Physiological responses and expression of VrDREB2A gene at different growth stages of mungbean (Vigna radiata L. Wilczek) under drought stress

8 Mungbean is an important pulse crop and commonly grown in Asia. Drought 9 affects mungbean growth and yield at at different growing stages and various levels 10 through physiological traits and gene expression. In this study, two mungbean cultivars, 11 DX208 and Tam Thanh Hoa, were exposed to drought at the vegetative and flowering 12 stages and assessed for various morphophysiological traits at 8, 12, 15 and 15 days post 13 withholding water and the plant recovery 7 days after re-watering. Differential 14 expression of VrDREB2A gene was observed in leaf and root of two mungbean cultivars 15 under drought condition. Plants used up water more quickly at the flowering stage than 16 the vegetative stage. Drought adversely affected the plant height, leaf number, above- 17 ground plant biomass and root weight with relative reduction to the control by 4.0 – 18 85%. Yield components and individual yield reduced significantly by around 50 – 60% 19 compared to the control. Relative expression of VrDREB2A gene was varied, with 20 stronger expression in leaves and roots when drought imposed at the flowering and 21 vegetative stages respectively. Increase in VrDREB2A expression occurred earlier at 8 22 days compared with 12 days for drought imposed at the flowering and vegetative stages 23 respectively, resulting in more tolerance of plants to drought at the flowering stage. The 24 results indicate that VrDREB2A functioned as an important transcriptional activator and 25 might help increase the drought stress tolerance of the mungbean plant at various 26 growing stages. Morphophysiological traits can also be used as indicators in screening 27 mungbean for drought tolerance. 28


INTRODUCTION
Drought is a common abiotic stress that is severe threats to crop growth and food 60 production worldwide and can lead to more than 50% yield loss (Singh et  GmDREB2A;2) were identified (Mizoi et al. 2013). Although the peptide sequences are 91 very similar to each other, the induction of GmDREB2A;2 was stronger than that of 92 GmDREB2A;1 and improved stress tolerance in Arabidopsis (Mizoi et al. 2013). The 93 DREB2A genes from different species, such as common wheat (Egawa et al. 2006), 94 pearl millet (Agarwal et al. 2007) and chrysanthemum (Liu et al. 2008 temporal and spatial distribution of rainfall, the crop often suffers from water stress at 105 any growth stages in its growing cycle leading to reduced growth and productivity. In     At the setup of the experiment, the pots were saturated with water and weighted.

154
All pots were weighted weekly to measure the water used by plants. After 8, 12, 15 or 155 20 days without water, all pots were also weighted to measure the water used. The 156 characteristics measured at 8, 12, 15 and 20 days post drought exposure included plant 157 height, leaf number, the fresh and dry weight of the above-ground mass and roots.

158
The recovery measurements included plant height, leaf number, yield 159 components and individual yield (g plant -1 ). The recovery was scored using 1 -4 scale:

Gene
Primer Sequence

227
Plant available water under water stress at vegetative and flowering stages 228 The plant available water (PAW) differed apparently among time duration 229 exposed to drought and mungbean cultivars (Table 2) (Table 3b). In general, relative reduction in plant growth was somewhat higher in G1.

248
Drought stress at vegetative as well as flowering stages significantly affected the 249 accumulation of plant biomass (Fig. 1). The fresh and dry plant weight, and fresh root 250 weight significantly reduced when exposed to drought at both vegetative and flowering 251 stage (Table 3 c-e). As soon as water was withheld, the plant and root fresh weights 252 dramatically reduced as compared to the control, with the rate of 5.3% to 16.1% at 8 253 days. The reduction was proportional with the time of exposure to drought with high 254 relative reduction, even up to 80% as observed for fresh root weight at 20 days.  Fig. 1 Severe symptoms of plants and roots after 20 days exposed to drought at the  (Table 4).     VrDREB2A when plants exposed to drought at flowering stage was ealier and higher in 312 comparison to the control both in leaves and roots (Fig. 3). Significant expression levels to recover after re-watering. Plants exposed to drought at the vegetative stage were able 356 to increase their height and leaf number after re-watering. However, the leaf number 357 was reduced after recovery 7 days since dropping and death of lower leaves near the 358 ground (Table 4). In addition, in this experiment, cultivar G2 seemed to be more 359 drought tolerant than cultivar G1 with smaller relative reduction in physiological traits, 360 and higher individual yields, recovery scores and drought resistance indices. VrSnRK2.6c, showed the highest level (12-fold) when 11-day-old mungbean plants in shoots in plants exposed to water stress. In contrast, drought stress led to increased 395 expression of GmRR34 in both soybean roots and shoots (Hoang et al. 2014).

396
The results of the present study clearly show that water stress adversely Severe symptoms of plants and roots after 20 days exposed to drought at the owering stage on two mung bean cultivars, DX208 (A) and Tam Thanh Hoa (B) (left: control, right: drought stress) Figure 2 Relative expression of VrDREB2A gene in leaves and roots of two mung bean cultivars, DX208 (G1) and Tam Thanh Hoa (G2), exposed to drought at the vegetative stage for (a) 8, (b) 12, (c) 15, (d) 20 days in comparison with the control Figure 3 Relative expression of VrDREB2A gene in leaves and roots of two mung bean cultivars, DX208 (G1) and Tam Thanh Hoa (G2), exposed to drought at the owering stage for (a) 8, (b) 12, (c) 15, (d) 20 days in comparison with the control