Virus-induced down-regulation of GmERA1A and GmERA1B genes enhances the stomatal response to abscisic acid and drought resistance in soybean

Abstract

Drought is a major threat to global soybean production. The limited transformation potential and polyploid nature of soybean have hindered functional analysis of soybean genes. Previous research has implicated farnesylation in the plant's response to abscisic acid (ABA) and drought tolerance. We therefore used virus-induced gene silencing (VIGS) to evaluate farnesyltransferase genes, GmERA1A and GmERA1B (Glycine max Enhanced Response to ABA1-A and -B), as potential targets for increasing drought resistance in soybean. Apple latent spherical virus (ALSV)-mediated GmERA1-down-regulated soybean leaves displayed an enhanced stomatal response to ABA and reduced water loss and wilting under dehydration conditions, suggesting that GmERA1A and GmERA1B negatively regulate ABA signaling in soybean guard cells. The findings provide evidence that the ALSV-VIGS system, which bypasses the need to generate transgenic plants, is a useful tool for analyzing gene function using only a single down-regulated leaf. Thus, the ALSV-VIGS system could constitute part of a next-generation molecular breeding pipeline to accelerate drought resistance breeding in soybean.

Fig 1. ALSV infection induces VIGS of GmERA1s in soybean.

Silencing efficiency was evaluated by examining the expression of GmERA1s using quantitative RT-PCR and total RNAs derived from the fourth trifoliate leaves of soybean plants inoculated with the indicated inoculums. A conserved region in the two GmERA1 genes was amplified by qRT-PCR. Relative values are presented as mean ± SD (n = 3) normalized to the expression of GmACT11 (Glyma15g05570) as a control. Asterisks indicate a significant difference from ALSV-VC (empty vector) by t-test (*P < 0.05).

Fig 2. GmERA1-down-regulated leaves show reduced water loss rates and stomatal conductance.

(A) Rates of water loss in the fourth or fifth trifoliate leaves of soybean plants inoculated with ALSV vectors. Each data point represents the mean of duplicate measurements (n = 3 for ALSV-VC and n = 6 for ALSV-GmERA1). Bars indicate SD. Asterisks at 0.3 and 0.5 h indicate a significant difference between ALSV-VC and ALSV-GmERA1 by t-test (*P < 0.05, **P < 0.01). (B) The surface temperature of detached fourth or fifth trifoliate leaflets of soybean plants inoculated with ALSV vectors was measured using thermography during the water-loss experiment shown in (A). Thermal images and photographs were taken 11 min after detachment. (C and D) Evaluation of leaf temperature (C) and stomatal conductance (D) in detached fourth or fifth trifoliate leaflets of soybean plants inoculated with ALSV vectors using a LI-6400XT portable photosynthesis system. Values are presented as mean ± SD (n = 3 for ALSV-VC and n = 4 for ALSV-GmERA1), and asterisks at 4, 8, and 12 min indicate a significant difference between ALSV-VC and ALSV-GmERA1 by t-test (**P < 0.01).

Fig 3. Down-regulation of GmERA1s enhances the stomatal closure response to ABA.

Leaf disks from the fourth trifoliate leaves of ALSV-infected soybean plants were treated with or without ABA for 4 h. (A) Representative photographs of leaflets after ABA treatment (scale bars = 20 μm). (B) Stomatal aperture size data (shown as dots) from six independent measurements (≥60 stomata each). Bars indicate mean values. *P = 0.010, by t-test.

Fig 4. Evaluation of drought resistance in ALSV-GmERA1-infected soybean plants.

Watering was withheld from plants at the V6 growth stage for 3 days. The drought status of the fourth to sixth trifoliate leaves was scored from 0 to 4 as follows: 0, no change; 1, wilting; 2, rolling; and 3, severe wilting. (A) Representative photographs of leaves with various scores. The leaves were scored at several time points after water withholding. (B) Leaf rolling score before rehydration (72–75 h after water withholding). The trial was repeated independently six times. (C) Plants were re-watered at 3 days after water withholding. Representative plants at one day after rehydration are shown.