Synergistic effects of melatonin and 24-epibrassinolide on chickpea water deficit tolerance.

BACKGROUND: Water deficiency stress reduces yield in grain legumes, primarily due to a decrease in the pods number. Melatonin (ML) and 24-epibrassinolide (EBL) are recognized for their hormone-like properties that improve plant tolerance to abiotic stresses. This study aimed to assess the impact of different concentrations of ML (0, 100, and 200 µM) and EBL (0, 3, and 6 µM) on the growth, biochemical, and physiological characteristics of chickpea plants under water-stressed conditions. RESULTS: The study's findings indicated that under water-stressed conditions, a decrease in seed (30%) and pod numbers (31%), 100-seed weight (17%), total chlorophyll content (46%), stomatal conductance (33%), as well as an increase in H2O2 (62%), malondialdehyde content (40%), and electrolyte leakage index (40%), resulted in a 40% reduction in chickpea plants grain yield. Our findings confirmed that under water-stressed conditions, seed oil, seed oil yield, and seed protein yield dropped by 20%, 55%, and 36%, respectively. The concurrent exogenous application of ML and EBL significantly reduces oxidative stress, plasma membrane damage, and reactive oxygen species (ROS) content. This treatment also leads to increased yield and its components, higher pigment content, enhanced oil and protein yield, and improved enzymatic and non-enzymatic antioxidant activities such as catalase, superoxide dismutase, polyphenol oxidase, ascorbate peroxidase, guaiacol peroxidase, flavonoid, and carotenoid. Furthermore, it promotes the accumulation of osmoprotectants such as proline, total soluble protein, and sugars. CONCLUSIONS: Our study found that ML and EBL act synergistically to regulate plant growth, photosynthesis, osmoprotectants accumulation, antioxidant defense systems, and maintain ROS homeostasis, thereby mitigating the adverse effects of water deficit conditions. ML and EBL are key regulatory network components in stressful conditions, with significant potential for future research and practical applications. The regulation metabolic pathways of ML and EBL in water-stressed remains unknown. As a result, future research should aim to elucidate the molecular mechanisms by employing genome editing, RNA sequencing, microarray, transcriptomic, proteomic, and metabolomic analyses to identify the mechanisms involved in plant responses to exogenous ML and EBL under water deficit conditions. Furthermore, the economical applications of synthetic ML and EBL could be an interesting strategy for improving plant tolerance.

Evaluation of different Kabuli chickpea genotypes against Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in relation to biotic and abiotic factors.

Background: The chickpea pod borer Helicoverpa armigera (Hübner) is a significant insect pest of chickpea crops, causing substantial global losses. Methods: Field experiments were conducted in Central Punjab, Pakistan, to investigate the impact of biotic and abiotic factors on pod borer population dynamics and infestation in nine kabuli chickpea genotypes during two cropping seasons (2020-2021 and 2021-2022). The crops were sown in November in both years, with row-to-row and plant-to-plant distances of 30 and 15 cm, respectively, following a randomized complete block design (RCBD). Results: Results showed a significant difference among the tested genotypes in trichome density, pod wall thickness, and leaf chlorophyll contents. Significantly lower larval population (0.85 and 1.10 larvae per plant) and percent damage (10.65% and 14.25%) were observed in genotype Noor-2019 during 2020-2021 and 2021-2022, respectively. Pod trichome density, pod wall thickness, and chlorophyll content of leaves also showed significant variation among the tested genotypes. Pod trichome density and pod wall thickness correlated negatively with larval infestation, while chlorophyll content in leaves showed a positive correlation. Additionally, the larval population positively correlated with minimum and maximum temperatures, while relative humidity negatively correlated with the larval population. Study results explore natural enemies as potential biological control agents and reduce reliance on chemical pesticides.

Arbuscular mycorrhizae reduced arsenic induced oxidative stress by coordinating nutrient uptake and proline-glutathione levels in Cicer arietinum L. (chickpea).

Accumulation of Arsenic (As) generates oxidative stress by reducing nutrients availability in plants. Arbuscular mycorrhizal (AM) symbiosis can impart metalloid tolerance in plants by enhancing the synthesis of sulfur (S)-rich peptides (glutathione- GSH) and low-molecular-weight nitrogenous (N) osmolytes (proline- Pro). The present study, therefore investigated the efficiency of 3 AM fungal species (Rhizoglomus intraradices-Ri, Funneliformis mosseae -Fm and Claroideoglomus claroideum- Cc) in imparting As (arsenate-AsV -40 at 60 mg kg-1 and arsenite- AsIII at 5 and 10 mg kg-1) tolerance in two Cicer arietinum (chickpea) genotypes (HC 3 and C 235). As induced significantly higher negative impacts in roots than shoots, which was in accordance with proportionately higher reactive oxygen species (ROS) in the former, with AsIII more toxic than AsV. Mycorrhizal symbiosis overcame oxidative stress by providing the host plants with necessary nutrients (P, N, and S) through enhanced microbial enzyme activities (MEAs) in soil, which increased the synthesis of Pro and GSH and established a redox balance in the two genotypes. This coordination between nutrient status, Pro-GSH levels, and antioxidant defense was stronger in HC 3 than C 235 due to its higher responsiveness to the three AM species. However, Ri was most beneficial in inducing redox homeostasis, followed by Fm and Cc, since the Cicer arietinum-Ri combination displayed the maximum ability to boost antioxidant defense mechanisms and establish a coordination with Pro synthesis. Thus, the results highlighted the importance of selecting specific chickpea genotypes having an ability to establish effective mycorrhizal symbiosis for imparting As stress tolerance.

Multifarious Plant Growth-Promoting Rhizobacterium Enterobacter sp. CM94-Mediated Systemic Tolerance and Growth Promotion of Chickpea (Cicer arietinum L.) under Salinity Stress.

BACKGROUND: Chickpea is one of the most important leguminous crops and its productivity is significantly affected by salinity stress. The use of ecofriendly, salt-tolerant, plant growth-promoting rhizobacteria (PGPR) as a bioinoculant can be very effective in mitigating salinity stress in crop plants. In the present study, we explored, characterized, and evaluated a potential PGPR isolate for improving chickpea growth under salt stress. METHODS: A potential PGPR was isolated from rhizospheric soils of chickpea plants grown in the salt-affected area of eastern Uttar Pradesh, India. The isolate was screened for salt tolerance and characterized for its metabolic potential and different plant growth-promoting attributes. Further, the potential of the isolate to promote chickpea growth under different salt concentrations was determined by a greenhouse experiment. RESULTS: A rhizobacteria isolate, CM94, which could tolerate a NaCl concentration of up to 8% was selected for this study. Based on the BIOLOG carbon source utilization, isolate CM94 was metabolically versatile and able to produce multiple plant growth-promoting attributes, such as indole acetic acid, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophore, hydrogen cyanide (HCN), and ammonia as well as solubilized phosphate. A polyphasic approach involving the analysis of fatty acid methyl ester (FAME) and 16S rRNA gene sequencing confirmed the identity of the isolate as Enterobacter sp. The results of greenhouse experiments revealed that isolate CM94 inoculation significantly enhanced the shoot length, root length, and fresh and dry weight of chickpea plants, under variable salinity stress. In addition, inoculation improved the chlorophyll, proline, sugar, and protein content in the tissues of the plant, while lowering lipid peroxidation. Furthermore, isolate CM94 reduced oxidative stress by enhancing the enzymatic activities of superoxide dismutase, catalase, and peroxidase compared to in the respective uninoculated plants. CONCLUSIONS: Overall, the results suggested that using Enterobacter sp. CM94 could significantly mitigate salinity stress and enhance chickpea growth under saline conditions. Such studies will be helpful in identifying efficient microorganisms to alleviate salinity stress, which in turn will help, to devise ecofriendly microbial technologies.

Exploring the molecular basis of resistance to Botrytis cinerea in chickpea genotypes through biochemical and morphological markers.

Chickpea (Cicer arietinum L.) is an important pulse crop around the globe and a valuable source of protein in the human diet. However, it is highly susceptible to various plant pathogens such as fungi, bacteria, and viruses, which can cause significant damage from the seedling phase until harvest, leading to reduced yields and affecting its production. Botrytis cinerea can cause significant damage to chickpea crops, especially under high humidity and moisture conditions. This fungus can cause grey mould disease, which can lead to wilting, stem and pod rot, and reduced yields. Chickpea plants have developed specific barriers to counteract the harmful effects of this fungus. These barriers include biochemical and structural defences. In this study, the defence responses against B. cinerea were measured by the quantification of biochemical metabolites such as antioxidant enzymes, malondialdehyde (MDA), proline, glutathione (GSH), H2O2, ascorbic acid (AA) and total phenol in the leaf samples of chickpea genotypes (one accession of wild Cicer species, viz. Cicer pinnatifidum188 identified with high level of resistance to Botrytis grey mould (BGM) and a cultivar, Cicer arietinumPBG5 susceptible to BGM grown in the greenhouse). Seedlings of both the genotypes were inoculated with (1 × 104 spore mL-1) inoculum of isolate 24, race 510 of B. cinerea and samples were collected after 1, 3, 5, and 7 days post-inoculation (dpi). The enhanced enzymatic activity was observed in the pathogen-inoculated leaf samples as compared to uninoculated (healthy control). Among inoculated genotypes, the resistant one exhibited a significant change in enzymatic activity, total phenolic content, MDA, proline, GSH, H2O2, and AA, compared to the susceptible genotype. The study also examined the isozyme pattern of antioxidant enzymes at various stages of B. cinerea inoculation. Results from scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy revealed that BGM had a more significant impact on susceptible genotypes compared to resistant ones when compared to the control (un-inoculated). In addition, SEM and FTIR spectroscopy analyses confirmed the greater severity of BGM on susceptible genotypes compared to their resistant counterparts. Our results suggest the role of antioxidant enzymes and other metabolites as defence tools and biochemical markers to understand compatible and non-compatible plant-pathogen interactions better. The present investigation will assist future plant breeding programs aimed at developing resistant varieties.

Induced defense responses in cultivated and wild chickpea genotypes against Helicoverpa armigera infestation.

Five desi (GL 12,021, GL 29,095, GL 29,078, H11 22 and CSJ 515) and three wild (GLW 22, GLW 58 and GLW 187) chickpea cultivars showed induced defense response against Helicoverpa armigera infestation as a result of enhanced activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione reductase, polyphenol oxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase in leaves, pod walls and seeds. Catalase activity increased in leaves of GL 12,021, H11 22, GL 29,095, CSJ 515, GLW 22, and GL 29,078 after infestation compared to resistant check; catalase and peroxidase activities in GL 29,095 and GL 29,078; ascorbate peroxidase and glutathione reductase activities in leaves of GLW 58. The increased activity of superoxide dismutase in pod wall of H1122; catalase in pod wall of 29,078, GL 29,095 and GL 22; ascorbate peroxidase and glutathione reductase in pod wall of GLW 58; phenylalanine ammonia lyase and tyrosine ammonia lyase in pod wall of GLW 187, H11 22, GL 20,978, GLW 22 and GLW 58 after infestation as compared to resistant check might be responsible for mitigating infestation induced oxidative stress. MDA content decreased in leaves, pod wall and seeds of GLW 187 and GL 12,021 after infestation. Lower percent pod damage (9.58-12.44%) in GL 12,021, GLW 187, GL 29,095, H11 22, GL 29,078, GLW 22 and GLW 58 as compared to resistant (16.18%) and susceptible (21.50) checks might be attributed to differential induced defense mechanism in them. The identified desi and wild genotypes might be used in breeding program to develop cultivars with improved resistance to herbivore.

The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat.

MAIN CONCLUSION: We identified 119 typical CaMYB encoding genes and reveal the major components of the proanthocyanidin regulatory network. CaPARs emerged as promising targets for genetic engineering toward improved agronomic traits in C. arietinum. Chickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein-protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.

Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L. ) varieties of North-West Pakistan / Caracterização bioquímica para determinação de distâncias genéticas entre diferentes variedades indígenas de grão-de-bico (Cicer arietinum L. ) do noroeste do Paquistão

Abstract Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.

Resumo As distâncias genéticas entre as diferentes variedades de grão-de-bico e a avaliação de seus perfis de aminoácidos livres foram determinadas com base na eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). As proteínas solúveis totais foram resolvidas em SDS-PAGE a 10%. Foi observada baixa variabilidade nas variedades testadas. O dendrograma fundamentado em dados eletroforéticos agrupou os genótipos em dois grupos. Os resultados mostraram que o teor médio de proteínas de todas as variedades foi de 26,01%, na faixa de 22,8% para Thal-2006 a 34,06% para Sheenghar-2000 do peso de sementes secas. Com base no conteúdo total de proteínas, Bittal-98, Dasht, Sheen Ghar-2000, Karak-3, CM-98, Paidar-91, Fakhr-e-Thal, C-44, Balaksar e KK-1 apresentaram concentrações semelhantes para o conteúdo de proteínas entre si, mas tiveram variação quanto ao restante das variedades. Diferentes proteínas foram separadas com base nas alterações de seus pesos moleculares por meio de eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). Dasht, CM-98 e Sheen Ghar mostraram 100% de similaridade. Balaksar, Fakhr-e-Thal, KK-2, Chattan e KC-98, KK-1 e Lawaghar foram 100% semelhantes entre si, mas apresentaram variação em relação ao restante dos acessos. O dendrograma geral mostrou alto e baixo nível de variação entre os acessos. A concentração de aminoácidos livres variou entre as 16 variedades de grão-de-bico. Foi encontrada uma diferença significativa entre os aminoácidos essenciais e não essenciais nas cultivares de grão-de-bico. A concentração total de aminoácidos essenciais foi registrada em 40,81 g / 100 g de proteína, enquanto a não essencial foi registrada em 59,18343 g / 100 g de proteína nas cultivares. A maior concentração de aminoácidos essenciais foi encontrada em C-44, seguida de KK-2, KK-1 e Fakhr-e-Thal, enquanto a menor concentração foi registrada em CM-98, Paidar-91 e Sheen Ghar-2000. As cultivares TAL-2006, Chattan e Karak-3 apresentaram concentração máxima de aminoácidos essenciais e endógenos. Em conclusão, para ampliar os pools genéticos em programas de melhoramento ou procurar caracteres exóticos, por exemplo, novos alelos de resistência a doenças, pode ser utilizada a adesão com baixos coeficientes de similaridade (Lawaghar e Battal-98). Além disso, as informações adquiridas neste estudo poderiam ser usadas para criar uma abordagem de criação eficiente, com o objetivo de melhorar a nutrição e ampliar a base genética dessa cultura alimentar essencial do Paquistão.

Characterization and differential expression of sucrose and starch metabolism genes in contrasting chickpea (Cicer arietinum L.) genotypes under low temperature.

Low temperature (LT) causes significant yield losses in chickpea (Cicer arietinum L.). The sucrose starch metabolism is associated with abiotic-stress tolerance or sensitivity in plants. The changes in sugars and starch contents under LT in chickpea have already been studied, however, no information is available on LT-induced alterations in transcription of carbohydrate metabolic pathway genes in chickpea. To understand the differences in the regulation of sucrose and starch metabolism under LT, the expression of sucrose and starch metabolism genes was studied in leaves of cold-sensitive (GPF2) and cold-tolerant (ICC 16349) chickpea genotypes. The mRNA sequences of chickpea genes were retrieved from the public databases followed by confirmation of identity and characterization. All the genes were functional in chickpea. Between the two paralogues of cell wall invertase, cell wall invertase 3×2 (CWINx2) was the truncated version of cell wall invertase 3×1 (CWINx1) with the loss of 241 bases in the mRNA and 67 amino acids at N terminal of the protein. Comparison of expression of the genes between control (22°C day / 16°C night) and LT treated (4°C; 72 h) plants revealed that granule bound starch synthase 2 (GBSS2) and ß-amylase 3 (BAM3) were upregulated in ICC 16349 whereas sucrose phosphate synthase 2 (SPS2), CWINx1, CWINx2 and ß-amylase 1 (BAM1) were downregulated. In contrast to this, SPS2, CWINx1, CWINx2 and BAM1 were upregulated and GBSS2 downregulated in GPF2 under LT. The gene expression data suggested that UGPase, CWINs, GBSS2 and BAM3 are important components of cold-tolerance machinery of chickpea.

Transcriptome analysis reveals key genes associated with root-lesion nematode Pratylenchus thornei resistance in chickpea.

The root-lesion nematode, Pratylenchus thornei, is one of the major plant-parasitic nematode species causing significant yield losses in chickpea (Cicer arietinum). In order to identify the underlying mechanisms of resistance to P. thornei, the transcriptomes of control and inoculated roots of three chickpea genotypes viz. D05253 > F3TMWR2AB001 (resistant advanced breeding line), PBA HatTrick (moderately resistant cultivar), and Kyabra (susceptible cultivar) were studied at 20 and 50 days post inoculation using the RNA-seq approach. On analyzing the 633.3 million reads generated, 962 differentially expressed genes (DEGs) were identified. Comparative analysis revealed that the majority of DEGs upregulated in the resistant genotype were downregulated in the moderately resistant and susceptible genotypes. Transcription factor families WRKY and bZIP were uniquely expressed in the resistant genotype. The genes Cysteine-rich receptor-like protein kinase 10, Protein lifeguard-like, Protein detoxification, Bidirectional sugar transporter Sugars Will Eventually be Exported Transporters1 (SWEET1), and Subtilisin-like protease were found to play cross-functional roles in the resistant chickpea genotype against P. thornei. The identified candidate genes for resistance to P. thornei in chickpea can be explored further to develop markers and accelerate the introgression of P. thornei resistance into elite chickpea cultivars.

Characterization of peptides with antioxidant activity and antidiabetic potential obtained from chickpea (Cicer arietinum L.) protein hydrolyzates.

Alcalase hydrolyzates were prepared from the albumin (AH) and globulin (GH) fractions of eight chickpea (Cicer arietinum L.) genotypes from Mexico and 10 from other countries. Protein content, antioxidant activity (AA) (ABTS, DPPH), and degree of hydrolysis were evaluated and the best genotype was selected by principal component analysis. The hydrolyzates of the chosen genotype were analyzed for its antidiabetic potential measured as inhibition of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP4). Peptide profiles were obtained by liquid chromatography-mass spectrometry (UPLC-DAD-MS), and the most active peptides were analyzed by molecular docking. The average antioxidant activity of albumin hydrolyzates was higher than that of globulin hydrolyzates. ICC3761 was the selected genotype and peptides purified from the albumin hydrolyzate showed the best antioxidant activity and antidiabetic potential (FEI, FEL, FIE, FKN, FGKG, and MEE). FEI, FEL, and FIE were in the same chromatographic peak and this mixture showed the best ABTS scavenging (78.25%) and DPP4 inhibition (IC50  = 4.20 µg/ml). MEE showed the best DPPH scavenging (47%). FGKG showed the best inhibition of α-amylase (54%) and α-glucosidase (56%) and may be a competitive inhibitor based on in silico-predicted interactions with catalytic amino acids in the active site of both enzymes. These peptides could be used as nutraceutical supplements against diseases related to oxidative stress and diabetes. PRACTICAL APPLICATION: This study showed that chickpea protein hydrolyzates are good sources of peptides with antidiabetic potential, showing high antioxidant activity and inhibition of enzymes related to carbohydrate metabolism and type 2 diabetes. These hydrolyzates could be formulated in functional foods for diabetes.

Calcium and ethylene glycol tetraacetic acid mitigate toxicity and alteration of gene expression associated with cadmium stress in chickpea (Cicer arietinum L.) shoots.

In the aim to estimate the protective role of calcium (Ca) and ethylene glycol tetraacetic acid (EGTA) against cadmium (Cd)-induced damage, chickpea (Cicer arietinum L.) seeds were exposed to 200 µM Cd stress for 6 days or 3 days then subjected to co-treatment of the metal with either 100 mM CaCl2 or 100 µM EGTA for 3 additional days. The addition of Ca and EGTA improved seedling growth. This protecting effect was correlated to the alleviation of the metal-induced oxidative stress, exemplified by the reduction of hydrogen peroxide (H2O2) contents. Besides, Ca and EGTA stimulated thioredoxin (Trx) and thioredoxin reductase (NTR) activities (2.75- and 1.75-fold increase when compared to Cd-stressed, respectively) protecting, thereby, protein -SH groups from the Cd-mediated oxidation, and modulated ferredoxin (Fdx) activity to a control level. Moreover, Ca and EGTA reinstated the glutathione redox steady state, mainly via preserving a high level of glutathione reduced form (GSH). This effect coincided with the maintaining of the Cd-stimulated glutathione reductase (GR) activity and the decline of glutathione peroxidase (GPX, 43% lower than Cd-stressed shoots) activity. Ca and EGTA counteracted the inhibitory effect of Cd on the activity and gene expression of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) isoenzyme and modulated the activities of catalase (CAT) and ascorbate peroxidase (APX). Overall, our results provided evidence that Ca and EGTA supplement could be a promising approach in the remediation of Cd-contaminated environment.

ABC Transporter-Mediated Transport of Glutathione Conjugates Enhances Seed Yield and Quality in Chickpea.

The identification of functionally relevant molecular tags is vital for genomics-assisted crop improvement and enhancement of seed yield, quality, and productivity in chickpea (Cicer arietinum). The simultaneous improvement of yield/productivity as well as quality traits often requires pyramiding of multiple genes, which remains a major hurdle given various associated epistatic and pleotropic effects. Unfortunately, no single gene that can improve yield/productivity along with quality and other desirable agromorphological traits is known, hampering the genetic enhancement of chickpea. Using a combinatorial genomics-assisted breeding and functional genomics strategy, this study identified natural alleles and haplotypes of an ABCC3-type transporter gene that regulates seed weight, an important domestication trait, by transcriptional regulation and modulation of the transport of glutathione conjugates in seeds of desi and kabuli chickpea. The superior allele/haplotype of this gene introgressed in desi and kabuli near-isogenic lines enhances the seed weight, yield, productivity, and multiple desirable plant architecture and seed-quality traits without compromising agronomic performance. These salient findings can expedite crop improvement endeavors and the development of nutritionally enriched high-yielding cultivars in chickpea.

Over-expression of CarMT gene modulates the physiological performance and antioxidant defense system to provide tolerance against drought stress in Arabidopsis thaliana L.

Drought is one of the major abiotic stresses which negatively affect plant growth and crop yield. Metallothionein (MTs) is a low molecular weight protein, mainly involved in metal homeostasis, while, its role in drought stress is still to be largely explored. The present study was aimed to investigate the role of MT gene against drought stress. The chickpea MT based on its up-regulation under drought stress was overexpressed in Arabidopsis thaliana to explore its role in mitigation of drought stress. The total transcript of MT gene was up to 30 fold higher in transgenic lines. Arabidopsis plants transformed with MT gene showed longer roots, better efficiency of survival and germination, larger siliques and higher biomass compared to WT. The physiological variables (A, WUE, G, E, qP and ETR) of WT plants were reduced during drought stress which recovered in transgenic Arabidopsis lines. The enzymatic and non-enzymatic antioxidant (APX, GPX, POD, GR, GRX, GST, CAT, MDHAR, ASc and GSH) levels were also enhanced in transgenic lines to provide tolerance. Simultaneously, drought responsive amino acids, i.e. proline and cysteine contents were higher in transgenic lines. Overall, the results suggest that MT gene is actively involved in the mitigation of drought stress and could be the choice for genetic engineering strategy to overcome drought stress.

RNA sequencing of leaf tissues from two contrasting chickpea genotypes reveals mechanisms for drought tolerance.

Chickpea (Cicer arietinum L.) is the second most important winter crop which is consumed globally due to its high nutritional value. Chickpea as one of the leguminous crop is important in crop rotation with cereal crops like wheat and barley. The main constraints for chickpea production are abiotic stresses such as drought, salinity, and heat. Among these, drought is a major cause of the decline in chickpea production in worldwide. Studies conducted so far have provided a limited insight into different genetic pathways associated with drought tolerance/response. In this study, the leaf tissue from shoots apical meristem stage of drought tolerant (ICC8261) and drought sensitive (ICC283) genotypes were analysed using RNA sequencing to identify genes/pathways associated with drought tolerance/sensitivity in both genotypes. It was observed that genes related to ethylene response, MYB-related protein, xyloglucan endotransglycosylase, alkane hydroxylase MAH-like, BON-1 associated, peroxidase 3, cysteine-rich and transmembrane domain, vignain and mitochondrial uncoupling were specifically up-regulated in the tolerant genotype whereas, same genes were down-regulated in sensitive genotype. The crosstalk between the different hormones and transcriptional factors involved in drought tolerance and sensitivity in both genotypes make them great candidates for future research.

mQTL-seq and classical mapping implicates the role of an AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family gene in Ascochyta blight resistance of chickpea.

Ascochyta blight (AB) caused by the fungal pathogen Ascochyta rabiei is a serious foliar disease of chickpea (Cicer arietinum L.). Despite many genetic studies on chickpea-Ascochyta interaction, genome-wide scan of chickpea for the identification of AB-associated quantitative trait loci (QTLs) and their gene(s) has not been accomplished. To elucidate narrow QTLs for AB resistance, here, we report the use of multiple QTL-sequencing approach on 2 sets of extreme AB phenotype bulks derived from Cicer intraspecific and interspecific crosses. Two major QTLs, qABR4.1 and qABR4.2, and a minor QTL, qABR4.3, were identified on assembled chickpea pseudomolecule 4. We narrowed qABR4.1 to a "robust region" at 4.568-4.618 Mb through mapping on a larger intraspecific cross-derived population and comparative analysis. Among 4 genes, the CaAHL18 gene showed higher expression under Ascochyta stress in AB resistant parent suggesting that it is the candidate gene under "robust qABR4.1." Dual-luciferase assay with CaAHL18 polymorphic cis-regulatory sequences showed that allelic variation is associated with higher expression. Thus, our findings on chickpea-Ascochyta interaction have narrowed down AB resistance associated QTLs on chickpea physical map. The narrowed QTLs and gene-associated markers will help in biotechnological and breeding programs for chickpea improvement.

Transcriptional regulation of chickpea ferritin CaFer1 influences its role in iron homeostasis and stress response.

Ferritin, ubiquitous among all living organisms except yeast, exhibits iron-regulated expression. In plants, this regulation is applied through transcriptional control. Previous studies established the presence of two types of cis-acting elements in the promoter region: the iron regulatory element (FRE) in soybean and the iron-dependent regulatory sequence (IDRS) in maize and Arabidopsis. Adverse environmental conditions (e.g. water-deficit and oxidative stress) are known to modulate the expression of phytoferritin genes. In this study, we cloned and investigated the promoter sequence of a chickpea ferritin, designated CaFer1. Phylogenetic analysis of the CaFer1 promoter revealed its evolutionary relationship with other phytoferritins. The CaFer1 promoter exhibited several putative regulatory elements including two known transcription factor (TF) binding sites, Athb-1 and Myb.Ph. Electrophoretic mobility shift assay confirmed the sequence-specific binding of Athb-1 and Myb.Ph on the CaFer1 promoter. The TF-binding dynamics of CaFer1 showed high induction under conditions of iron-deficiency and water-deficit. We also demonstrated the possible interaction of CaFer1 with IRT1, a key component of the iron uptake system in plants, indicating its involvement in maintaining cellular iron levels. These results provide new insights into the underlying mechanisms of function of these interacting factors in CaFer1-mediated iron homeostasis and the stress response in plants.

Chickpea WRKY70 Regulates the Expression of a Homeodomain-Leucine Zipper (HD-Zip) I Transcription Factor CaHDZ12, which Confers Abiotic Stress Tolerance in Transgenic Tobacco and Chickpea.

Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stress-induced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response.

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.

Simulated herbivory in chickpea causes rapid changes in defense pathways and hormonal transcription networks of JA/ethylene/GA/auxin within minutes of wounding.

Chickpea (C. arietinum L.) is an important pulse crop in Asian and African countries that suffers significant yield losses due to attacks by insects like H. armigera. To obtain insights into early responses of chickpea to insect attack, a transcriptomic analysis of chickpea leaves just 20 minutes after simulated herbivory was performed, using oral secretions of H. armigera coupled with mechanical wounding. Expression profiles revealed differential regulation of 8.4% of the total leaf transcriptome with 1334 genes up-regulated and 501 down-regulated upon wounding at log2-fold change (|FC| ≤ -1 and ≥1) and FDR value ≤ 0.05. In silico analysis showed the activation of defenses through up-regulation of genes of the phenylpropanoid pathway, pathogenesis, oxidases and CYTP450 besides differential regulation of kinases, phosphatases and transcription factors of the WRKY, MYB, ERFs, bZIP families. A substantial change in the regulation of hormonal networks was observed with up-regulation of JA and ethylene pathways and suppression of growth associated hormone pathways like GA and auxin within 20 minutes of wounding. Secondary qPCR comparison of selected genes showed that oral secretions often increased differential expression relative to mechanical damage alone. The studies provide new insights into early wound responses in chickpea.