Overexpression of AtDREB1A causes a severe dwarf phenotype by decreasing endogenous gibberellin levels in soybean [Glycine max (L.) Merr]

Abstract

Gibberellic acids (GAs) are plant hormones that play fundamental roles in plant growth and developmental processes. Previous studies have demonstrated that three key enzymes of GA20ox, GA3ox, and GA2ox are involved in GA biosynthesis. In this study, the Arabidopsis DREB1A gene driven by the CaMV 35S promoter was introduced into soybean plants by Agrobacterium- mediated transformation. The results showed that the transgenic soybean plants exhibited a typical phenotype of GA-deficient mutants, such as severe dwarfism, small and dark-green leaves, and late flowering compared to those of the non-transgenic plants. The dwarfism phenotype was rescued by the application of exogenous GA(3) once a week for three weeks with the concentrations of 144 µM or three times in one week with the concentrations of 60 µM. Quantitative RT-PCR analysis revealed that the transcription levels of the GA synthase genes were higher in the transgenic soybean plants than those in controls, whereas GA-deactivated genes except GmGA2ox4 showed lower levels of expression. The transcript level of GmGA2ox4 encoding the only deactivation enzyme using C(20)-GAs as the substrates in soybean was dramatically enhanced in transgenic plants compared to that of wide type. Furthermore, the contents of endogenous bioactive GAs were significantly decreased in transgenic plants than those of wide type. The results suggested that AtDREB1A could cause dwarfism mediated by GA biosynthesis pathway in soybean.

Figure 1. Herbicide tolerance and qRT-PCR analysis of transgenic plants.

A: Detection of herbicide tolerance on AtDREB1A-transformed plants. B: Expression analysis of the AtDREB1A gene in transgenic plants. Data represents six biological replications, and error bars represent SE. WT, wide type; L1: AtDREB1A transgenic line 1; L2: AtDREB1A transgenic line 2; CK: control check, without GA₃ treatment; GA₃: 144 µM GA₃ treatment.

Figure 2. Phenotypes of AtDREB1A-overexpressing soybean plants.

A: Phenotypes of 3-week-old T₃ transgenic and WT soybean plants. B: Leaf phenotype of 3-week-old T₃ transgenic and WT soybean plants. C: Seeds of transgenic and WT soybean plants. D: The first to fourth leaf acreage of 3-week-old transgenic and WT soybean plants. E: The chlorophyll content of first and second leaf in transgenic and WT soybean plants. WT: wide type; L1: AtDREB1A transgenic line 1; L2: AtDREB1A transgenic line 2. Values are the mean of six biological replicates ± SE, the same letter on each column set indicates no significant difference and different letters are significantly different by the analysis of variance (ANOVA), p<0.05.

Figure 3. Effects of GA₃ on phenotypic restoration.

A: Phenotypes of transgenic plants under 0 µM, 60 µM, 144 µM or 288 µM GA₃ treatments for 7 days and 14 days. B: The plant height of eight weeks of WT and transgenic plants after treated with or without 144 µM GA₃ for two weeks. WT: wide type; L1: AtDREB1A transgenic line 1; L2: AtDREB1A transgenic line 2; L1+L2: AtDREB1A transgenic plants line1 and 2. Values are the mean of six biological replicates ± SE, the same letter on each column set indicates no significant difference and different letters are significantly different by the analysis of variance (ANOVA), p<0.05.

Figure 4. Expression levels of GA metabolism related genes.

β-Tubulin was used as an internal control. GA20ox5: GmGA20ox5 (Glyma13g09460); GA3ox6: GmGA3ox6 (Glyma17g30800); GA2ox1: GmGA2ox1 (Glyma02g01330); GA2ox2: GmGA2ox2 (Glyma10g01380); GA2ox4: GmGA2ox4 (Glyma11g00550); GA2ox6: GmGA2ox6 (Glyma13g33290); GA2ox7: GmGA2ox7 (Glyma13g33300); GA2ox8: GmGA2ox8 (Glyma15g10070). Values are the mean of four biological replicates ± SE.