Two-dimensional Gel Electrophoresis Method Research Articles

EFFECT OF TEMPERATURE ON THE PROTEIN PROFILE OF MACROVIPERA LEBETINUS (BLUNT-NOSED VIPER) VENOM: A PRELIMINARY STUDY

Snake venom is a complex biological fluid consisting mainly of proteins and peptides possessing diverse biological activities. Snake venoms draw attention due to their bioactive proteins/peptides with therapeutic and diagnostic potential. Testing the stability of snake venom proteins under different conditions including temperature provides useful data for venom research. Macrovipera lebetinus, blunt-nosed viper, is the biggest viper species of Türkiye distributed mainly in eastern and southeastern Anatolia. Although its venom components were investigated before, there is limited data regarding the effect of temperature on its venom proteins. The present study aimed to investigate the effect of temperature on the venom proteins of M. lebetinus. For this purpose, venom samples were incubated at 25, 37, and 50°C. Thereafter, venom proteins were separated by two-dimensional gel electrophoresis (2D-PAGE) method. Some qualitative and quantitative differences in the protein profile indicating structural changes and degradation were observed especially after 50°C treatment. It has been found that the protein spots most affected by temperature will most likely contain metalloproteinase, phospholipase A2 and ʟ-amino acid oxidase enzymes, by comparing the experimental molecular weight and pI values with those in the literature. Detailed studies including enzyme activities and toxicity assays will provide more data on the stability of M. lebetinus venom under different conditions.

Exploring the potential of Lactobacillus and Saccharomyces for biofunctionalities and the release of bioactive peptides from whey protein fermentate

Whey protein concentrate-80 (WPC-80) fermented with L. fermentum (KGL4) (37 °C) and S. cerevisiae (WBS2A) (25 °C) was tested for ACE-inhibitory and antioxidant activities over different periods (12, 24, 36 and 48 h). Proteolytic activity (OPA method) was used to optimize the growth conditions (inoculation rate, i.e. at 1.5%, 2.0%, and 2.5% and incubation time, i.e. 12, 24, 36, and 48 h) for peptide production. Results indicated that the highest amount of peptides was obtained at 7.24 mg/mL for KGL4 (37 °C, 48 h) and 8.59 mg/mL for WBS2A (25 °C, 48 h). The whey protein fermentate inhibited the LPS-induced NO production, while enhanced production concentrations of TNF-α, IL-6, and IL-1β. Subsequently, SDS-PAGE, as well as Two-Dimensional (2D) gel electrophoresis methods, were applied for protein purification using water-soluble extracts (WSEs) of WPC-80 fermented by a combination of L. fermentum and S. cerevisiae. On SDS-PAGE, protein bands were observed in the range of 10–55 kDa, whereas on the 2D page, protein spots were in the range of 10–70 kDa. All the 2D spots were analyzed using RPLC/MS. WSEs of 3 kDa and 10 kDa permeates were used in RP-HPLC to identify distinct peptide fractions. The data from LC/MS was also characterized by utilizing ProteinPilot software. Further, different functional groups were also analyzed using FTIR investigation. The research aims to isolate and characterize novel ACE-inhibitory and antioxidative peptides from fermented WPC-80 produced by Lactobacillus fermentum and S. cerevisiae.

Heating Effect on Protein’s Aggregation in Goat and Bovine Milk Based on One and Two-Dimensional Polyacrylamide Gel Electrophoresis

Goat milk has been considered as a possible alternative of bovine milk because of hypoallergenic. The milk protein is the major allergenic substances, however, the differences of proteins have been few demonstrated in processing between goat and bovine milk. To explore the influence of three heat treatment and ultrahigh pressure (UHP, at 500 MPa and 17 °C) on milk, we investigated the differences of proteins in the two types of milk by one and two-dimensional polyacrylamide gel electrophoresis (1D and 2D PAGE) methods. β-lactoglobulin (β-LG) from goat milk showed one big band and migrated slower than α-lactalbumin (α-LA) on the native gel; while bovine milk showed two β-LG bands that had higher mobility ratio than other protein bands. The pasteurizing (63 °C, 30 min) and UHT (143 °C, 15 s) led to no obvious missing of protein bands, but the boiling treatment (100 °C, 3 min) caused binding of whey protein with κ-CN and αs2-CN in both types of milk. UHP treatment of milk also causes β-LG aggregates with casein by forming disulfide-bonded. All of the treatments indicated that goat milk was more sensitive to heat and pressure than bovine milk to form the aggregates and heat processing had the greatest effect on β-LG. This study could be helpful in understanding the difference in technological-functional properties of the two types of milk.

Diagnosis of Sepsis by AI-Aided Proteomics Using 2D Electrophoresis Images of Patient Serum Incorporating Transfer Learning for Deep Neural Networks

An accuracy of ≥98% was achieved in sepsis diagnosis using serum samples from 30 sepsis patients and 68 healthy individuals and a high-performance two-dimensional polyacrylamide gel electrophoresis (HP-2D-PAGE) method developed here with deep learning and transfer learning algorithms. In this method, small-scale target domain data, which are collected to achieve our objective, are inputted directly into a model constructed with source domain data which are collected from a different domain from the target; target vectors are estimated with the outputted target domain data and applied to refine the model. Recognition performance of small-scale data is improved by reusing all layers, including the output layers of the neural network. Proteomics is generally considered the ultimate bio-diagnostic technique and provides extremely high information density in its two-dimensional electrophoresis images, but extracting the data has posed a basic problem. The present study is expected to solve that problem and will be an important breakthrough for practical utilization and future perspectives of proteomics in clinics after evaluation in clinical settings.

Comparative Two-dimensional Gel Electrophoresis Maps for Amastigote-like Proteomes of Iranian Leishmania Tropica and Leishmania major Isolates

Background: Leishmania major and Leishmania tropica are the main causative agents of cutaneous leishmaniasis. Proteomics as a novel approaches could be used to evaluate protein expression levels in different stages of Leishmania species. We compare the protein contents of amastigote-like forms in L. tropica and L. major using two-dimensional gel electrophoresis (2-DE) and bioinformatics methods. Materials and Methods: Leishmania parasites were isolated from the lesions of Iranian patients and identified using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Five isolates of each two species were cultured in specific media to obtain amastigote-like forms to be prepared for proteomics study. Total protein contents were separated using 2-DE. The gels were stained by silver nitrate and scan was imaged. The protein spots with different expression changes in each gel were analyzed using Progenesis SameSpots software. Results: A total of 354 protein spots were detected in both amastigote-like forms. Comparative analysis of protein spots with different expressions in the two amastigote-like form species showed 173 highly expressed spots of which 74 L. tropica and 99 L. major proteins were spotted with fold≥2. Also, 16 and 20 new protein spots were uniquely found in L. tropica and L. major, respectively. Clustering of different detected proteins using correlation analysis divided the proteins into two clusters based on their expression level. Furthermore, clustering results were confirmed by principal component analysis. Conclusion: Using proteomics methods specially 2-DE and statistical analysis demonstrated significant changes in protein expression levels in amastigote-like forms of L. tropica and L. major isolates. [GMJ.2019;8:e1520]

Comparative Two-dimensional Gel Electrophoresis Maps for Amastigote-like Proteomes of Iranian Leishmania Tropica and Leishmania Major Isolates.

Background: Leishmania major and Leishmania tropica are the main causative agents of cutaneous leishmaniasis. Proteomics as a novel approaches could be used to evaluate protein expression levels in different stages of Leishmania species. We compare the protein contents of amastigote-like forms in L. tropica and L. major using two-dimensional gel electrophoresis (2-DE) and bioinformatics methods. Materials and Methods:Leishmania parasites were isolated from the lesions of Iranian patients and identified using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Five isolates of each two species were cultured in specific media to obtain amastigote-like forms to be prepared for proteomics study. Total protein contents were separated using 2-DE. The gels were stained by silver nitrate and scan was imaged. The protein spots with different expression changes in each gel were analyzed using Progenesis SameSpots software. Results: A total of 354 protein spots were detected in both amastigote-like forms. Comparative analysis of protein spots with different expressions in the two amastigote-like form species showed 173 highly expressed spots of which 74 L. tropica and 99 L. major proteins were spotted with fold≥2. Also, 16 and 20 new protein spots were uniquely found in L. tropica and L. major, respectively. Clustering of different detected proteins using correlation analysis divided the proteins into two clusters based on their expression level. Furthermore, clustering results were confirmed by principal component analysis. Conclusion: Using proteomics methods specially 2-DE and statistical analysis demonstrated significant changes in protein expression levels in amastigote-like forms of L. tropica and L. major isolates.

Stress Response and Virulence Potential Modulating Effect of Peppermint Essential Oil in Campylobacter jejuni.

Campylobacter jejuni is one of the most common food-borne bacteria that causes gastrointestinal symptoms. In the present study we have investigated the molecular basis of the anti-Campylobacter effect of peppermint essential oil (PEO), one of the oldest EO used to treat gastrointestinal diseases. Transcriptomic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and proteomic, two-dimensional polyacryl amid gel electrophoresis (2D-PAGE) methods have revealed that, in the presence of a sublethal concentration of PEO, the expression of several virulence-associated genes was decreased (cheY 0.84x; flhB 0.79x; flgE 0.205x; cadF 0.08x; wlaB 0.89x; porA 0.25x; cbf2 4.3x) while impaired motility was revealed with a functional analysis. Scanning electron micrographs of the exposed cells showed that, unlike in the presence of other stresses, the originally curved C. jejuni cells straightened upon PEO exposure. Gaining insight into the molecular background of this stress response, we have revealed that in the presence of PEO C. jejuni dominantly exerts a general stress response that elevates the expression of general stress genes like dnaK, groEL, groES (10.41x, 3.63x, and 4.77x). The most important genes dps, sodB, and katA involved in oxidative stress responses showed however moderate transcriptional elevations (1,58x, 1,55x, and 1,85x).

Detection and discrimination of Shigella sonnei and Shigella flexneri based on vacuolar responses in Saccharomyces cerevisiae

This study provided a system for bacteria detection based on a lysosome-like-vacuole response in the yeast Saccharomyces cerevisiae. Vacuoles are factors known to activate the immune system in the presence of foreign substances. Here, Shigella sonnei and Shigella flexneri were exposed to yeast to analyze the alteration of vacuolar enzymes. The ability to detect the bacteria was evaluated by confocal microscopy after exposing and staining vacuoles with LysoTracker. Results showed that the treatment of yeast with these bacteria increased the number of red vacuole-like organelles surrounding yeast nuclei. Thus, vacuole alteration can be used as a biomarker for bacteria detection. Next, the expression of vacuolar enzymes under the influence of bacteria was examined using two-dimensional gel electrophoresis (2-DE) method for screening specific biomarkers for each Shigella strain. Finally, the recombinant yeasts that contained biomarkers fused to different fluorescent proteins confirmed the ability of yeast to detect these two Shigella strains at concentrations ranging from 10 to 100 CFU/mL.

Impact of imipramine on proteome of rat primary glial cells

Microglia and astrocytes, two types of glial cells are known to be important targets for antidepressant drugs. Here we used a comprehensive proteomic analysis to examine the effect of imipramine on rat primary mixed glial culture. The two-dimensional differential gel electrophoresis method allowed us to identify 62 proteins that were altered by imipramine. Functional analysis revealed that imipramine influenced the level of proteins involved in oxidative stress; in particular, it elevated the level of glutathione transferases. Imipramine upregulated proteins related to glycolysis but down-regulated many mitochondrial proteins including enzymes involved in oxidative phosphorylation. Mitochondrial dysfunction, especially decrease of mitochondrial membrane potential can be counted as a side effect triggered by imipramine. Imipramine induced lowering of chaperone level and alterations suggesting impaired protein synthesis could be associated with increased apoptosis. One of the most pronounced effect of imipramine is the reduction of vimentin level, this protein is engaged in majority of biological processes which were found to be affected by imipramine. Many imipramine regulated proteins, including chaperones, cathepsins and annexins are involved in immune responses. Additionally, imipramine influenced proteins associated with phagocytosis and cell migration. Overall these findings indicate that imipramine produces complex effect on glial cells, primarily on microglia and suggest their transition towards a more quiescent, metabolically less demanding phenotype.

DIGE Analysis Software and Protein Identification Approaches.

DIGE is a high-resolution two-dimensional gel electrophoresis method, with excellent dynamic range obtained by fluorescent tag labeling of protein samples. Scanned images of DIGE gels show thousands of protein spots, each spot representing a single or a group of protein isoforms. By using commercially available software, each protein spot is defined by an outline, which is digitized and correlated with the quantity of proteins present in each spot. Software packages include DeCyder, SameSpots, and Dymension 3. In addition, proteins of interest can be excised from post-stained gels and identified with conventional mass spectrometry techniques. High-throughput mass spectrometry is performed using sophisticated instrumentation including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), MALDI-TOF/TOF, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem MS (MALDI-TOF/TOF or LC-MS/MS), analyzes fragmented peptides, resulting in amino acid sequence information, especially useful when protein spots are low abundant or where a mixture of proteins is present.

2D-DIGE in Proteomics.

The two-dimensional difference gel electrophoresis method is a valuable approach for proteomics. The method, using cyanine fluorescent dyes, allows the co-migration of multiple protein samples in the same gel and their simultaneous detection, thus reducing experimental and analytical time. 2D-DIGE, compared to traditional post-staining 2D-PAGE protocols (e.g., colloidal Coomassie or silver nitrate), provides faster and more reliable gel matching, limiting the impact of gel to gel variation, and allows also a good dynamic range for quantitative comparisons. By the use of internal standards, it is possible to normalize for experimental variations in spot intensities and gel patterns. Here we describe the experimental steps we follow in our routine 2D-DIGE procedure that we then apply to multiple biological questions.

Physiological, Ultrastructural and Proteomic Responses in the Leaf of Maize Seedlings to Polyethylene Glycol-Stimulated Severe Water Deficiency.

After maize seedlings grown in full-strength Hoagland solution for 20 days were exposed to 20% polyethylene glycol (PEG)-stimulated water deficiency for two days, plant height, shoot fresh and dry weights, and pigment contents significantly decreased, whereas malondialdehyde (MDA) content greatly increased. Using transmission electron microscopy, we observed that chloroplasts of mesophyll cells in PEG-treated maize seedlings were swollen, with a disintegrating envelope and disrupted grana thylakoid lamellae. Using two-dimensional gel electrophoresis (2-DE) method, we were able to identify 22 protein spots with significantly altered abundance in the leaves of treated seedlings in response to water deficiency, 16 of which were successfully identified. These protein species were functionally classified into signal transduction, stress defense, carbohydrate metabolism, protein metabolism, and unknown categories. The change in the abundance of the identified protein species may be closely related to the phenotypic and physiological changes due to PEG-stimulated water deficiency. Most of the identified protein species were putatively located in chloroplasts, indicating that chloroplasts may be prone to damage by PEG stimulated-water deficiency in maize seedlings. Our results help clarify the molecular mechanisms of the responses of higher plants to severe water deficiency.

Proteomics analysis of human oligodendroglioma proteome

Proteomics analyses enable the identification and quantitation of proteins. From a purely clinical perspective, the application of proteomics based on innovations, may greatly affect the future management of malignant brain tumors. This optimism is based on four main reasons: diagnosis, prognosis, selection of targeted therapy based on molecular profile of the brain tumor and monitoring therapeutic response, or resistance. We extracted the proteins of tumor and normal brain tissues, and then evaluated the protein purity by Bradford test. In this study, we separated the proteins by two-dimensional (2DG) gel electrophoresis methods. Then spots were analyzed, compared using statistical data and specific software and were identified by pH isoelectric, molecular weights and data banks. The protein profiles were determined using 2D gel electrophoresis and MALDI TOF/TOF mass spectrometry approaches. Simple statistical tests were used to establish a putative hierarchy in which the change in protein level was ranked according to a cut-off point with p<0.05. The 2D gel showed a total of 1328 spots among which 157 spots were under-expressed and 276 spots were overexpressed. Most proteins are subjects to post-translational modifications, where amino acid residues may be chemically modified or conjugated by small proteins like ubiquitin. Proteomics is a powerful way to identifying multiple proteins which are altered following a neuropharmacological intervention in a CNS disease.

The study of fibroblast proteome changes in the presence of Ethanol

Ethanol known as ethyl alcohol is being widely used around the world. Many serious diseases are related with its consumption. Alcohol posses many divers effects on human body including risk of cirrhosis and/or hepatocellular carcinoma. Therefore, analysis of this component is prominent. Fibroblast cells were cultured in various dosages of ethanol. The effective dosage was then investigated by proteomic methods. Separated proteins of fibroblast cells by Two-Dimensional Gel (2DG) Electrophoresis method based on pI and MW were analyzed based on spots alteration by Same Spot Software. Furthered analysis was carried out with vigorous statistical analysis based on significant folding changes and one-way ANOVA. About 372 protein spots were identified and among them 65 of them were having significant expression profile, which is evaluated as p <0.05. Therefore, ethanol can induce a great impact on protein profile of fibroblast. It is concluded that altering morphologic features and viability, as well as protein expression changes, confirm toxic properties of ethanol in human body.

Metalloproteomic Profile Determination of Muscle Samples from Nile Tilapia (Oreochromis niloticus) Using AAS and ESI-MS/MS after 2D-PAGE Separation

This study evaluated the metalloproteomic profile of muscle tissue samples from Nile tilapia (Oreochromis niloticus). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods was used for protein fractionation and identification based on image analysis. Determination of Ca, Cu, Fe, Mn and Zn was performed in the protein spots by flame and/or graphite furnace atomic absorption spectrometry (FAAS, GFAAS), the protein spots were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). FAAS and GFAAS determinations have indicated the presence of calcium in seven protein spots, iron in only one spot, manganese in six spots, and zinc in two spots. Of the seventeen protein spots where the presence of metal ions was detected, ten were characterized by ESI-MS/MS.

The investigation of changes in proteins expression (Apolipoprotein A1 and albumin) in malignant astrocytoma brain tumor.

Angiogenesis performs a critical role in the development of cancer. Angiogenesis research is a cutting-edge field in cancer research. Proteomics is a powerful tool in identifying multiple proteins that are altered following a neuropharacological intervention in a disease of the central nervous system. Diagnostic oncoproteomics is the application of proteomic techniques for the diagnosis of malignancies. We extracted proteins of tumor and normal brain tissues and then evaluated the protein purity by Bradford test and spectrophotometery method. In this study, we separated proteins by two-dimensional (2D) gel electrophoresis method and the spots were then analyzed and compared using statistical data and specific software, after providing three-dimensional images of spots alteration. Spots were identified by pH isoelectric, molecular weights, and data banks. Simple statistical test were used to establish a putative hierarchy in which the change in protein level were ranked according a cutoff point with P < 0.05. Apolipoprotein A1 (apo A1) protein and albumin were consistently upregulated in astrocytoma brain tumors. The vascular microenvironment of glioma play a major role in determining the pathophysiological character is tics of the tumor. apo A1 and albumin are very significant due to their functional consequences in glioma tumor growth, migration and angiogenesis.

Proteomics analysis of human brain glial cell proteome by 2D gel.

Proteomics is increasingly employed in both neurological and oncological research, and applied widely in every area of neuroscience research including brain cancer. Astrocytomas are the most common glioma and can occur in most parts of the brain and occasionally in the spinal cord. Patients with high-grade astrocytomas have a life expectancy of <1 year even after surgery, chemotherapy, and radiotherapy. We extracted proteins from tumors and normal brain tissues and then evaluated the protein purity by Bradford test and spectrophotometry method. In this study, we separated proteins by the two-dimensional (2DG) gel electrophoresis method, and the spots were analyzed and compared using statistical data. On each analytical 2D gel, an average of 800 spots was observed. In this study, 164 spots exhibited up-regulation of expression level, whereas the remaining 179 spots decreased in astrocytoma tumor relative to normal tissue. RESULTS demonstrate that functional clustering and principal component analysis (PCA) has considerable merits in aiding the interpretation of proteomic data. Proteomics is a powerful tool in identifying multiple proteins that are altered following a neuropharmacological intervention in a disease of the central nervous system (CNS). 2-D gel and cluster analysis have important roles in the diagnostic management of astrocytoma patients, providing insight into tumor biology. The application of proteomics to CNS research has invariably been very successful in yielding large amounts of data.

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).

Identification of cellular proteome using two-dimensional difference gel electrophoresis in ST cells infected with transmissible gastroenteritis coronavirus

BackgroundTransmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs, which is correlated with high morbidity and mortality in suckling piglets. Information remains limited about the comparative protein expression of host cells in response to TGEV infection. In this study, cellular protein response to TGEV infection in swine testes (ST) cells was analyzed, using the proteomic method of two-dimensional difference gel electrophoresis (2D DIGE) coupled with MALDI-TOF-TOF/MS identification.Results33 differentially expressed protein spots, of which 23 were up-regulated and 10 were down-regulated were identified. All the protein spots were successfully identified. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and the mitochondrial pathway. Western blot analysis was used to validate the changes of alpha tubulin, keratin 19, and prohibitin during TGEV infection.ConclusionsTo our knowledge, we have performed the first analysis of the proteomic changes in host cell during TGEV infection. 17 altered cellular proteins that differentially expressed in TGEV infection were identified. The present study provides protein-related information that should be useful for understanding the host cell response to TGEV infection and the underlying mechanism of TGEV replication and pathogenicity.

Proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanoLC-ESI–MS/MS

To elucidate differentially expressed proteins and to further understand post-translational modifications of transcripts, full leaf proteome profiles of two wild emmer (Triticum turgidum ssp. dicoccoides TR39477 and TTD22) and one modern durum wheat (Triticum turgidum ssp. durum cv. Kızıltan) genotypes were compared upon 9-day drought stress using two-dimensional gel electrophoresis and nano-scale liquid chromatographic electrospray ionization tandem mass spectrometry methods. The three genotypes compared exhibit distinctive physiological responses to drought as previously shown by our group. Results demonstrated that many of the proteins were common in both wild emmer and modern wheat proteomes; of which, 75 were detected as differentially expressed proteins. Several proteins identified in all proteomes exhibited drought regulated patterns of expression. A number of proteins were observed with higher expression levels in response to drought in wild genotypes compared to their modern relative. Eleven protein spots with low peptide matches were identified as candidate unique drought responsive proteins. Of the differentially expressed proteins, four were selected and further analyzed by quantitative real-time PCR at the transcriptome level to compare with the proteomic data. The present study provides protein level differences in response to drought in modern and wild genotypes of wheat that may account for the differences of the overall responses of these genotypes to drought. Such comparative proteomics analyses may aid in the better understanding of complex drought response and may suggest candidate genes for molecular breeding studies to improve tolerance against drought stress and, thus, to enhance yields.