Identification of drought stress-responsive proteins in common bean

Proteomics has emerged as a vital tool for high throughput systematic analysis of protein expression which helps to unlock various biochemical pathways. Keeping in view the relevance of differential expression studies at the post transcriptional level, we attempted to explore the candidate proteins that are triggered under low water stress conditions at vegetative and reproductive stages in common bean. The evaluation of drought-responsive proteins was performed by resolving the quantified protein extracted from leaf samples using SDS PAGE for detection of differential bands followed by trypsin digestion and MALDI-MS analysis. We report the presence of seven significant proteins where five proteins (Glucan endo-1.3 beta-glucosidase, Auxin-responsive protein, Endochitinase, Endochitinase CH5B and L-type lectin domain containing receptor kinase IV.3) were upregulated and two proteins (Ribulose bisphosphate carboxylase/oxygenase activase, ADP-ribosylation factor-like protein 8a) were down regulated. The study conducted herein indicates the strong interaction and varied activation of proteins at both the developmental stages. The identified proteins are primarily localized in nucleus, cell membrane, cytoskeleton, vacuole and chloroplast and play significant roles in plant defense, photosynthesis, and various molecular regulatory processes.


Introduction
Common bean (Phaseolus vulgaris L.) is appraised for being the most important food legume holding tremendous medicinal potential coupled with substantial amount of micronutrients (Fe, Zn, Ca, Mg, P, K, Na) and vitamins (Vitamin A, B6 and B7). Commercially cultivated across the world, the crop represents 50 percent of the total grain legumes consumption (McClean et al. 2004) with most of the production concentrated in Latin America and Eastern Africa (Varshney et al. 2012). Among various developing countries, India secures the first rank in dry bean production, whereas fourth in green been production (FAO database 2015). However, various biotic and abiotic factors limit the productivity of the crop to a great extent where reductions in common bean yields have been observed to be as high as 88% due to severity of the drought (Singh 2007). The situation has become more drastic on account of frequent and prolonged drought prevailing conditions with higher temperatures that adversely affect the adaptation of bean genotypes and curtailed root growth (Thorton et al. 2009). Previous reports indicating the significant role of the crop in terms of nutrition and livelihood (WMO 1991) and measureable reductions in grain yield (Pimentel et al. 1999;Nielsen and Nelson, 1998;Ramirez-Vallejo and Kelly 1998), strongly suggest that there is dire need to take immediate action for making the crop adaptable to drought-prone environments. Amidst several strategies, massive reports have been communicated that approach to understand the molecular regulation in response to various biotic and abiotic environmental conditions by means of proteomics approach Staudinger et al. 2012;Zadraznik et al. 2012;Zargar et al. 2013;Muneer et al. 2014;Irar et al. 2014). For elucidating the biological mechanisms more efficiently, proteomics study is proven advantageous over genomic and transcriptomic studies as it takes into account several post transcriptional modifications as well which can further advance our understanding of the molecular regulators involved in various catabolic and anabolic pathways. In this regard, an experiment for detection of proteins actuated during water deficiency and understanding their possible strategies of action in common bean plant was executed using MALDI-MS approach.

Materials and methods
To examine various drought-responsive proteins in common bean, we used VLR-125 variety procured from Almorha, VPKAS, India. The seeds were germinated in greenhouse and low water stress was imposed at two developmental stages (i.e., vegetative as well as reproductive stages). The detailed procedure followed during the course of study where isolated protein samples from different treatments were subjected to SDS PAGE electrophoresis followed by trypsin digestion of differential bands and analysis for the presence of proteins using MALDI-MS technique are discussed in the following sections.

Protein isolation
Viable seeds checked by incubation at 25 °C temperature with 16 h light/8 h dark under BOD incubator were germinated in pots kept in greenhouse where 25 °C temperature and 80% relative humidity were maintained. Water deficiency stress was imposed after 22 days and 42 days of germination during vegetative stage and flowering stage, respectively. Half of the plants were well watered in both the developmental stages by providing the steady amount of water at regular intervals, whereas the other plants were deprived of water supply in both the developmental phases for 15 days. Total protein was isolated from the leaves collected from plants subjected under different treatments using TCA-acetone-based extraction method. This method is comparatively simple and preferred over phenol extraction protocol when high-quality resolution of proteins is desired (Bhardwaj and Yadav 2013). Sodium-phosphate buffer (0.1 M, pH 7.6) was poured in centrifuge tubes and the fine powder obtained by grinding 1 g of leaf sample in liquid nitrogen was transferred to it. Supernatant obtained from the homogenous mixture by performing the centrifugation for 15 min at 12,000 rpm and 4 °C was collected in other tube discarding the debris and treated with chilled 10% TCA-acetone solution (10 g of TCA dissolved in 100 ml of acetone containing 0.08% β-mercaptoethanol) and kept overnight at − 20 °C. The pellet obtained after centrifugation was then washed with pure acetone (chilled) supplemented with a Roche tablet for protease activity inhibition, EDTA (20 mM) and 0.08% β-mercaptoethanol, allowed to air dry for solubilization in extraction buffer and stored at − 80 °C. BSA standard curve was prepared and used as reference to estimate the concentration of unknown protein samples.

Proteomic analysis
For resolution of proteins, each sample (65 µg) was mixed with 1X Tris-glycine buffer (0.025 M Tris base, 0.912 M Glycine and 0.1% SDS), exposed to 80 °C for 2 min and immediately shifted to ice for 5 min for preventing the renaturation of proteins. The samples and the molecular ladder were loaded onto acrylamide gel (5% stacking gel, pH 6.8 and 10% resolving gel, pH 8.8) and subjected to electrophoresis performed at 60 V using Tris-glycine tank buffer. The gel was stained (50% methanol, 10% GAA and 0.05% coomassie brilliant blue R-250), placed at rotating platform for 2 h, further rinsed with ddH 2 O for 1 min and finally treated with destaining solution (30% methanol and 10% glacial acetic acid) to make the background clear. The differential expression of the protein samples at both the stages was examined by observing the distinct banding patterns (Fig. 1). For trypsin digestion, the differentially expressed bands prominent in both the stages were excised from the gel, cut into very small cubes using sharp scalpel and transferred into sterilized 1.5 mL microcentrifuge tubes using forceps. To remove the excessive stain from the gel slices, 500 µl of destaining solution (25 mM ammonium bicarbonate, 50% acetonitrile) was poured to the tubes, agitated and incubated at room temperature for 15 min. The solution was removed and the process was repeated two to three times for complete disappearance of the stain. The slices were then treated with 100 µl acetonitrile (40%), gently swirled and allowed to dry after careful removal of acetonitrile using pipette. 100 µl of reducing solution (10 mM DTT, 50 mM ammonium bicarbonate) was added to the samples followed by incubation at 56 °C for 30 min. The reducing solution was discarded and 100 µl of alkyl solution containing 55 mM iodoacetamide and 50 mM ammonium bicarbonate was added to the tubes and incubated in the dark for 30 min. The wash solution was discarded and the samples were mixed and incubated for 5 min with 100 µl acetonitrile (40%) for dehydration, again rehydrated and finally trypsin digestion was performed for MALDI-TOF MS analysis. The significance of a particular protein was determined by calculating protein score, i.e., 10*Log (p) where the probability value (p ≤ 0.05) ensures that the observed match is not a random event.

Results and discussion
The concentration of protein samples was determined using standard BSA and found to be ranging from 4.31 to 4.87 µg/ µl. Resolution of protein samples in SDS PAGE showed the presence of twenty-one differential bands. At flowering stage, six bands were expressed distinctively in case of drought stress-treated sample and a single differential band was observed for well-watered sample. At vegetative stage, stress-treated sample generated five differential bands in contrast to nine bands that were found to be expressed in the well-watered sample. We also found varied expression between drought-responsive proteins that belonged to two different developmental stages. The bands that were most prominently expressed in vegetative as well as flowering stages were analyzed using MALDI-MS. Among the six selected bands, four bands were expressed in low water stress conditions (CB1, CB2, CB3, and CB4) and two bands belonged to the samples that were well watered (CB5 and CB6). CB1 band showed the presence of two proteins, i.e., mediator of RNA polymerase II transcription subunit 19a and glucan endo1, 3-beta-glucosidase. However, only the later was found significant in which thirty-six peaks were obtained for mass to charge ratio (m/z) and nine matches corresponded to known peptides reported in the database. Two proteins namely Auxin-responsive protein IAA6 and F-box/LRR-repeat protein 16 were detected in case of CB2 band, where only the former protein was considered significant which yielded a total of 25 peaks for m/z ratio and eight matches for the reference peptides. Three proteins were identified for CB3 band where endochitinase and endochitinase CH5B yielded eight and seven matches, respectively, for m/z ratio were found significant and ferredoxin-thioredoxin reductase catalytic chain, chloroplastic protein was non-significant. In CB4 band, two proteins were found to be expressed ,i.e., L-type lectin domain containing receptor kinase IV.3 and pentatricopeptide repeat-containing protein At1g06140, mitochondrial where the former presented significant results with twenty-two peaks and nine matches.
Two bands from each of the developmental stage were examined for well-watered conditions. In the flowering stage, out of the two proteins found to be present in CB5 band, i.e., protein ribulose bisphosphate carboxylase/oxygenase activase, chloroplastic and protein Ras-related protein RABH1c, only the former was considered significant with nine matches from a total of thirty peaks. CB6 band detected in case of well-watered conditions at vegetative stage showed the presence of two proteins, i.e., ADP-ribosylation factor-like protein 8a and ADP-ribosylation factorlike protein 8b where only the former was found significant with six matches among thirty-two peaks generated for m/z ratio. The cellular location and biological roles of the proteins identified and regulated under drought stress conditions are illustrated in Table 1.

Glucan endo-1, 3-β-glucosidase
The protein Glucan endo-1,3 -beta-glucosidase is mainly present in vacuole and plays a significant role in carbohydrate metabolism and plant defense mechanism (Nakatani et al. 2010). In rice seedlings, β-glucosidase was found to be expressed in response to abscisic acid, methyl jasmonate, salt and submergence stress (Oppasiri et al. 2007). Prior reports indicate the involvement of the gene encoding glucan endo-1,3-beta-glucosidase in pathogen infection (Vogeli-Lange et al. 1994;Meirinho et al. 2010), cold stress (Hincha et al.1997;Soltanian et al. 2014) and ozone treatment (Pell et al. 1997). Moreover, a study conducted in Pinus pinaster for drought stress responses using PEG treatment and cDNA-AFLP technique also showed the expression of genes encoding glucan endo-1, 3-β-glucosidase protein (Dubos et al. 2003). Further, the protein has been previously reported to be expressed under water deficiency stress in cotton (Cui et al. 2000), Lupinus albus (Alves et al. 2006) and common bean (Yang et al. 2011). Thus, the expression of the aforementioned protein in various reports strongly suggests its role in biological mechanisms triggered under the influence of biotic as well as abiotic stresses.

Auxin-responsive protein
Auxin-responsive proteins are mainly the transcription factors that regulate the transcription and translation processes in the auxin-activated signaling pathway where they repress the auxin response genes at low auxin concentrations 1 3 (Weijers et al. 2016). Drought stress-responsive upregulation of the protein in our study indicates that there is considerable repression of gene expression mechanisms. ARFs were reported to be mainly responsible for regulating auxin signaling pathway during root development when subjected to low water stress in soybean (Quach et al. 2014). Further, the function of auxin response factors (ARFs) in Dendrobium officinale L. has been detected where the proteins were found to intervene auxin signaling pathways for adaptation to the unfavorable ambience (Chen et al. 2017). The upregulation of auxin-responsive protein in our study strongly supports the interrelationship existing between defense mechanisms and auxin signaling pathways and that these mechanisms coupled with many pathways have a unified effect on molecular regulation during stress conditions.

Endochitinase and endochitinase CH5B
Endochitinase proteins belong to the family GH19 (glycoside hydrolase family), mainly present in the vacuole and considered critical in defensive responses (Mellacheruvu et al. 2016a). Sarjaz et al. (2011) traced the impact of critical atmospheric conditions on productivity of the crop. It was reported that the plant isotopic composition and water-use efficiency were strongly influenced by the expression of chitinase gene in transgenic strawberry. The function of pigeonpea hybrid-proline-rich protein encoding gene (CcHyPRP) was demonstrated in transgenic rice (Mellacheruvu et al. 2016b). The study revealed an enhanced expression of genes encoding catalase, superoxide dismutase (SOD) enzymes and endochitinase with reduction in malondialdehyde (MDA) synthesis. Further, the activation of molecular chaperone CsHSP17.5 and the endochitinase CsCh3 demonstrated in two chestnut seed proteins when subjected to thermal stress and microbial infection further substantiate the contribution of endochitinases during various adverse conditions (Gomez and Aragoncillo 2001).

L-type lectin domain containing receptor kinase IV.3
The protein is an intrinsic component of cell and plasma membrane which acts as receptors and transferases that play an active part in regulation mediated by serine/threonine kinases. Previous reports indicate strong evidences for the close involvement of kinases and phosphatases in activation or inactivation of ion channels under various stress conditions (Schroeder and Hagiwara 1989;Negi et al. 2008;Pei et al. 2000;Lee et al. 2009;Vahisalu et al. 2008;Ward et al. 2009;Chen et al. 2010). Under low water conditions, ABA triggers stomatal closure through signaling processes mediated by secondary messengers (ROS, nitric oxide, Ca 2 + , and protein kinases) targeting ion channels. Also, ABA binding stimulates structural changes in various proteins which target kinases and anion channels for transcriptional regulation (Geiger et al. 2009;Lee et al. 2013). In addition, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase were expressed in susceptible sunflower genotypes (Castillejo et al. 2008) and functional redundant kinases for abscissic acid and stress signaling have been communicated (Fujii et al. 2009;Nakashima et al. 2009).

Ribulose bisphosphate carboxylase/oxygenase activase
Our study depicted comparatively less expression of ribulose bisphosphate carboxylase/oxygenase activase protein in stressed plants. Previous reports communicated that environmental stress has immense impact on metabolism of plants.
The exposure of stress to several glycophytic plants and crops leads to reduced CO 2 assimilation rate which limits the abundance of RubisCO activase and their subunits, components of OEC complex and carbonic anhydrase by enhancing their disintegration (Aghaei et al. 2008;Pang et al. 2010;Sobhanian et al. 2010;Bandehagh et al. 2011;Chattopadhyay et al. 2011). Moreover, drought stress promoted the down regulation of proteins and enzymes that actively participate in photosynthetic pathways which is attributed to the fact that less water uptake triggers stomatal closure thereby restricting CO 2 uptake by leaves to a great extent (Yokota et al. 2002;Chaves et al. 2003;Medici et al. 2007;Castillejo et al. 2008). Further, downregulation of RubisCO carboxylase activity and RuBP protein synthesis has been observed in rice subjected to salt stress due to inadequate expression of transketolase (TK) as they catalyze the regeneration of ribulose-1,5-bisphosphate (Kim et al. 2005).

ADP-ribosylation factor-like protein 8a
The protein forms cellular component of the endosomal, lysosomal and plasma membranes, primarily observed in azurophil and ficolin-1-rich granule membrane and extracellular exosome (Nei et al. 2002;Lee et al. 2002). A genomewide survey performed in rice and foxtail millet depicted the presence of thirty-six putative ARF proteins and conserved ADP-ribosylation factor using ScanProsite analysis (Muthamilarasan et al. 2016). The impact of biotic stress on eight different plant species studied by conducting microarray experiments revealed the downregulation of photosynthesis and corresponding upregulation of genes involved in the jasmonic acid, salicylic acid, and ethylene signaling pathways (Bilgin et al. 2010). Moreover, the photosynthetic genes that were downregulated were also found to be involved in defense responses which strongly suggest the interference of biochemical pathways. Another study conducted in common bean also revealed the expression of proteins closely 1 3  intricated with mechanisms related to energy metabolism, photosynthesis, ATP interconversion, protein synthesis and defense mechanisms in response to water scarcity (Zadraznik et al. 2012). Maize plants subjected to PEG-mediated drought stress demonstrated the presence of proteins that are known to have critical role in signaling, plant defense, carbohydrate and protein metabolism as well as suggested strong interrelationship among these proteins (Shao et al. 2015). In addition, the altered reduced photosynthetic gene transcription has been noticed due to drought, salinity and low temperatures (Saibo et al. 2009). Further, the influence of nitrogen deficiency on photosynthetic machinery and its regulation was observed and supported the fact that induction of defensive proteins or compounds may prompt the unavailability of nitrogen in Rubisco and vice versa (Stitt and Schulze 1994;Pauland Foyer 2001) Thus, the prior reports on drought stress responses in various crops further validate and strengthen our results.

Conclusion and future prospects
In summary, the study aimed to detect low water stressresponsive proteins expressed during vegetative as well as flowering stage in common bean, a brief outline of which has been shown in Fig. 2. Relying on MALDI-TOF MS approach, we were able to identify seven proteins out of which five were upregulated and two were downregulated when drought stress was imposed. The differentially expressed proteins were involved in vital processes such as plant defense, photosynthesis and various vital molecular mechanisms. The downregulation of proteins involved in photosynthetic processes under drought stress conditions indicates a strong interdependency of these proteins in essential biochemical pathways and physiological processes. This study presents inclusive perception pertaining to highly correlated expression between photosynthetic and defense responsive proteins. Further, investigation can bring new insights and unravel the metabolic pathways that conjoin these two crucial mechanisms triggered upon stress response. Thus, the identified proteins can be used as molecular regulators for understanding the very basis of biological processes which in turn will pave the way for developing drought resistant crops.