*Article* **Distinctive Physio-Biochemical Properties and Transcriptional Changes Unfold the Mungbean Cultivars Differing by Their Response to Drought Stress at Flowering Stage**

**Gunasekaran Ariharasutharsan 1,2,†, Adhimoolam Karthikeyan 3,†, Vellaichamy Gandhimeyyan Renganathan 2,3 , Vishvanathan Marthandan 3,4, Manickam Dhasarathan <sup>5</sup> , Ayyavoo Ambigapathi <sup>2</sup> , Manoharan Akilan 2,6 , Subramani Palaniyappan 1,2, Irulappan Mariyammal <sup>2</sup> , Muthaiyan Pandiyan 7,\* and Natesan Senthil 8,\***


**Abstract:** Mungbean is a nutritionally and economically important pulse crop cultivated around Asia, mainly in India. The crop is sensitive to drought at various developmental stages of its growing period. However, there is limited or almost no research on a comparative evaluation of mung-bean plants at the flowering stage under drought conditions. Hence, the aim of this research was to impose the drought stress on two mungbean cultivars VRM (Gg) 1 and CO6 at the flowering stage and assess the physio-biochemical and transcriptional changes. After imposing the drought stress, we found that VRM (Gg) 1 exhibited a low reduction in physiological traits (Chlorophyll, relative water content, and plant dry mass) and high proline content than CO6. Additionally, VRM (Gg) 1 has a low level of H2O<sup>2</sup> and MDA contents and higher antioxidant enzymes (SOD, POD, and CAT) activity than CO6 during drought stress. The transcriptional analysis of photosynthesis (*PS II-PsbP*, *PS II-LHC*, *PS I-PsaG/PsaK*, and *PEPC 3*), antioxidant (*SOD 2*, *POD*, *CAT 2*), and drought-responsive genes (*HSP-90, DREB2C, NAC 3* and *AREB 2*) show that VRM (Gg) 1 had increased transcripts more than CO6 under drought stress. Taken together, VRM (Gg) 1 had a better photosynthetic performance which resulted in fewer reductions in chlorophyll, relative water content, and plant dry mass during drought stress. In addition, higher antioxidative enzyme activities led to lower H2O<sup>2</sup> and MDA levels, limiting oxidative damage in VRM (Gg) 1. This was positively correlated with increased transcripts of photosynthesis and antioxidant-related genes in VRM (Gg) 1. Further, the increased transcripts of drought-responsive genes indicate that VRM (Gg) 1 has a better genetic basis against drought stress than CO6. These findings help to understand the mungbean response to drought stress and will aid in the development of genotypes with greater drought tolerance by utilizing natural genetic variants.

**Keywords:** abiotic stresses; biochemical response; drought; legumes; mungbean

**Citation:** Ariharasutharsan, G.; Karthikeyan, A.; Renganathan, V.G.;

Marthandan, V.; Dhasarathan, M.; Ambigapathi, A.; Akilan, M.; Palaniyappan, S.; Mariyammal, I.; Pandiyan, M.; et al. Distinctive Physio-Biochemical Properties and Transcriptional Changes Unfold the Mungbean Cultivars Differing by Their Response to Drought Stress at Flowering Stage. *Horticulturae* **2022**, *8*, 424. https://doi.org/10.3390/ horticulturae8050424

Academic Editors: Stefania Toscano, Giulia Franzoni and Sara Álvarez

Received: 13 February 2022 Accepted: 26 April 2022 Published: 10 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

With the effects of global warming and drastic climate changes, drought is the major abiotic stress that affects crop production in arid and semi-arid regions of the world. It is brought about by a scarcity of rain or a vast difference in rainfall quantity [1,2]. Drought impairs plant growth and development and accounts for over 70% of agriculture yield losses worldwide. However, it relies on the drought intensity, duration, phenophases of the crop, and environmental stress factors. An increasingly warming climate and decreased water availability are likely to upsurge the occurrence and severity of drought in the near future. Therefore, boosting the tolerance to drought is a major aim of crop improvement programs. Much progress has been made in understanding the effect of drought stress on plants. Decoding the molecular mechanisms underpinning plant response during drought is not easy due to the intricacy of drought behavior, environmental factors, and their interactions [3]. When subject to drought, plants undergo a series of morphological, physiological, and biochemical changes that seriously reduce plant growth and development [4,5] Physiological responses include (i) a reduction in the content of chlorophyll, rate of photosynthesis, and transpiration, (ii) stomatal closure, (iii) dehydration of cells [6–8]. Drought stress causes increased peroxidation of lipid membranes and mass accumulation of reactive oxygen species (ROS) [9–11]. The augmented ROS accumulation causes damage to proteins, lipids, cell membranes, carbohydrates, and nucleic acids and leads to disruption of cellular homeostasis and subsequently cellular death [12]. Both enzymatic and non-enzymatic antioxidant systems are fundamental to protecting the cells against toxic ROS and minimizing the oxidative stress effects [13–17]. Previously, it was shown that ROS production under drought stress can be minimized by increasing the antioxidant enzymatic activities in mungbean [18]. Masoumi et al. [19] reported that tolerant soybean plants enhanced their antioxidant enzyme activities and antioxidant contents in response to drought stress, whereas drought-sensitive plants were unable to do so. The lower level of MDA along with enhanced activities of SOD and CAT in black gram plants can be linked to its ability to cope up with water scarcity by limiting the damaging effects of drought through up-regulation of antioxidant enzymes [20].

Physiological and transcriptome responses of soybean to drought stress were investigated by Xu et al. [21]. Physiological traits such as photosynthetic rate, stomatal conductance, transpiration, and water potential were reduced, while SOD and CAT activities were enhanced, and POD activity remained unchanged. Furthermore, in drought-stressed plants, a total of 2771 differentially expressed genes were identified, and they were involved in different biochemical and molecular pathways, including ABA biogenesis, compatible compound accumulation, secondary metabolite synthesis, fatty acid desaturation, and transcription factors. In another study, Mahdavi Mashaki, et al. [22] employed RNA-Seq to investigate transcriptome profiles in drought-responsive contrasting genotypes of Iranian kabuli chickpea under drought stress in root and shoot tissues at the early flowering stage. Of these, 261 and 169 drought stress-responsive genes were identified in the shoots and the roots, respectively, and 17 genes were common in the shoots and the roots. Several molecular mechanisms are involved in the stress response and their corresponding drought-related pathway, (i.e., ABA, proline, and flavonoid biosynthesis). Lopez et al. [23] showed the importance of phosphorous homeostasis, as well as several other key factors, in response to drought stress in the common bean. Upregulation of several key transcription factors, remodeling of cell walls, synthesis of osmoprotectant oligosaccharides, protection of the photosynthetic apparatus, and downregulation of genes involved in cell expansion were all revealed by RNA-seq analysis of the drought-tolerant landrace PHA-683 in response to drought, but there was a significant proportion of DEGs related to phosphate starvation response.

Mungbean (*Vigna radiata*), native to India, is a short-duration grain legume and is extensively cultivated in Asia. This crop mainly features high protein (25%) and nutrient (carbohydrates, lipids, minerals, and vitamins) contents. It also improves soil fertility by fixing atmospheric nitrogen [24]. India is the world's leading mungbean grower, with 2.17 million tonnes of grains from a 4.32 m ha area. Mungbean yield in India is still low (502 kg/ha), considerably lower than the productivity of other main pulse crops [25]. In India, mungbean is mainly grown under rainfed conditions at high temperatures, with low humidity and rainfall. Thus, the mungbean is exposed to drought at various developmental stages of its growing period [26,27]. This crop is comparatively surviving under drought conditions. However, in comparison to other developmental stages, mungbean growth is sensitive to drought during flowering and post-flowering. Drought stress during these stages can decrease the grain yield ranging from 30% to 80%. Regardless of their importance, the studies that investigated the impacts of drought-influenced mungbean are limited [28,29]. Under drought, comparative evaluation of physio-biochemical and transcriptional changes between mungbean cultivars at the flowering stage lacks existing information in the literature. Taking into account the above, in this research, we aimed at revealing the physio-biochemical and transcriptional alterations in two mungbean cultivars (VRM (Gg) 1 and CO6) at the flowering stage under drought conditions.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Drought Treatment*

Seeds of mungbean varieties CO6 and VRM (Gg) 1 were provided by Agricultural Research Station, Tamil Nadu Agricultural University, Virinjipuram, Tamil Nadu, India. The seeds for two varieties were sown in pots (15 L, 30 cm height, 33.0 cm diameter at top and 25.5 cm bottom diameter) containing 3:1 ratio of soil and compost in a greenhouse at Agricultural College and Research Station, Madurai, Tamil Nadu, India. The experimental design was a completely randomized design (CRD) with three replications of 15 plants (5 plants per pot). During the experiment period, the temperature was maintained at 28 ◦C, and relative humidity was 65%. Three different sets of plants were maintained. Watering was done regularly until the flowering started. When the flowering was observed, drought stress for 6 days (soil moisture, 50%) and 12 days (soil moisture, 25%) were imposed in two sets; the third set was kept as the control 0 days (soil moisture, >80%). Soil moisture was measured using Lutron PMS-*714* soil moisture meter.

#### *2.2. Plant Sampling*

The fully expanded leaves from three to five plants were sampled following the 0, 6, and 12 days of drought stress and immediately frozen into liquid nitrogen and then stored at −80 ◦C. Leaf relative water content (RWC), chlorophyll content, leaf gas exchange parameters, and plant dry mass were used to estimate at 0 and 12 days of drought stress plants. Proline, protein, hydrogen peroxide (H2O2), malondialdehyde (MDA), enzymatic (SOD, POD, and CAT), and non-enzymatic (Ascorbic acid) antioxidants assays were conducted at 0, 6, and 12 days of drought stress plants. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted at 0 and 12 days.

#### *2.3. Physiological Traits*

Total chlorophyll content was measured according to Arnon [30]. Plant dry mass was measured after drying the plant at 80 ◦C to a constant weight. RWC was measured as the standard method described by Barrs and Weatherly [31]. RWC = (FW − DW)/(TW − DW) × 100, where FW is the fresh leaf weight, DW is the dry leaf weight, and TW is the turgid weight of the leaves. Photosynthetic gas exchange parameters were measured by portable photosynthesis system Li-6400 (LiCor, Lincoln, NE, USA).

#### *2.4. Proline Estimation*

Proline content was estimated according to the methodology described by Bates et al. [32]. Leaf samples (1 g) were taken and homogenized in 3% aqueous sulphosalicylic acid and centrifuged at 10,000 rpm for 15 min at 4 ◦C. The supernatant was carefully taken, and then the acid-ninhydrin solution (1.25 g of ninhydrin in 30 mL glacial acetic acid) was added. The mixture was then incubated for an hour at 100 ◦C and cooled in ice to stop

the reaction. For extracting the reaction mixture 4 mL of toluene was added and vortexed thoroughly for 2 min. The solute is then measured for absorbance at 520 nm. Toluene was considered as a blank, and the content of proline was calculated giving to the formula: [(µg proline/mL × mL toluene)/115.5 µg/µmole]/[(g sample)/5] = µg proline/g FW.
