Similar findings confirmed that Cicer arietinum gene family was closely related to Medicago tranculata while soybean, pigeonpea and common bean sharing a common ancestor grouped in a separate cluster [ 54 ].
Results confirmed that drought stress significantly increased the ASR gene expression that possibly elicited increased responsiveness towards drought tolerance in chickpea. Expression patterns revealed high and significant expression of ASR gene in all the genotypes under drought stress. It has been proposed ASR1 gene expression increases under stress in a variety of species [ 55 ]. Transgenic Brachypodium distachyon L. Physiological studies also proved the involvement of ASR1 gene in stress tolerance in transgenic tobacco, tomato, maize and rice [ 57 ].
The instability index value of ASR1 proteins may be nuclear or may be dispersed in the cytoplasm. In general, KDa molecules diffuse passively through nuclear pores depending on their concentration gradient, whereas other molecules are transported actively [ 59 ] through the nuclear localization signals.
These NLS rich in basic amino acids are recognized by certain docking molecules viz. Hybrid assays and sub-cellular fractionation studies confirmed the role of ZmASR proteins as transcription factors or molecular chaperons in different plant species [ 61 ]. The Cicer arietinum ASR protein was found to be closely related to monocotyledonous Gramineae species, Zea mays and Oryza sativa , Sorghum bicolor in comparison to other plant species including cottonwood, banana species.
Similar phylogenetic relationships were found while comparing the apple ASR genes with those of other plant species [ 58 ].
ASR proteins adapt two different conformations viz. This structural plasticity is critical for plant stress resistance, facilitating their response to drought and interaction with target proteins [ 62 ].
The predicted model was then evaluated through Ramachandran plot using Swiss-Pdb Viewer v4. The Psi-Phi pairs had Protein Structure Analysis ProSA tool identifies the regions that contribute to an overall bad score in the predicted model and has long been used for their refinement and validation.
The energy plots and the Z-scores indicate the problems in the predicted structure of the target protein. The positive values indicate the erroneous parts of the model in general. Residual error plots indicating reliability of the local model were also plotted and visualized by color gradients.
Blue colored regions indicate more reliable regions and red regions indicate the probable unreliable regions. Models that slide towards blue regions from light red color are considered to be of high quality Fig 12B. Software packages available online offer great opportunities for analyzing biological systems. AraNet, GeneMania, and STRING being user-friendly [ 63 — 65 ] have been used for studying the protein interactions, regulatory networks, gene associations and their biological pathways [ 66 — 68 ].
The hypothetical protein was found to interact with ten different proteins Fig 13 for its functioning viz. Integrated high-throughput approaches employing molecular networks with phenomics together may provide assumptions and address precise biological queries [ 69 ].
Drought QTLs have been identified using different approaches viz. Ten genes were found to express under abiotic stresses [ 36 ].
Functional validation was also done for these genes using the already reported genes from model plants. Linkage analysis and association mapping approach was also used to identify and validate the chickpea genes and QTLs for moisture stress tolerance based on sequence similarity approach [ 72 ].
Candidate genes identified may be used to develop cultivars with desired tolerance to drought and ensure greater genetic gains and also enhance the probability of breeding widely adapted high yielding hybrids in chickpea.
ASR is the most widely reported drought stress responsive gene [ 73 ]. The ASR gene family has evolved from Spermatophyta. ASR gene is regulated by water deficit, salinity stress and hormone Abscisic acid ABA , low temperature and intensity of light [ 74 ]. ASR genes were first recognized in tomato [ 75 ] and consequently, in different plant species Zea mays nine , Sorghum bicolor seven , Oryza sativa six , Brachypodium distachyon six , Pinus taeda four , Phaseolus vulgaris two and Vitis vitifera one [ 76 , 77 , 18 ].
Transgenic studies confirmed ASR genes could be involved in ABA signalling pathways enabling the plants to respond to external stresses [ 78 , 79 ] and transgenic plants with over-expressed ASR gene were found to be more tolerance to water and salt stress [ 26 ].
During late embryogenesis accumulation of tomato ASR1 was observed. Electrophoretic assays and direct visualization also confirmed formation of homodimers in DNA by ASR1 in response to water stress [ 23 , 80 ]. In contrast, activity of tomato ASR2 promoter was enhanced in response to ABA in papaya and tobacco, while reduction was observed in tomato and potato [ 81 ].
Transgenic Arabidopsis lines over-expressing maize ASR genes exhibited better growth performance and higher survival rates as compared to wild type under drought conditions. These lines had lower malondialdehyde content and higher ABA and proline content improving their drought tolerance.
Their precise role in conferring improved tolerance to drought and salt has also been established in tomato, rice and lily [ 33 , 34 , 26 ].
Consequently, reports on involvement of ASR gene in legumes in drought responses are insufficient. Seven promising genotypes of chickpea Cicer arietinum viz. The amalgamated soil peat to vermiculite, with pH 7. These pots were irrigated with ml water on daily basis. Drought stress was imposed on 12 day old plants by withholding water for 6 days and 12days respectively [ 35 ].
Control plants were watered regularly for the same duration. Leaf tissues of the control and drought stressed plants were collected at different time points viz. These genotypes were identified on their relative basis of tolerance to drought [ 83 ]. Relative water content RWC of selected chickpea genotypes was measured at different time points 0 day, 6 th day and 12 th day as per the standard method [ 84 ]. Young leaf tissues of chickpea were collected and their fresh weights were recorded.
The leaf tissues were then incubated in petriplates containing distilled water for 4 hours for calculating their turgid weights. Konica Minolta SPAD Plus chlorophyll meter was used for measuring the chlorophyll index of the selected chickpea genotypes at three different time points 0 day, 6 th day and 12 th day.
For estimating protein content leaf at different time points, crushing of leaves was done in 50mM phosphate buffer with pH 7. The protein content was estimated by colorimetric method [ 85 ]. The MSI was calculated using formula:. The mean values and coefficient of variation CV were calculated for each parameter. The standard errors of the mean were presented in the figures as error bars.
Genomic DNA and other contaminants were removed by precipitation. The cDNA from leaf tissues were used as template. PCR amplicons of the seven chickpea genotypes were purified using BigDye terminator v3. Sequencing data was analyzed in Sequencing Analysis v 5. Raw sequences were assembled using the forward and reverse sequences of each genotype in KB v 1. Common characteristics of the predicted protein including molecular weight, isoelectric point pI , amino acid composition, aliphatic and instability index were assessed using protparam tool.
The target protein model was further refined by the Program structure Analysis ProSA program that predicts the structure of the target protein using the molecule viewer Jmol in order to find out the regions that contribute to errors in the protein model.
Present study reveals that increased expression of ASR gene under drought stress possibly enabled the tolerant chickpea genotypes to perform better under stressed conditions. Modifications have occurred at various nucleotides in gene sequence of ASR genes during evolution. This hypothetical ASR protein might have enhanced the ASR gene activity as a transcription factor mediating drought responses in chickpea. The authors would like to thank the Division of Genetics for providing all the facilities to carry out genotyping and software analysis.
Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Chickpea has a profound nutritional and economic value in vegetarian society. Introduction Chickpea Cicer arietinum L. Results Relative water content, chlorophyll, protein content and membrane stability index in control and water stressed plants Ten genotypes were used to examine drought responses at different time points viz.
Download: PPT. Fig 1. Drought responses of selected chickpea genotypes measured after different periods of stress 0, 6 and 12 days after drought stress treatment. Table 1. ANOVA for all the four physiological traits under study viz. Table 2. Fig 2. Differential expression of chickpea ASR genes under drought stress conditions.
Fig 3. Fig 4. Jalview of multiple sequence alignment of ASR gene homologues of seven chickpea genotypes. Neighbour joining analysis Multiple sequence alignment of ASR gene was done using MUSCLE software and a phylogenetic tree was constructed by Neighbour joining method with replications in bootstrap test using Treedyne software.
Fig 6. Target protein sequence analysis Blastx results showed Fig 7. Fig 9. Structural prediction and validation The three-dimensional model of the hypothetical protein was constructed using Phyre2 database Fig 10A. Fig Discussion Pulses have a great potential to improve human health as a rich source of protein, soil health through nitrogen fixation and helps in attaining food and nutritional security.
Material and methods Experimental material, soil selection, drought stress treatment Seven promising genotypes of chickpea Cicer arietinum viz.
Determination of relative water content Relative water content RWC of selected chickpea genotypes was measured at different time points 0 day, 6 th day and 12 th day as per the standard method [ 84 ]. Estimation of chlorophyll index and protein content Konica Minolta SPAD Plus chlorophyll meter was used for measuring the chlorophyll index of the selected chickpea genotypes at three different time points 0 day, 6 th day and 12 th day. Determination of membrane stability index mg fresh leaf sample was taken and added to test tubes containing 10ml of distilled water.
Nucleotide sequence analysis PCR amplicons of the seven chickpea genotypes were purified using BigDye terminator v3. Conclusion Present study reveals that increased expression of ASR gene under drought stress possibly enabled the tolerant chickpea genotypes to perform better under stressed conditions.
Supporting information. S1 File. S1 Raw image. Acknowledgments The authors would like to thank the Division of Genetics for providing all the facilities to carry out genotyping and software analysis. References 1. Impact of genomic technologies on chickpea breeding strategies.
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Okay, I'll say it: It was useless. During the beta test period of Windows , a feature called Automated System Recovery was available, but those of us who tried to use it faced repeated failures and transmitted our resulting bug reports to Microsoft. Before the retail version of Windows was released, ASR had been dropped from the feature set. It's back, and it works and I use the term loosely. It's the function you should think of after you think "It looks as if I have to reformat my drive and reinstall Windows," and then reject that solution.
However, don't reject that solution too quickly; I've found it to be a better solution than using ASR sometimes, because ASR reinstates a registry that's no longer accurate.
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