Chickpea Tissues Research Articles

A comprehensive analysis of the B3 superfamily identifies tissue-specific and stress-responsive genes in chickpea (Cicer arietinum L.).

The aim of this study was to provide a comprehensive analysis of the plant-specific B3 domain-containing transcription factors (TFs) in chickpea. Scanning of the chickpea genome resulted in the identification of 51 B3 domain-containing TFs that were located on seven out of eight chickpea chromosomes. Based on the presence of additional domains other than the B3 domain, the candidates were classified into four subfamilies, i.e., ARF (24), REM (19), LAV (6) and RAV (2). Phylogenetic analysis classified them into four groups in which members of the same group had similar intron-exon organization and motif composition. Genome duplication analysis of the candidate B3 genes revealed an event of segmental duplication that was instrumental in the expansion of the B3 gene family. Ka/Ks analysis showed that the B3 gene family was under purifying selection. Further, chickpea B3 genes showed maximum orthology with Medicago followed by soybean and Arabidopsis. Promoter analyses of the B3 genes led to the identification of several tissue-specific and stress-responsive cis-regulatory elements. Expression profiling of the candidate B3 genes using publicly available RNA-seq data of several chickpea tissues indicated their putative role in plant development and abiotic stress response. These findings were further validated by real-time expression analysis. Overall, this study provides a comprehensive analysis of the B3 domain-containing proteins in chickpea that would aid in devising strategies for crop manipulation in chickpea.

Non rhizobial endophytic bacteria from chickpea (Cicer arietinum L.) tissues and their antagonistic traits

Bacteria that colonize plant tissues other than rhizobia and are beneficial for plant growth referred to non rhizobial plant growth-promoting endophytic bacteria (PGPEB). This study was designed to assay the biocontrol activity of plant growth promoting endophytic bacterial isolates those found positive for P. solubilization, ACC deaminase, Indole acetic acid and Gibberelic acid production. These bacterial isolates were obtained from chickpea (Cicer arietinum L.) tissues (roots and nodules). In a previous study a total of 263 non rhizobial endophytic bacterial isolates were isolated. Out of 263 isolates, 64.5% and 34.5% were Gram positive and negative, respectively. Further for biochemical characterization, catalase, oxidase, citrate utilization, nitrate reduction, methyl red and Voges Proskauer’s tests, were performed. On the basis of P solubilization, ACC deaminase, Indole acetic acid and Gibberelic acid production 75 potential isolates were selected and screened for their biocontrol activity viz. (production of cell wall degrading enzymes, production of HCN and fluorescent pigment). Out of 75 isolates, only 29 isolates produced cellulase, 64 isolates were able to produce protease and 28 were positive for both cellulose and protease. Of 75 endophytic isolates 12 isolates (7 from root tissue and 5 from nodules tissue, respectively) were positive for HCN production and 16 isolates were found to be fluorescent pigment producer under µv ligh. As chemical fertilizers and pesticides have detrimental effects on the environment. So these bacterial endophytic isolates will be used not only as a biofertilizer because of their plant growth promotional activities but also used as an alternative of synthetic chemicals for control of several plant diseases.

Pretreatment of seeds with thidiazuron delimits its negative effects on explants and promotes regeneration in chickpea (Cicer arietinum L.)

Thidiazuron (TDZ) is an important cytokinin-like substance used in plant tissue culture. However, long exposure of plant tissues to TDZ results in abnormal regeneration. The present study reports an efficient regeneration protocol that avoids the TDZ overexposure to chickpea (Cicer arietinum L.) tissues. Two separate experiments were designed for seed pretreatment with TDZ, of which the first one involved short-term pretreatment with various concentrations of TDZ (15, 20 and 25 µM), whereas the second one lacked TDZ pretreatment. Axillary meristem explants prepared from TDZ-pretreated and TDZ-non-pretreated seeds were then analyzed in shoot induction medium (SIM) with or without 4 µM TDZ. Thus, four conditions were chosen to analyze the effect of TDZ: (i) TDZ pretreatment only without TDZ in SIM, (ii) TDZ pretreatment along with TDZ in SIM, (iii) no TDZ pretreatment but there was TDZ in SIM, and (iv) there was neither TDZ pretreatment nor TDZ supplied in SIM (control). The response in terms of percent regeneration (69%), number of shoots per explant (20.66 ± 0.5), minimum number of days taken for multiple shoot induction (7.3 ± 0.5) and effective rooting was the highest under the condition (i) with 20 μM TDZ pretreatment without subsequent TDZ supplementation into the SIM. In addition, shoot elongation medium (SEM-3) having 5 μM benzyladenine, 2 μM kinetin and 2 μM gibberellic acid showed the highest branching and maximum shoot length. In conclusion, we reported an easy and efficient regeneration method in chickpea using TDZ pretreatment only, which showed qualitative multiple shooting and effective rooting.

Genome-wide analysis of the CCCH zinc finger family identifies tissue specific and stress responsive candidates in chickpea (Cicer arietinum L.).

The CCCH zinc finger is a group of proteins characterised by a typical motif consisting of three cysteine residues and one histidine residue. These proteins have been reported to play important roles in regulation of plant growth, developmental processes and environmental responses. In the present study, genome wide analysis of the CCCH zinc finger gene family was carried out in the available chickpea genome. Various bioinformatics tools were employed to predict 58 CCCH zinc finger genes in chickpea (designated CarC3H1-58), which were analysed for their physio-chemical properties. Phylogenetic analysis classified the proteins into 12 groups in which members of a particular group had similar structural organization. Further, the numbers as well as the types of CCCH motifs present in the CarC3H proteins were compared with those from Arabidopsis and Medicago truncatula. Synteny analysis revealed valuable information regarding the evolution of this gene family. Tandem and segmental duplication events were identified and their Ka/Ks values revealed that the CarC3H gene family in chickpea had undergone purifying selection. Digital, as well as real time qRT-PCR expression analysis was performed which helped in identification of several CarC3H members that expressed preferentially in specific chickpea tissues as well as during abiotic stresses (desiccation, cold, salinity). Moreover, molecular characterization of an important member CarC3H45 was carried out. This study provides comprehensive genomic information about the important CCCH zinc finger gene family in chickpea. The identified tissue specific and abiotic stress specific CCCH genes could be potential candidates for further characterization to delineate their functional roles in development and stress.

Determination of Ascochyta blight disease in chickpea using real-time PCR

Ascochyta blight is a devastating disease of chickpea caused by Ascochyta rabiei. In this article, we described a real-time PCR assay for the determination and quantification of A. rabiei infection in chickpea tissues and accurate monitoring of disease progression in plant materials inoculated with different inoculation methods. The primer pairs HEF1/HEF2 were designed to anneal to conserved regions of translation elongation factor 1 alpha (EF) gene for specific amplification of 82-bp fragment of A. rabiei based on SYBR Green I technology. The detection limit of assay was determined as 0.1 pg DNA. PCR specificity was confirmed by testing against uninfected chickpea tissues and another fungal species associated with chickpea. The chickpea plants were inoculated by the methods of whole-plant and detached leaflet inoculation. Disease progression in resistant and susceptible cultivars was evaluated at certain time intervals after pathogen inoculation by real-time PCR. The results revealed a good correlation between visual assessments of disease reaction and pathogen quantification in infected chickpea tissues. The target DNA sequence was also amplified from the samples of DNA extracts from artificially infested seed. This technique could provide a useful approach for efficient selection of resistant breeding material in an early stage of infection as an alternative to the visual disease assessment and will be also used for the determination and quantification of A. rabiei infection.

The influence of iron chelate and zinc sulfate on the growth and nutrient composition of chickpea grown on a calcareous soil

ABSTRACT- The effects of iron (Fe) and zinc (Zn) treatments on the growth and nutrient composition of chickpea were studied in a greenhouse experiment arranged in a completely randomized design. While the application of Fe decreased mean shoot dry weight of chickpea, that of Zn had no significant effect on chickpea shoot dry weight. Increasing Fe levels drastically decreased Mn concentration and uptake in chickpea shoot. Addition of Zn, however, had no significant effect on Mn concentration and uptake in chickpea shoot. Although the addition of 10 mg Fe kg-1 significantly increased mean Fe concentration in chickpea shoot, the effect of Fe application on mean Fe uptake was negligible. Although the application of Zn had no significant effect on mean Fe concentration or uptake, it increased mean Zn uptake in chickpea shoot. While the application of Fe increased mean Zn concentration, application of 5 mg Fe kg-1 had no significant effects on mean Cu concentration or uptake in chickpea tissues although 10 mg Fe kg-1 increased mean Cu concentration and uptake in chickpea tissues. Addition of Zn, however, had no significant effect on Cu uptake. A significant negative correlation was observed between Mn uptake and Fe rates demonstrating a reduction in shoot Mn uptake following Fe application. Although shoot dry weight of chickpea was negatively correlated with Fe rates, it showed significant positive correlations with the uptakes of Zn and Mn indicating that the patterns of changes in dry matter was in coordination with changes in Zn and Mn uptakes. Since Fe chelate addition may cause nutrient imbalance and growth reduction in chickpea, it appears that the use of Fe and Zn efficient genotypes should be considered as an appropriate practice for chickpea grown on calcareous soils low in available Fe and Zn.

Root endophytes modify the negative effects of chickpea on the emergence of durum wheat

Secondary metabolite production in plants is influenced by biotic and abiotic environmental factors. We hypothesized that (1) resident root endophytes and (2) water limitation can modify the negative effects of chickpea on durum wheat. A Canadian prairie-resident root endosphere community was used to test these hypotheses. Microcosms were constructed with pasteurized field soil, inoculated or not with surface sterilized roots containing an endophytic community from the same soil, and one of four chickpea cultivars. Microcosms were maintained under conditions of water sufficiency or limitation (70% and 30% of field capacity) until chickpea plants reached maturity. The negative effects of the shoot and root tissues of these chickpea plants was then assessed in the same microcosm, using durum wheat as test plant. The impact of chickpea shoot tissues on durum wheat emergence was as negative as the impact of the positive control, black mustard, but chickpea roots had no effect. Initial introduction of root endophytes in the microcosms modified the pattern of durum wheat seedling emergence in chickpea tissues-amended soil, generally reducing the negative effect of chickpea, except for cultivar CDC Nika whose tissues impacted durum wheat maximum emergence rate more negatively in inoculated than non-inoculated microcosms. Water limitation had no effect on the negative properties of chickpea tissues. We conclude that unfavourable and favourable combinations of chickpea cultivars and resident root-associated microorganisms may explain at least partly, the erratic yield of wheat obtained after a chickpea crop.

A new set of ESTs from chickpea (Cicer arietinum L.) embryo reveals two novel F-box genes, CarF-box_PP2 and CarF-box_LysM, with potential roles in seed development.

Considering the economic importance of chickpea (C. arietinum L.) seeds, it is important to understand the mechanisms underlying seed development for which a cDNA library was constructed from 6 day old chickpea embryos. A total of 8,186 ESTs were obtained from which 4,048 high quality ESTs were assembled into 1,480 unigenes that majorly encoded genes involved in various metabolic and regulatory pathways. Of these, 95 ESTs were found to be involved in ubiquitination related protein degradation pathways and 12 ESTs coded specifically for putative F-box proteins. Differential transcript accumulation of these putative F-box genes was observed in chickpea tissues as evidenced by quantitative real-time PCR. Further, to explore the role of F-box proteins in chickpea seed development, two F-box genes were selected for molecular characterization. These were named as CarF-box_PP2 and CarF-box_LysM depending on their C-terminal domains, PP2 and LysM, respectively. Their highly conserved structures led us to predict their target substrates. Subcellular localization experiment revealed that CarF-box_PP2 was localized in the cytoplasm and CarF-box_LysM was localized in the nucleus. We demonstrated their physical interactions with SKP1 protein, which validated that they function as F-box proteins in the formation of SCF complexes. Sequence analysis of their promoter regions revealed certain seed specific cis-acting elements that may be regulating their preferential transcript accumulation in the seed. Overall, the study helped in expanding the EST database of chickpea, which was further used to identify two novel F-box genes having a potential role in seed development.

Genome-wide survey and expression analysis of F-box genes in chickpea.

BackgroundThe F-box genes constitute one of the largest gene families in plants involved in degradation of cellular proteins. F-box proteins can recognize a wide array of substrates and regulate many important biological processes such as embryogenesis, floral development, plant growth and development, biotic and abiotic stress, hormonal responses and senescence, among others. However, little is known about the F-box genes in the important legume crop, chickpea. The available draft genome sequence of chickpea allowed us to conduct a genome-wide survey of the F-box gene family in chickpea.ResultsA total of 285 F-box genes were identified in chickpea which were classified based on their C-terminal domain structures into 10 subfamilies. Thirteen putative novel motifs were also identified in F-box proteins with no known functional domain at their C-termini. The F-box genes were physically mapped on the 8 chickpea chromosomes and duplication events were investigated which revealed that the F-box gene family expanded largely due to tandem duplications. Phylogenetic analysis classified the chickpea F-box genes into 9 clusters. Also, maximum syntenic relationship was observed with soybean followed by Medicago truncatula, Lotus japonicus and Arabidopsis. Digital expression analysis of F-box genes in various chickpea tissues as well as under abiotic stress conditions utilizing the available chickpea transcriptome data revealed differential expression patterns with several F-box genes specifically expressing in each tissue, few of which were validated by using quantitative real-time PCR.ConclusionsThe genome-wide analysis of chickpea F-box genes provides new opportunities for characterization of candidate F-box genes and elucidation of their function in growth, development and stress responses for utilization in chickpea improvement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1293-y) contains supplementary material, which is available to authorized users.

Genome-Scale Transcriptomic Insights into Molecular Aspects of Abiotic Stress Responses in Chickpea

Chickpea is an important legume crop plant and various abiotic stresses are the major constraints affecting its overall productivity. For discovery of candidate genes involved in abiotic stress responses, we employed RNA sequencing for transcriptome profiling of roots and shoots of chickpea seedlings subjected to desiccation, salinity, and cold stresses. In total, we generated more than 250 million high-quality reads from non-stressed and stressed tissue samples. Data analyses provided a comprehensive view of the dynamic transcriptional response of chickpea tissues to different abiotic stresses. Differential expression analysis identified a total of 11,640 chickpea transcripts showing response to at least one of the stress conditions. The reference-based transcriptome assembly was generated and at least 3,536 previously unannotated gene loci differentially expressed under abiotic stress conditions were identified. We observed extensive transcriptional reprogramming of genes involved in transcription regulation, energy metabolism, photosynthesis, hormonal responses, secondary metabolite biosynthesis and osmoprotectant metabolism under stress conditions. In addition, genes involved in post-translational modifications, RNA metabolic processes, and epigenetic regulation were also significantly highlighted. The comprehensive transcriptome analyses presented in this study revealed several potential key regulators of plant response to abiotic stresses and open avenues to carry out functional and applied genomic studies for improving abiotic stress tolerance in chickpea.

Differences between easy- and difficult-to-mill chickpea (Cicer arietinum L.) genotypes. Part II: Protein, lipid and mineral composition

Part I introduced the concept of easy- and difficult-to-mill chickpea genotypes, the broad chemical composition of their seed fractions and proposed mechanistic explanations for physical differences consistent with observed variation in milling ease. Part II continues this research by delving deeper into the amino acid, fatty acid and mineral components. No association between fatty acid composition and ease of milling was observed. However, particular amino acids and mineral elements were identified that further support roles of lectins, pectins and mineral-facilitated binding in the adhesion of chickpea seed coat and cotyledons. These differences suggest underlying mechanisms that could be exploited by breeding programmes to improve milling performance. This study shows that the content and composition of amino acids, fatty acids and minerals within different chickpea tissues vary with seed type (desi and kabuli) and within desi genotypes in ways that are consistent with physical explanations of how seed structure and properties relate to milling behaviour.

LC-ESI-MS characterisation of phytoalexins induced in chickpea and pea tissues in response to a biotic elicitor of Hypnea musciformis (red algae)

A simple extraction procedure and HPLC method was developed to analyse the major and minor components of induced phytoalexins of elicited tissues (seeds) of chickpeas (Cicer arietinum L.) and peas (Pisum sativum L.) treated with a biotic elicitor (k-carrageenan) of Hypnea musciformis (red algae) from the Karachi coast. The level and timing of the induced phytoalexin production were estimated on the basis of various elicitor dilutions and as a function of time; the results are presented and discussed. A LC-ESI-MS/MS technique has been employed for the detection and characterisation of the induced phytochemical components (flavonoids and their glyco-conjugates). Nine flavonoids were identified from chickpeas: naringin, naringin malonate, liquiritigenin, naringenin, biochanin A, daidzein, formononetin, maackiain and medicarpin, while five flavonoids were identified from peas: afrormosin, anhydropisatin, pisatin, pseudobaptigenin and maackiain. These compounds play a vital role as phytoalexins because of their antimicrobial activity.

In Planta and Soil Quantification of Fusarium oxysporum f. sp. ciceris and Evaluation of Fusarium Wilt Resistance in Chickpea with a Newly Developed Quantitative Polymerase Chain Reaction Assay

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.

Characterization of a chickpea (Cicer arietinum L.) NAC family gene, CarNAC5, which is both developmentally- and stress-regulated

It has been documented that the plant-specific NAC (for NAM, ATAF1,2 and CUC2) transcription factors play an important role in plant development and stress responses. In this study, a chickpea NAC gene CarNAC5 (for Cicer arietinum L. NAC gene 5) was isolated from a cDNA library from chickpea leaves treated by polyethylene glycol (PEG). CarNAC5, as a single/low copy gene, contained three exons and two introns within genomic DNA sequence and encoded a polypeptide with 291 amino acids. CarNAC5 protein had a conserved NAC domain in the N-terminus and showed high similarity to other NACs, especially ATAF subgroup members. The CarNAC5:GFP fusion protein was localized in the nucleus of onion epidermal cells. Furthermore, CarNAC5 protein activated the reporter genes LacZ and HIS3 in yeast. The transactivation activity was mapped to the C-terminal region. The transcripts of CarNAC5 appeared in many chickpea tissues including seedling leaves, stems, roots, flowers, seeds and pods, but mostly accumulated in flowers. Meanwhile, CarNAC5 was strongly expressed during seed maturation and in embryos of the early germinating seeds. It was also significantly induced by drought, heat, wounding, salicylic acid (SA), and indole-3-acetic acid (IAA) treatments. Our results suggest that CarNAC5 encodes a novel NAC-domain protein and acts as a transcriptional activator involved in plant developmental regulation and various stress responses.

Phytotoxicity of solanapyrones A and B produced by the chickpea pathogen Ascochyta rabiei(Pass.) Labr. and the apparent metabolism of solanapyrone A by chickpea tissues

When chickpea shoots were placed in solanapyrone A, the compound could not be recovered from the plant and symptoms developed. These consisted of loss of turgor, shrivelling and breakage of stems and flame-shaped, chlorotic zones in leaflets. In similar experiments with solanapyrone B, only 9.4% (22μ g) of the compound taken up was recovered and stems remained turgid but their leaflets became twisted and chlorotic and some abscized.Cells isolated from leaflets of 12 chickpea cultivars differed by up to five-fold in their sensitivity to solanapyrone A and this compound was 2.6–12.6 times more toxic than solanapyrone B, depending on cultivar.Glutathione reacted with solanapyrone A in vitro reducing its toxicity in a cell assay and forming a conjugate. Measurement of reduced glutathione concentration and glutathione-S-transferase (GST) activity among cultivars showed that the differences of their means were highly significant and both were negatively and significantly correlated with their sensitivity to solanapyrone A. Treatment of shoots with solanapyrone A enhanced total, reduced and oxidized glutathione content as well as GST activity 1.26-, 1.23-, 1.50- and 1.94-fold, respectively. Similarly, treatment of shoots with the safener, dichlormid, also raised total, oxidized and reduced glutathione levels and GST activity 1.42-, 1.07-, 1.43-, 1.42-fold, respectively. Cells isolated from shoots treated with dichlormid at 150 and 300μ g per shoot were 2.45 and 2.66 times less sensitive to solanapyrone A, with LD50values of 71.5 and 77.8μ g ml−1, respectively, as compared to 29.2μ g ml−1for controls.

Associative effect of Azospirillum lipoferum and Azotobacter chroococcum with Rhizobium spp. on mineral composition and growth of chickpea (Cicer arietinum) on sandy soils

Summary The interactions between Azospirillum lipoferum, Azotobacter chroococcum and Rhizobium spp. were assessed by the growth pattern and mineral concentration in chickpeas (Cicer arietinum cv. Giza 2) on loamy sand soil and on sandy soil. Seeds were inoculated with Rhizobium and with either Azospirillum or Azotobacter or with a mixture of both inoculants. Growth of chickpea was improved by adding Azospirillum and/or Azotobacter with Rhizobium in both soil types used. However, Rhizobium with both inoculants was found to be most effective. Cross sections of chickpea root nodules showed that the 2 diazotrophs plus Rhizobium improved nodule branching compared to Rhizobium inoculated treatments in both soil types. Generally the values of nutrient concentration in chickpea tissues were higher in plants grown in loamy sand soil than those grown in sandy soil inspite of the plant age and the applied treatment.