Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean

Abstract

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

Keywords: Glycine max; Phytophthora sojae; antioxidant properties; gene expression; isoflavone reductase; isoflavonoid.

Figure 1

Expression patterns of the GmIFR genes in various tissues of “Suinong” 10 soybean under normal condition. The roots, stems or leaves were prepared from 14-day-old seedlings, and the cotyledons from 7-day-old seedlings. The amplification of the soybean EF1 (GmEF1) gene was used as an internal control to normalize all the data. For each sample, three biological replicates were analyzed with their respective three technical replicates, bars indicate standard error of the mean.

Figure 2

Relative quantities of GmIFR mRNA at various time points post-treatment with ET, SA, MeJA, ABA, wounding, and P. sojae. Fourteen-day-old plants were used for treatments and analyses. Water treatment were used as control for P. sojae treatments. The amplification of the soybean Actin (GmActin4) gene was used as an internal control to normalize all the data. Relative transcript levels of GmIFR were quantified compared with mock plants at the same time point. The experiment was performed on three biological replicates with their respective three technical replicates and statistically analyzed using Student's t-test (^*P < 0.05; ^**P < 0.01). Bars indicate standard error of the mean.

Figure 3

Subcellular localization of the IFR-GFP fusion protein in Soybean protoplasts. GmIFR-GFP expression was driven by the cauliflower mosaic virus 35S promoter and transiently expressed in Soybean protoplasts. The images of bright-field (A,E), the GFP fluorescence (green) only (B,F), the chlorophyll autofluorescence (red) only (C,G), cytoplasmic marker fluorescence localization, and combined ones (D,H) are shown. Bars = 10 mm.

Figure 4

Analysis of GmIFR activity by HPLC. (A) After IPTG induction, Transetta cells containing pET28a-IFR were grown at 37°Cfor 1, 2, 4, 6 h. Lane 1 protein of total cells without IPTG induction, lane 2 protein of total cells with IPTG induction for 1 h, lane 3 protein of total cells with IPTG induction for 2 h, lane 4 induction for 4 h, lane 5 induction for 6 h, lane 6 purified recombinant GmIFR protein with Nickel-CL agarose affinity chromatography and used for enzyme activity assay; M, molecular marker; Lane Western blot western blotting of the purified recombinant GmIFR protein with an anti-His tag primary antibody probe. (B) The reaction with 2′-hydroxyformononetin. (C) The reaction harboring the GmIFR protein and with 2′-hydroxyformononetin as the substrate. S, substrate; P, product.

Figure 5

Resistance analysis of GmIFR transgenic soybean plants. (A) qRT-PCR determining the relative abundance of GmIFR (lines T8-80, T8-88, and T8-96) in the transgenic soybean plants. The non-transgenic soybean plants were used as controls. For each sample, three biological replicates were analyzed with their respective three technical replicates and statistically analyzed using Student's t-test (^**P < 0.01). Bars indicate standard error of the mean. (B) Disease symptoms after infection with P. sojae. Lesions on living cotyledon at 72 h with P. sojae isolate race 1. For infection assays, three biological replicates were analyzed with their respective technical replicates. And do the same with control. (C) Relative lesion area of transgenic soybean cotyledon infection with P. sojae after 72 h. Seventy-two hours a represents the cotyledon of transgenic soybean and non-transgenic soybean infected with V8 juice agar, and 72 h-b represents the cotyledon were treated with a P. sojae inoculum. The average lesion area of each independent transgenic line (n = 3) was calculated and their relative lesion areas are shown in columns after comparison with the average lesion area on non-transgenic soybean. The statistically analyzed using Student's t-test (^**P < 0.01). Bars indicate standard error of the mean.

Figure 6

Relative expression levels of reactive oxygen species (ROS) in transgenic soybean plants and non-transgenic soybean plants at 0, 3, 6, 12, 24, 48 h after P. sojae infection. Values are relative to the value of mock plants at the same time point. Statistically significant differences were performed between the overexpression transgenic lines and non-transgenic lines. Three biological replicates with their three technical replicates were averaged and statistically analyzed using Student's t-test (^*P < 0.05; ^**P < 0.01). Bars indicate standard error of the mean.

Figure 7

The content of isoflavone components and the relative content of glyceollins in seeds of transgenic and non-transgenic soybeans. (A) The daidzein levels in seeds of transgenic and non-transgenic soybeans. (B) The glycitein levels in seeds of transgenic and non-transgenic soybeans. (C) The genistein levels in seeds of transgenic and non-transgenic soybeans. (D) The relative content of glyceollins in the seeds of transgenic and non-transgenic soybeans. The experiment was performed three biological replicates with their respective three technical replicates and statistically analyzed using Student's t-test (^**P < 0.01). Bars indicate standard error of the mean.

Figure 8

The transcript levels of the three genes (GmPAL, Gm4CL, GmCHS) in GmIFR transgenic and non-transgenic soybean plants after P. sojae infection using quantitative RT-PCR analysis. The amplification of the soybean Actin (GmActin4) gene was used as an internal control to normalize all the data. The relative transcript levels of the genes were quantified compared with mock plants at the same time point. The experiment was performed on three biological replicates with their respective three technical replicates and statistically analyzed using Student's t-test (^*P < 0.05; ^**P < 0.01). Bars indicate standard error of the mean.