**4. Response to drought on the molecular level**

Understanding the changes in metabolic pathways in plants under the influence of drought, as well as the molecular mechanisms regulating their adaptation to this stress, is very important in identifying key molecular markers that could help distinguish between genotypes with different tolerance. On the molecular level, drought affects plant cells in different ways—through changes in gene expression and/or translation of transcripts to proteins, through posttranslational modification leading to protein activation and by further direct action on the protein itself. It is important to underline that these ways are interdependent and that only active key proteins enable a response beneficial for the plant.

Screening genes with differential expression and proteins with changed abundance or activity in plants exposed to drought is greatly facilitated by modern transcriptomic and proteomic tools which have, together with other approaches, enabled rapid development of the field. Identification of detected genes and proteins is greatly facilitated by the recent advances in sequencing and the publication of full genome sequences of model legumes *Medicago truncatula* [48] and *Lotus japonicus* [49] and of crop legumes, such as, common bean [50], soybean [51], chickpea (*Cicer arietinum*) [52] and peanut [53]. Comprehensive lists of genes and proteins obtained from screening studies are then classified according to their

*Drought - Detection and Solutions*

for further breeding applications.

**3. The physiology of drought response**

as well as having an additional toxic effect on the plant [34–36].

as well as supporting breeding programs with genotypes showing different environmental adaptations. Characterization and evaluation of this germplasm are an ongoing process and have confirmed the very broad genetic diversity of common bean in Eastern Europe. Our recent proceedings have resulted in formation of a core collection having applicative value for direct breeding purposes [32]. Screening for representative genotypes for core collection included initial evaluation of basic multi-crop passport descriptors (e.g., geographic origin, biological status, and ancestral data), phenotypic seed characteristics and phaseolin type, as well as assessment of genetic structure by genotyping with genetic markers. The resulting core collection encompasses 63 accessions representing the global genetic diversity and 14 standard genotypes with desirable traits from the East European region (unpublished data) and was evaluated under field conditions as well as for the presence of genetic markers associated with traits of interest and biochemical analysis. Core collection was further evaluated for agronomic traits in field conditions (response to abiotic stress), genetic markers for desirable traits and nutritional traits of importance (multi-elemental composition, fats, proteins, and phytic acid). These results enabled selection of superior genotypes in core collection

The observed physiological changes in plants exposed to drought can be a direct consequence of drought, as well as of the response of the plant, in order to mitigate the stress. Drought typically occurs as a result of low and non-frequent precipitation, resulting in reduced soil water content that is first detected by plant roots [33]. Depending on drought duration and severity the water status of plants can be affected by insufficient water absorption due to low soil water availability, as well as to increased water loss in the process of transpiration. Water deficit in plants affects their normal physiological processes and hinders the development, growth and yield, ultimately resulting in wilting, senescence and plant death [33]. Drought can be potentiated by heat stress, which propagates the water loss from the plant by increased water evaporation from the leaves, and by soil salinity stress, together reducing soil water availability

Drought responsive traits have been studied in common bean by evaluating various traits in field experiments as well as in more controlled environments, such as, greenhouses [37]. Phenological and yield-associated traits have been studied frequently because they are affected by drought stress, are an important indicator of yield output and are also measurable in a large phenotyping population. Phenological traits can also represent the adaptation of the life cycle of a plant to specific drought conditions in the environment. Breeding for earliness is an effective strategy for increasing the yield stability in regions such as the Mediterranean, where plants are exposed to increased drought in the summer time. Early flowering can help in drought avoidance; however, it is not effective in mitigating the drought

The response to drought in different common bean genotypes has been characterized in several studies based on physiological measurements such as photosynthesis and photosynthate acquisition as well as on partitioning indices [5, 38–44]. Photosynthesis and cell growth are primary processes influenced by drought due to decreased stomata conductivity in the early drought phases that limits evaporation and CO2 diffusion in the leaf mesophyll. The surplus of energy on the thylakoids in the photosynthesis apparatus results in photo inhibition—reduced photochemical efficiency [45]. Stomata closure is an effective strategy for shorter drought periods and for mild drought where photosynthesis is not affected in such a way as to reduce

**112**

stress once it occurs [9].

known ontologies in order to further investigate their interactions and connections, by methods of bioinformatics and systems biology, into metabolic pathways. A combination of these approaches has enabled the identification of thousands of genes with differential expression and hundreds of proteins with changed abundance in common bean under drought [15, 17, 54–56] as well as other model, crop and forage legumes [57].

#### **4.1 Transcriptomic profiling of drought response**

Early transcriptomic profiling methods employed over the past decades utilized polymerase chain reaction (PCR) and hybridization techniques and allowed for detection of a smaller number of transcripts with a large difference in mRNA abundance between compared samples [58, 59]. Today these methods are being replaced by genome-wide profiling techniques, such as, microarrays and whole-genome mRNA sequencing (RNA-seq). Further intricacies of gene regulation are explored by profiling miRNAs, small non-coding RNAs that regulate gene expression [55, 56, 60–62].

The transcriptomic response of common bean has been investigated in various plant organs of different genotypes with respect to different stages of drought severity [56]. One of the first studies focused on roots, the first plant organ in which to detect changes in soil water content [55]. Several dehydration-related genes were identified that are associated with signaling, protein homeostasis and root growth modulations, among which a gene *PvOCT1*, encoding a new type of organic cation transporter in plants, has been reported [63]. The response in leaves is equally important since the regulation of transpiration plays an important role in the plant response to drought. We showed that in leaves of eight common bean genotypes at different levels of dehydration, up-regulation of transcription factors and genes encoding osmoprotectants, late embryogenesis abundant (LEA) proteins, protein kinases, aldehyde dehydrogenases and cell and carbohydrate metabolism-associated genes occurs, while several photosynthesis-related genes were down-regulated [58]. Only minor differences in expression of 15 studied genes were found between the studied cultivars. The similarity in the gene expression of different cultivars tested in the growth chamber and under greenhouse conditions supports the conclusion that the genes identified in response to water withdrawal constitute a general and intrinsic response of common bean to drought and strengthens the relevance of the experimental results to field conditions [58]. In a study on drought tolerant 'Long 22-0579' and drought sensitive 'Naihua' Chinese common bean cultivars, *de novo* assembly of transcriptome data enabled detection of more than nine thousand drought-responsive candidate genes differentially expressed between the drought and control treatments or between both cultivars exposed to drought [56]. Detected genes include those associated with drought-related metabolic processes (cell metabolism, cell wall and carbohydrate biosynthesis), osmoprotectants (proline), transcription factors (MYB, WRKY, DREB, and NAC), plant hormone regulation, signaling, and cell communication. The expression data enabled further characterization of drought responsive NAC transcription factors [64]. In the same two cultivars, 49 novel and 120 known miRNA were detected, 24 of them showing either increased or decreased expression during drought, and only four sharing the same expression pattern between the cultivars [58]. Among the target genes were genes encoding transcription factors, protein kinases and nuclear transcription factors.

Examples of studies aimed at identifying differences in gene expression of particular groups of genes are those focused on aquaporins (AQPs). These are membrane proteins controlling transcellular water movement from the roots and throughout the plant to assimilating tissues. For this reason, they are involved in controlling the ability of plants to regulate their water supply and transport which is closely related

**115**

*Drought Stress Response in Agricultural Plants: A Case Study of Common Bean...*

to their ability to tolerate or withstand drought. In higher plants, AQPs form a large and diverse protein family with 35 homologs in Arabidopsis (*Arabidopsis thaliana*) and up to 71 homologs in cotton (*Gossypium hirsutum* L.) [65, 66]. In common bean AQPs with the highest mean expression during drought, as well as under normal conditions, were identified [67]. Expression of their genes has been investigated in genotypes with different responses to drought [47, 59, 68]. Increased expression of *PvTIP2;3* was reported in drought-stressed roots of the tolerant genotype [68] and up-regulation of *PvPIP2;5* in leaves of bean exposed to drought was correlated with a reduction in the transpiration rate [69]. In our recent study, physiological measurements indicate greater prevention of water loss in more drought tolerant cultivars, which may be associated with rapid and adequate down-regulation of AQPs in the

Although studies of drought stress at the gene expression level provide many important data and indications, changes in the transcriptome do not necessarily mean that they will be translated into the proteome level. Studies using proteomic methods are therefore essential for revealing, not only the role of proteins in complex mechanisms of drought response in common bean, but also for pointing out possible molecular markers of drought tolerance. These methods not only enable identification of proteins with abundance changed in response to environmental stress, but also the detection of protein complexes and protein localization, as well as of post-translational protein modifications related to a specific stress factor [70–72] as reported later in this chapter. As underlined above, this approach has experienced rapid development by the recent publishing of full genome sequences of many

One of the first studies of drought induced changes in common bean on the proteome was our research focusing on leaves and stems of two cultivars differing in their response to drought [15–17]. In 'Tiber,' we identified 58 proteins whose abundance changed significantly and in 'Starozagorski čern' 64 [15]. Most of the identified proteins were classified into functional categories that include energy metabolism, photosynthesis, ATP interconversion, protein synthesis and proteolysis, stress and defense-related proteins. Significant changes in abundance were observed in large proportion of proteins associated with photosynthesis, such as Rubisco, carbonic anhydrase, oxygen evolving enhancer proteins and chlorophyll a/b binding proteins. While Rubisco small subunit showed lower abundances in drought in both cultivars, carbonic anhydrase was reduced in 'Starozagorski čern,' and in 'Tiber' we detected both increased and reduced abundance. Abundance of chlorophyll a/b binding proteins increased in 'Tiber' and was reduced in 'Starozagorski čern.' The most outstanding contrasting abundance between the two cultivars was the oxygen evolving enhancer proteins, OEE1 and OEE2. Significant changes in abundance were observed in case of a few of the proteins involved in response to stress (e.g., superoxide dismutase, ascorbate peroxidase, and dehydrin) and in case of proteins associated with proteolysis and protein folding (e.g., cysteine proteinase CP2, precursors of cysteine proteinase, proteasome subunit beta type, peptidyl-prolyl cis-trans isomerase, and 20 kDa chaperonin). For peptidyl-prolyl cis-trans isomerase we detected higher abundance in 'Tiber' and reduced abundance in 'Starozagorski čern' under drought conditions. From the category ATP interconversion, in both cultivars nucleoside diphosphate kinase (NDPK) significantly increased under drought whereas ATP synthase decreased in abundance. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological

pathways and molecular functions affected by drought stress.

*DOI: http://dx.doi.org/10.5772/intechopen.86526*

plasma membrane and tonoplast [47].

plants, among them common bean [50].

**4.2 Proteome analysis of drought response**

*Drought Stress Response in Agricultural Plants: A Case Study of Common Bean... DOI: http://dx.doi.org/10.5772/intechopen.86526*

to their ability to tolerate or withstand drought. In higher plants, AQPs form a large and diverse protein family with 35 homologs in Arabidopsis (*Arabidopsis thaliana*) and up to 71 homologs in cotton (*Gossypium hirsutum* L.) [65, 66]. In common bean AQPs with the highest mean expression during drought, as well as under normal conditions, were identified [67]. Expression of their genes has been investigated in genotypes with different responses to drought [47, 59, 68]. Increased expression of *PvTIP2;3* was reported in drought-stressed roots of the tolerant genotype [68] and up-regulation of *PvPIP2;5* in leaves of bean exposed to drought was correlated with a reduction in the transpiration rate [69]. In our recent study, physiological measurements indicate greater prevention of water loss in more drought tolerant cultivars, which may be associated with rapid and adequate down-regulation of AQPs in the plasma membrane and tonoplast [47].

### **4.2 Proteome analysis of drought response**

*Drought - Detection and Solutions*

and forage legumes [57].

**4.1 Transcriptomic profiling of drought response**

known ontologies in order to further investigate their interactions and connections, by methods of bioinformatics and systems biology, into metabolic pathways. A combination of these approaches has enabled the identification of thousands of genes with differential expression and hundreds of proteins with changed abundance in common bean under drought [15, 17, 54–56] as well as other model, crop

Early transcriptomic profiling methods employed over the past decades utilized polymerase chain reaction (PCR) and hybridization techniques and allowed for detection of a smaller number of transcripts with a large difference in mRNA abundance between compared samples [58, 59]. Today these methods are being replaced by genome-wide profiling techniques, such as, microarrays and whole-genome mRNA sequencing (RNA-seq). Further intricacies of gene regulation are explored by profiling miRNAs, small non-coding RNAs that regulate gene expression [55, 56, 60–62]. The transcriptomic response of common bean has been investigated in various plant organs of different genotypes with respect to different stages of drought severity [56]. One of the first studies focused on roots, the first plant organ in which to detect changes in soil water content [55]. Several dehydration-related genes were identified that are associated with signaling, protein homeostasis and root growth modulations, among which a gene *PvOCT1*, encoding a new type of organic cation transporter in plants, has been reported [63]. The response in leaves is equally important since the regulation of transpiration plays an important role in the plant response to drought. We showed that in leaves of eight common bean genotypes at different levels of dehydration, up-regulation of transcription factors and genes encoding osmoprotectants, late embryogenesis abundant (LEA) proteins, protein kinases, aldehyde dehydrogenases and cell and carbohydrate metabolism-associated genes occurs, while several photosynthesis-related genes were down-regulated [58]. Only minor differences in expression of 15 studied genes were found between the studied cultivars. The similarity in the gene expression of different cultivars tested in the growth chamber and under greenhouse conditions supports the conclusion that the genes identified in response to water withdrawal constitute a general and intrinsic response of common bean to drought and strengthens the relevance of the experimental results to field conditions [58]. In a study on drought tolerant 'Long 22-0579' and drought sensitive 'Naihua' Chinese common bean cultivars, *de novo* assembly of transcriptome data enabled detection of more than nine thousand drought-responsive candidate genes differentially expressed between the drought and control treatments or between both cultivars exposed to drought [56]. Detected genes include those associated with drought-related metabolic processes (cell metabolism, cell wall and carbohydrate biosynthesis), osmoprotectants (proline), transcription factors (MYB, WRKY, DREB, and NAC), plant hormone regulation, signaling, and cell communication. The expression data enabled further characterization of drought responsive NAC transcription factors [64]. In the same two cultivars, 49 novel and 120 known miRNA were detected, 24 of them showing either increased or decreased expression during drought, and only four sharing the same expression pattern between the cultivars [58]. Among the target genes were genes encoding transcription factors, protein kinases and nuclear transcription factors. Examples of studies aimed at identifying differences in gene expression of particular groups of genes are those focused on aquaporins (AQPs). These are membrane proteins controlling transcellular water movement from the roots and throughout the plant to assimilating tissues. For this reason, they are involved in controlling the ability of plants to regulate their water supply and transport which is closely related

**114**

Although studies of drought stress at the gene expression level provide many important data and indications, changes in the transcriptome do not necessarily mean that they will be translated into the proteome level. Studies using proteomic methods are therefore essential for revealing, not only the role of proteins in complex mechanisms of drought response in common bean, but also for pointing out possible molecular markers of drought tolerance. These methods not only enable identification of proteins with abundance changed in response to environmental stress, but also the detection of protein complexes and protein localization, as well as of post-translational protein modifications related to a specific stress factor [70–72] as reported later in this chapter. As underlined above, this approach has experienced rapid development by the recent publishing of full genome sequences of many plants, among them common bean [50].

One of the first studies of drought induced changes in common bean on the proteome was our research focusing on leaves and stems of two cultivars differing in their response to drought [15–17]. In 'Tiber,' we identified 58 proteins whose abundance changed significantly and in 'Starozagorski čern' 64 [15]. Most of the identified proteins were classified into functional categories that include energy metabolism, photosynthesis, ATP interconversion, protein synthesis and proteolysis, stress and defense-related proteins. Significant changes in abundance were observed in large proportion of proteins associated with photosynthesis, such as Rubisco, carbonic anhydrase, oxygen evolving enhancer proteins and chlorophyll a/b binding proteins. While Rubisco small subunit showed lower abundances in drought in both cultivars, carbonic anhydrase was reduced in 'Starozagorski čern,' and in 'Tiber' we detected both increased and reduced abundance. Abundance of chlorophyll a/b binding proteins increased in 'Tiber' and was reduced in 'Starozagorski čern.' The most outstanding contrasting abundance between the two cultivars was the oxygen evolving enhancer proteins, OEE1 and OEE2. Significant changes in abundance were observed in case of a few of the proteins involved in response to stress (e.g., superoxide dismutase, ascorbate peroxidase, and dehydrin) and in case of proteins associated with proteolysis and protein folding (e.g., cysteine proteinase CP2, precursors of cysteine proteinase, proteasome subunit beta type, peptidyl-prolyl cis-trans isomerase, and 20 kDa chaperonin). For peptidyl-prolyl cis-trans isomerase we detected higher abundance in 'Tiber' and reduced abundance in 'Starozagorski čern' under drought conditions. From the category ATP interconversion, in both cultivars nucleoside diphosphate kinase (NDPK) significantly increased under drought whereas ATP synthase decreased in abundance. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways and molecular functions affected by drought stress.

The further study on stem [16] carried out on cultivar 'Tiber' showed changed abundances under drought of proteins that can be classified in the same categories as leaf proteins. The proteins with increased abundance indicate the importance of maintaining protein homeostasis to mitigate this stress. There was increased abundance of proteins involved in protein synthesis, proteolysis and protein folding. Among them, the protein with the greatest abundance was 70 kDa heat shock protein that chaperones the correct folding of proteins [16].

#### **4.3 Postranslational modifications in the response to drought**

The complexity of the response of plants to drought is further emphasized by reports indicating that posttranslational modifications (PTMs) of proteins also play an important role. These include covalent modifications of a number of cell proteins that follow protein biosynthesis and are usually catalyzed by enzymes. There are different types of PTMs, among them glycosylation, that are recognized as being very important in plants and in their response to stress [73, 74]. This type of PTM affects protein stability, interaction with other proteins, protein trafficking and, as a consequence, protein activity.

Only a small number of the proteomic studies that have been carried out address specifically protein glycosylation and changes in abundance of glycoproteins in crops under abiotic stress [17]. One of them is our study of glycosylated proteins in leaves of common bean stressed by drought [17]. 'Tiber,' previously identified as relatively tolerant to drought [46, 47], was investigated. Thirty-five glycoproteins with changed abundance were detected. Their structures showed high mannose, complex and hybrid types of N-glycans, most of them being associated with the cell wall (many cell wall-degrading enzymes, such as, β-glucosidase, α-arabinofuranosidase and β-xylosidase, were more abundant under drought), with the stress response (such as, ascorbate oxidase, purple acid phosphatase and reticulin oxidase-like protein that were also more abundant) and with proteolysis and protein folding (such as, the precursor of subtilisin-like serine protease, nicastrin, the precursor of cysteine protease and protein disulfide isomerase that were less abundant) [17].

It follows from the studies reported above that proteome analysis of common bean under drought has revealed the participation of proteins involved in proteolysis [15, 17]. Many proteins, after their synthesis, need to be activated by highly regulated proteolytic cleavage of specific peptide bonds that removes parts of their peptide chains. Activation of regulated proteolysis and simultaneous inhibition of uncontrolled proteolysis are vital for cell survival under dehydration stress. All beneficial changes in metabolism under drought require the active involvement of controlled proteolysis that regulates the turnover rates of specific enzymes and/or proteins involved in cell signaling, and ensures degradation of oxidatively damaged, improperly folded and irreversibly denatured proteins [75, 76]. On the other hand non-specific, uncontrolled proteolysis can be damaging to cells, leading to random breakdown of the majority of cell proteins. Such protein degradation, provoked by drought, results mainly in the disruption of cell membranes and exhibits many features in common with plant senescence [77].

Proteolysis is catalyzed by proteases whose activity is regulated mainly by specific plant protease inhibitors both detected by transcriptomics and/or proteomics [76]. The latter are important, not only for inhibiting proteases activated on drought, but also for osmoprotection, since many of them are highly hydrophilic. The striking diversity of plant proteases and of their inhibitors in each species [76] coupled with the fact that very few of their natural substrates are known [78], complicates research in this field. In addition, it appears that the changes in abundance of many proteases in plants stressed by drought have not been detected by

**117**

**tolerance**

**5.1 QTL mapping**

*Drought Stress Response in Agricultural Plants: A Case Study of Common Bean...*

types of protease, such as serine and aspartic endopeptidases [81, 82].

this response depending on the developmental stage of the leaves [83].

is stronger in the cultivar more susceptible to drought.

We have further isolated and characterized, at the protein and gene levels, a protease from the leaves of a common bean that is influenced by drought [81]. It has been classified as a new plant subtilisin-like serine protease. While its gene expression did not change on water deficit, its proteolytic activity did. Further, in common bean leaves an aspartic protease was characterized whose activity was strongly induced on water deficit [82, 84]. It was shown that proteolytic processing of its precursor form was induced by drought, and this, together with the effect of stress on the level of its transcript, led to the suggestion that water deficit regulates activity at both the transcriptional and PTM levels. This response occurs earlier and

**5. Applications of QTL and molecular markers in breeding for drought** 

Quantitative trait locus (QTL) mapping is an established approach for detecting loci associated with complex quantitative traits, such as, plant tolerance to drought. In common bean multiple populations derived from crosses of susceptible and tolerant parental genotypes, belonging to either a single gene pool, or both Andean and Mesoamerican gene-pools, have been genotyped and genetic linkage maps constructed [18, 85]. Their precision and resolution have been greatly improved by novel sequencing technology and genetic markers, such as, SNPs. For instance, two inter-gene pool populations of 'BAT93' × 'JaloEEP558' and 'DOR364' × 'G19833' have been genotyped repeatedly using a variety of marker systems, ranging from SSR and amplified fragment length polymorphism (AFLP) to SNP [18, 85]. The efforts have culminated in consensus linkage map generation joining both major inter-gene pool maps as well as serving as a core for integration with Mesoamerican linkage map [18]. These approaches have enabled identification of numerous QTLs, controlling resistance to various viral, bacterial and fungal pathogens as well as multigenic traits such as tolerance to drought, biomass production, yield partitioning, and micronutrient accumulation [86, 87].

proteomics, due to their low abundance. A combination of proteome analysis and measurement of activities is therefore needed. The proteases most often reported to be involved in the response to drought are cysteine endopeptidases [79, 80], although research on legume plants has indicated participation of other catalytic

The response to drought at the level of leaf proteases has been relatively extensively investigated in common bean. Different types of protease have been studied at levels ranging from gene expression to proteolytic activity. In several cases, cultivars with different sensitivities to water deficit have been investigated and changes in proteolytic activity correlated with cultivar sensitivity [46, 54, 82]. In leaf extracts from Brazilian cultivars several endoproteolytic activities with different pH optima were higher in plants under drought. This effect correlated with the level of sensitivity to drought of cultivars [54, 82]. Our research, carried out on cultivars of European origin differing in sensitivity, showed the involvement in response to this stress of different classes of endopeptidases [46]. Increased activities with pH optima in the acid region were observed in leaf extracts of the more sensitive cultivars and were assigned to cysteine and serine proteases. It should be emphasized that differential analysis of leaf proteomes indicated higher abundances of cysteine proteinase precursors in stressed samples [15]. In addition, we have found that the activities of five aminopeptidases in leaves of common bean changed when plants were subjected to drought,

*DOI: http://dx.doi.org/10.5772/intechopen.86526*

*Drought Stress Response in Agricultural Plants: A Case Study of Common Bean... DOI: http://dx.doi.org/10.5772/intechopen.86526*

proteomics, due to their low abundance. A combination of proteome analysis and measurement of activities is therefore needed. The proteases most often reported to be involved in the response to drought are cysteine endopeptidases [79, 80], although research on legume plants has indicated participation of other catalytic types of protease, such as serine and aspartic endopeptidases [81, 82].

The response to drought at the level of leaf proteases has been relatively extensively investigated in common bean. Different types of protease have been studied at levels ranging from gene expression to proteolytic activity. In several cases, cultivars with different sensitivities to water deficit have been investigated and changes in proteolytic activity correlated with cultivar sensitivity [46, 54, 82]. In leaf extracts from Brazilian cultivars several endoproteolytic activities with different pH optima were higher in plants under drought. This effect correlated with the level of sensitivity to drought of cultivars [54, 82]. Our research, carried out on cultivars of European origin differing in sensitivity, showed the involvement in response to this stress of different classes of endopeptidases [46]. Increased activities with pH optima in the acid region were observed in leaf extracts of the more sensitive cultivars and were assigned to cysteine and serine proteases. It should be emphasized that differential analysis of leaf proteomes indicated higher abundances of cysteine proteinase precursors in stressed samples [15]. In addition, we have found that the activities of five aminopeptidases in leaves of common bean changed when plants were subjected to drought, this response depending on the developmental stage of the leaves [83].

We have further isolated and characterized, at the protein and gene levels, a protease from the leaves of a common bean that is influenced by drought [81]. It has been classified as a new plant subtilisin-like serine protease. While its gene expression did not change on water deficit, its proteolytic activity did. Further, in common bean leaves an aspartic protease was characterized whose activity was strongly induced on water deficit [82, 84]. It was shown that proteolytic processing of its precursor form was induced by drought, and this, together with the effect of stress on the level of its transcript, led to the suggestion that water deficit regulates activity at both the transcriptional and PTM levels. This response occurs earlier and is stronger in the cultivar more susceptible to drought.
