 h before imaging. DIC, differential interference contrast. Signals from GmMATE-YFP and FM^TM4-64 were illustrated in green and magenta, respectively.

Figure 4

Relative expressions of GmMATE1 and GmMATE2 in different tissues within the developing soybean seed pod. The relative expression of (A) GmMATE1 and (B) GmMATE2 in different tissues of the developing pod, normalized to the reference gene, VPS, using the 2^−ΔCt method. DAF, days after flowering. Values shown are the averages of three technical replicates ± SEM. Similar expression trends were obtained in another biological repeat. (C,D) Photos showing the GUS staining of (C) transgenic soybean pods and (D) transgenic A. thaliana expressing β-glucuronidase (GUS) driven by the native GmMATE1^C08 promoter. Similar results were obtained from two independent transgenic lines. Scale bar = 1 cm.

Figure 5

GmMATE1 is responsible for determining seed isoflavone contents. (A) The total isoflavone contents in the CRISPR/Cas9-edited GmMATE1 mutant was significantly lower than those in its wild-type, W82. Values shown were the means of at least three independently collected batches of seeds ± SEM. Significant differences were determined by Mann-Whitney test. *, p < 0.05. (B) The mutation site in the GmMATE1^C08 gene, with the single-nucleotide deletion marked with an asterisk. (C) The levels of isoflavones in the transgenic soybean overexpressing GmMATE1^C08 were significantly higher than those in the empty vector control, both in the W82 background. Values shown were the means of four independently collected batches of seeds ± SEM. Significant differences were determined by Mann-Whitney test. *, p < 0.05. (D) Relative expression levels of GmMATE1 in the GmMATE1^C08-overexpressor and the empty vector control in the W82 background. The expression levels were normalized to the reference gene, VPS, using the 2^−ΔΔCt method.