MicroRNA397 regulates tolerance to drought and fungal infection by regulating lignin deposition in chickpea root.

Plants deposit lignin in the secondary cell wall as a common response to drought and pathogen attacks. Cell wall localised multicopper oxidase family enzymes LACCASES (LACs) catalyse the formation of monolignol radicals and facilitate lignin formation. We show an upregulation of the expression of several LAC genes and a downregulation of microRNA397 (CamiR397) in response to natural drought in chickpea roots. CamiR397 was found to target LAC4 and LAC17L out of twenty annotated LACs in chickpea. CamiR397 and its target genes are expressed in the root. Overexpression of CamiR397 reduced expression of LAC4 and LAC17L and lignin deposition in chickpea root xylem causing reduction in xylem wall thickness. Downregulation of CamiR397 activity by expressing a short tandem target mimic (STTM397) construct increased root lignin deposition in chickpea. CamiR397-overexpressing and STTM397 chickpea lines showed sensitivity and tolerance, respectively, towards natural drought. Infection with a fungal pathogen Macrophomina phaseolina, responsible for dry root rot (DRR) disease in chickpea, induced local lignin deposition and LAC gene expression. CamiR397-overexpressing and STTM397 chickpea lines showed more sensitivity and tolerance, respectively, to DRR. Our results demonstrated the regulatory role of CamiR397 in root lignification during drought and DRR in an agriculturally important crop chickpea.

Biochemical analysis of anthocyanin and proanthocyanidin and their regulation in determining chickpea flower and seed coat colour.

Flower and seed coat colour are important agronomic traits in chickpea (Cicer arietinum L.). Cultivated chickpeas are of two types namely, desi (dark seeded, purple flowered) and kabuli (light seeded, white flowered). There has been limited information about the molecular mechanism underlying colour variation of flower and seed coats in desi and kabuli chickpea. We profiled the anthocyanin and proanthocyanidin (PA) contents in chickpea flowers and seed coats. Tissue-specific silencing of two genes encoding a basic helix-loop-helix (CabHLH) protein and a tonoplast-localized multidrug and toxic compound extrusion (CaMATE1) transporter in a desi genotype resulted in the reduction in expression of anthocyanin and PA biosynthetic genes and anthocyanin and PA contents in the flower and seed coat, and produced flowers and seeds with kabuli characteristics. Transcriptional regulation of a subset of anthocyanin and PA biosynthetic genes by a natural CabHLH variant and transport assay of a natural CaMATE1 variant explained the association of these alleles with the kabuli phenotype. We carried out a detailed molecular characterization of these genes, and provided evidence that kabuli chickpea flower and seed colour phenotypes can be derived by manipulation of single genes in a desi chickpea background.

In-depth analysis of the xanthine oxidase inhibitors of Cicer arietinum L.-based receptor-ligand affinity coupled with complex chromatography.

INTRODUCTION: Cicer arietinum L. is the choice of health food for people with diabetes, hypertension, and hyperlipidemia. As an essential source of high-nutrition legumes, it is also an important source of dietary isoflavones. OBJECTIVES: In order to improve the preparation efficiency of natural plants, a rapid biological activity screening and preparation of xanthine oxidase inhibitors from C. arietinum L. was established. METHODS: Xanthine oxidase (XOD) inhibitors were rapidly screened using ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) based on receptor-ligand affinity. The change in XOD activity was evaluated by enzymatic reaction kinetics measurement. The potential bioactive compounds were verified through molecular docking. In addition, the biological activity of ligands screened was separated and purified by complex chromatography. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Three active ingredients, namely daidzin, daidzein, calycosin with XOD binding affinities were identified and isolated from the raw plant materials via semi-preparative high-performance liquid chromatography (HPLC), 0-60 min, 5-50% B and countercurrent chromatography (CCC) (ethyl acetate/acetic acid/water [5:0.8:10, v/v/v]). CONCLUSION: This study will help to elucidate the mechanisms of action of natural plants of interest at the molecular level and could also provide more opportunities for the discovery and development of new nutritional value from other natural resources.

Relationships of frequencies of extreme low temperatures with grain yield of some Australian commercial chickpea cultivars.

In this study, we examined the relationships between extremes of low temperatures and chickpea yield in 12 field experiments conducted at six sites in the subtropical environment of southeast Queensland (SEQ) from 2014 to 2019. Three commercial chickpea cultivars, PBA-Boundary, PBA-HatTrick and PBA-Seamer, were grown in all the experiments. Cultivars PBA-Pistol, PBA-Monarch and Kyabra were also included in three of these experiments conducted in 2015. In these experiments, the crop experienced a total of 8 to 41 frosts (minimum temperature < = 0 °C), 2 to 41 pre-flowering frosts, 2 to 19 frosts during the critical period, 0 to 13 frosts and 2 to 71 low-temperature days (< = 15 °C) after flowering. The mean yield, which varied from 1 to 3 t/ha, was negatively related to post-flowering frosts (r = - 0.74, p < 0.01) and low-temperature days (r = - 0.76, p < 0.01), and positively related to pre-flowering frosts (r = 0.67, p < 0.05). Each post-flowering frost was associated with a 5% decrease and a low-temperature day with a 1% decrease in yield. The cultivar × site interaction was significant only in the three experiments with six commercial cultivars. This interaction was most likely due to an increase in the sensitivity range with additional cultivars, as indicated by frost damage scores and their relationships with yield. The results imply that extreme low-temperature events after flowering could negatively impact chickpea yield in SEQ and similar subtropical environments. Overcoming these effects through management and breeding should increase and stabilise chickpea yield.

Chickpeas from a Chilean Region Affected by a Climate-Related Catastrophe: Effects of Water Stress on Grain Yield and Flavonoid Composition.

The Valparaiso region in Chile was decreed a zone affected by catastrophe in 2019 as a consequence of one of the driest seasons of the last 50 years. In this study, three varieties ('Alfa-INIA', 'California-INIA', and one landrace, 'Local Navidad') of kabuli-type chickpea seeds produced in 2018 (control) and 2019 (climate-related catastrophe, hereafter named water stress) were evaluated for their grain yield. Furthermore, the flavonoid profile of both free and esterified phenolic extracts was determined using liquid chromatography-mass spectrometry, and the concentration of the main flavonoid, biochanin A, was determined using liquid chromatography with diode array detection. The grain yield was decreased by up to 25 times in 2019. The concentration of biochanin A was up to 3.2 times higher in samples from the second season (water stress). This study demonstrates that water stress induces biosynthesis of biochanin A. However, positive changes in the biochanin A concentration are overshadowed by negative changes in the grain yield. Therefore, water stress, which may be worsened by climate change in the upcoming years, may jeopardize both the production of chickpeas and the supply of biochanin A, a bioactive compound that can be used to produce dietary supplements and/or nutraceuticals.

Nutritional and textural properties and antioxidant activity of breads prepared from immature, mature, germinated, fermented and black chickpea flours.

BACKGROUND: Chickpea is a rich source of proteins with well-balanced amino acids, dietary fibers, vitamins, minerals and phytochemicals. In the present study, immature, mature, germinated, fermented and black chickpea flours at a 20% ratio were used in breadmaking to reduce the glycemic index and enhance nutritive value. The effects of chickpea flours on the physical, chemical and textural characteristics, as well as the antioxidant properties and in vitro glycemic index of bread were compared. RESULTS: Compared to the control (100% wheat bread), the inclusion of chickpea flours at a 20% ratio generally showed greater ash, fat and protein contents in bread. The use of immature, germinated and fermented chickpea flours in bread elicited a lower phytic acid concentration than that of bread containing mature and black chickpea flours. On the other hand, the inclusion of immature and germinated chickpea flours presented the highest total phenolic content in bread. Moreover, in vitro glycemic index values of loaves made with chickpea flours were markedly lower (at least 11%) compared to the control. The specific volume values of bread samples formulated with chickpea flours (except for fermented chickpea flour) were similar (P > 0.05) to each other. Bread samples containing immature and germinated chickpea flours exhibited lower hardness and chewiness than those of other samples containing chickpea flours. CONCLUSION: The findings showed that immature, germinated and black chickpea flours are a good alternative to mature chickpea flour with respect to producing healthy bread. © 2022 Society of Chemical Industry.

Enzyme pre-milling treatments improved milling performance of chickpeas by targeting mechanisms of seed coat and cotyledon adhesion with various effects on dhal quality.

BACKGROUND: Dehulling and splitting are important elements of the milling process to produce dhal from pulses. However, grain that is difficult-to-mill because of tightly adhered seed coats or cotyledons that resist separation makes it difficult to achieve high quality dhal. Milling yields are reduced, energy inputs into the milling process are increased, and the resulting dhal can be of poorer quality, chipped or abraded. RESULTS: Eight enzyme pre-treatments were chosen based on the hypothesised mechanisms of seed coat and cotyledon adhesion established previously. Using a difficult-to-mill chickpea (Cicer arietinum L.) genotype, we examined the effects of these pre-treatments, over time, on laboratory-scale milling performance and dhal quality. We pioneered a texture analyser method to measure the flex of the cotyledons and the force required to cleave the cotyledons. The enzyme-induced changes ranged from negative (tough seed coat, weight loss, deleterious colour and texture, increased visual damage to cotyledons and increased kibble loss, concave cotyledons, increased flex, and changes in taste) to positive (brittle seed coat, increased seed volume, improved dehulling efficiency and splitting yield, reduced cotyledon cleavage force, and acceptable dhal quality and taste). CONCLUSION: All pre-treatments improved milling performance compared to milling the raw seed, although there was considerable variation between them. Two pre-treatments showed no improvement in milling yields compared to the water control, and several pre-treatments resulted in unacceptable qualities. Three pre-treatments, endo-polygalacturonanase, α-galactosidase and cellulase, show potential for commercial milling applications and could assist pulse millers globally to achieve high quality dhal at the same time as minimising milling effort. © 2021 Society of Chemical Industry.

Root-specific expression of chickpea cytokinin oxidase/dehydrogenase 6 leads to enhanced root growth, drought tolerance and yield without compromising nodulation.

Cytokinin group of phytohormones regulate root elongation and branching during post-embryonic development. Cytokinin-degrading enzymes cytokinin oxidases/dehydrogenases (CKXs) have been deployed to investigate biological activities of cytokinin and to engineer root growth. We expressed chickpea cytokinin oxidase 6 (CaCKX6) under the control of a chickpea root-specific promoter of CaWRKY31 in Arabidopsis thaliana and chickpea having determinate and indeterminate growth patterns, respectively, to study the effect of cytokinin depletion on root growth and drought tolerance. Root-specific expression of CaCKX6 led to a significant increase in lateral root number and root biomass in Arabidopsis and chickpea without any penalty to vegetative and reproductive growth of shoot. Transgenic chickpea lines showed increased CKX activity in root. Soil-grown advanced chickpea transgenic lines exhibited higher root-to-shoot biomass ratio and enhanced long-term drought tolerance. These chickpea lines were not compromised in root nodulation and nitrogen fixation. The seed yield in some lines was up to 25% higher with no penalty in protein content. Transgenic chickpea seeds possessed higher levels of zinc, iron, potassium and copper. Our results demonstrated the potential of cytokinin level manipulation in increasing lateral root number and root biomass for agronomic trait improvement in an edible legume crop with indeterminate growth habit.

Cicer arietinum L. Sprouts' Influence on Mineralization of Saos-2 and Migration of MCF-7 Cells.

In the present study, we investigated the biological activity of four extracts obtained from Cicer arietinum L. sprouts. The fermentation of the sprouts with Lactobacillus casei and their incubation with ß-glucosidase elevated the concentrations of isoflavonoids, especially coumestrol, formononetin and biochanin A. To study the biological activity of C. arietinum, the human osteosarcoma Saos-2 and human breast cancer MCF-7 cell lines were used. The extracts obtained from fermented sprouts exhibited the strongest ability to decrease intracellular oxidative stress in both types of cells. They augmented mineralization and alkaline phosphatase activity in Saos-2 cells, as well as diminished the secretion of interleukin-6 and tumor necrosis factor α. Simultaneously, the extracts, at the same doses, inhibited the migration of MCF-7 cells. On the other hand, elevated concentrations of C. arietinum induced apoptosis in estrogen-dependent MCF-7 cells, while lower doses stimulated cell proliferation. These results are important for carefully considering the use of fermented C. arietinum sprouts as a dietary supplement component for the prevention of osteoporosis.

Immunomodulatory Activity in vitro and in vivo of Polysaccharides from Kabuli Chickpea (Cicer arietinum L.) Hull.

RESEARCH BACKGROUND: Polysaccharides isolated from plants, fungi and bacteria are associated with immunomodulatory effects. Chickpea hull, which is regarded as food industrial waste, contains considerable amount of antioxidants and bioactive compounds. EXPERIMENTAL APPROACH: In the present study, we investigated the immunomodulatory activity of polysaccharides from kabuli chickpea (Cicer arietinum L.) hull (CHPS). In vitro study was conducted with RAW264.7 cell line while in vivo study was carried out using specific pathogen-free BALB/c mouse animal model. RESULTS AND DISCUSSION: In in vitro test with RAW264.7 murine macrophage cells, the three purified fractions of chickpea hull polysaccharides showed potent immunomodulatory activity. Sample CHPS-3 showed stronger effect on cell viability, promoted the phagocytosis index to a greater extent and had the best effect on acid phosphatase activity. Moreover, it was found that CHPS-3 significantly (p<0.05) enhanced the secretion of nitrogen monoxide and cytokine (interleukins IL-6, IL-1ß and tumor necrosis factor-alpha (TNF-α)) levels. In in vivo study, CHPS-3 improved thymus and spleen indices in cyclophosphamide-induced immunodeficient mice. Increased activities of lysozyme, catalase, superoxide dismutase and glutathione peroxidase, serum haemolysin content and total antioxidant capacity were observed, while the amount of malondialdehyde in the liver decreased. NOVELTY AND SCIENTIFIC CONTRIBUTION: The results suggest that chickpea hull polysaccharides enhanced the immune activity and could be developed as the ingredient of functional foods.

Salinity tolerance in chickpea is associated with the ability to 'exclude' Na from leaf mesophyll cells.

Salinity tolerance is associated with Na 'exclusion' from, or 'tissue tolerance' in, leaves. We investigated whether two contrasting chickpea genotypes, salt-tolerant Genesis836 and salt-sensitive Rupali, differ in leaf tissue tolerance to NaCl. We used X-ray microanalysis to evaluate cellular Na, Cl, and K concentrations in various cell types within leaflets and also in secretory trichomes of the two chickpea genotypes in relation to photosynthesis in control and saline conditions. TEM was used to assess the effects of salinity on the ultrastructure of chloroplasts. Genesis836 maintained net photosynthetic rates (A) for the 21 d of salinity treatment (60 mM NaCl), whereas A in Rupali substantially decreased after 11 d. Leaflet tissue [Na] was low in Genesis836 but had increased markedly in Rupali. In Genesis836, Na was accumulated in epidermal cells but was low in mesophyll cells, whereas in Rupali cellular [Na] was high in both cell types. The excessive accumulation of Na in mesophyll cells of Rupali corresponded to structural damage to the chloroplasts. Maintenance of photosynthesis and thus salinity tolerance in Genesis836 was associated with an ability to 'exclude' Na from leaflets and in particular from the photosynthetically active mesophyll cells, and to compartmentalize Na in epidermal cells.

SA and AM symbiosis modulate antioxidant defense mechanisms and asada pathway in chickpea genotypes under salt stress.

Salt stress disturbs redox homeostasis by perturbing equilibrium between generation and removal of reactive oxygen species (ROS), which alters the normal metabolism of plants through membrane damage, lipid peroxidation and denaturation of proteins. Salicylic acid (SA) seed priming and arbuscular mycorrhizal (AM) fungi impart salt tolerance in legumes by maintaining redox balance. The present investigation focused on the relative and combined applications of SA and Rhizoglomus intraradices in scavenging ROS in Cicer arietinum L. (chickpea) genotypes (salt tolerant-PBG 5, relatively sensitive-BG 256) subjected to salt stress. Despite the enhanced antioxidant mechanisms under salt stress, ROS (superoxide, O2- and hydrogen peroxide, H2O2) accumulation increased significantly and induced lipid peroxidation and lipoxygenase (LOX) activities, which disrupted membrane stability, more in BG 256 than PBG 5. Salt stress also caused redox imbalance by lowering ascorbate/dehydroascorbate (ASA/DHA) and reduced glutathione/oxidized glutathione (GSH/GSSG) ratios, indicating that redox-homeostasis was crucial for salt-tolerance. Exogenous SA was more promising in reducing ROS-generation and lipid-peroxidation, which provided higher membrane stability as compared to AM inoculation. Although, the enzymatic antioxidants were more active in SA treated plants, yet, AM inoculation outperformed in increasing reformative enzyme activities of Foyer-Halliwell-Asada cycle, which resulted in higher plant biomass in a genotype-dependent manner. SA increased AM root colonization and provided functional complementarity to R. intraradices and thereby strengthening antioxidant defense mechanisms through their cumulative contribution. The study suggested the use of +SA+AM as an eco-friendly tool in imparting salt tolerance in chickpea genotypes subjected to long-term salinity.

Genotypic variation in the response of chickpea to arbuscular mycorrhizal fungi and non-mycorrhizal fungal endophytes.

Plant roots host symbiotic arbuscular mycorrhizal (AM) fungi and other fungal endophytes that can impact plant growth and health. The impact of microbial interactions in roots may depend on the genetic properties of the host plant and its interactions with root-associated fungi. We conducted a controlled condition experiment to investigate the effect of several chickpea (Cicer arietinum L.) genotypes on the efficiency of the symbiosis with AM fungi and non-AM fungal endophytes. Whereas the AM symbiosis increased the biomass of most of the chickpea cultivars, inoculation with non-AM fungal endophytes had a neutral effect. The chickpea cultivars responded differently to co-inoculation with AM fungi and non-AM fungal endophytes. Co-inoculation had additive effects on the biomass of some cultivars (CDC Corrine, CDC Anna, and CDC Cory), but non-AM fungal endophytes reduced the positive effect of AM fungi on Amit and CDC Vanguard. This study demonstrated that the response of plant genotypes to an AM symbiosis can be modified by the simultaneous colonization of the roots by non-AM fungal endophytes. Intraspecific variations in the response of chickpea to AM fungi and non-AM fungal endophytes indicate that the selection of suitable genotypes may improve the ability of crop plants to take advantage of soil ecosystem services.

Phenolic profiles and their contribution to the antioxidant activity of selected chickpea genotypes from Mexico and ICRISAT collections.

Chickpea (Cicer arietinum L.) genotypes, nine kabuli from Mexico and 9 desi from other countries, were investigated for their phenolic profiles and antioxidant activity (AA). Phenolics in methanol extracts (ME) were analyzed by ultra-performance liquid chromatography coupled to diode array detection and mass spectrometry (UPLC-DAD-MS), whereas the AA was measured as Trolox equivalents (TE) by ABTS, DPPH and FRAP methods. Twenty phenolic compounds were identified in the ME and their levels showed a great variability among the chickpea genotypes. Phenolic acids and flavonoids were the most abundant compounds in kabuli and desi genotypes, respectively. The AA values (µmol TE/ 100 g dw) by ABTS (278-2417), DPPH (52-1650), and FRAP (41-1181) were mainly associated with the content of sinapic acid hexoside, gallic acid, myricetin, quercetin, catechin, and isorhamnetin, suggesting they are the main compounds responsible for the AA. The sum of the AA obtained for standards of these compounds evaluated at the concentration found in the extracts accounted for 34.3, 69.8, and 47.0% of the AA in the extract by ABTS, DPPH, and FRAP, respectively. In the AA by DPPH, most of the mixtures of these compounds resulted in synergistic interactions. Three desi genotypes with black seeds (ICC 4418, ICC 6306, and ICC 3761) showed the highest AA and flavonoids content, whereas the most promising kabuli genotypes were Surutato 77, Bco. Sin. 92, and Blanoro that showed the highest values of phenolic acids. These genotypes represent good sources of antioxidants for the improvement of nutraceutical properties in chickpea.

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.

Vegetative and reproductive growth of salt-stressed chickpea are carbon-limited: sucrose infusion at the reproductive stage improves salt tolerance.

Reproductive processes of chickpea (Cicer arietinum L.) are particularly sensitive to salinity. We tested whether limited photoassimilate availability contributes to reproductive failure in salt-stressed chickpea. Rupali, a salt-sensitive genotype, was grown in aerated nutrient solution, either with non-saline (control) or 30mM NaCl treatment. At flowering, stems were either infused with sucrose solution (0.44M), water only or maintained without any infusion, for 75 d. The sucrose and water infusion treatments of non-saline plants had no effect on growth or yield, but photosynthesis declined in response to sucrose infusion. Salt stress reduced photosynthesis, decreased tissue sugars by 22-47%, and vegetative and reproductive growth were severely impaired. Sucrose infusion of salt-treated plants increased total sugars in stems, leaves and developing pods, to levels similar to those of non-saline plants. In salt-stressed plants, sucrose infusion increased dry mass (2.6-fold), pod numbers (3.8-fold), seed numbers (6.5-fold) and seed yield (10.4-fold), yet vegetative growth and reproductive failure were not rescued completely by sucrose infusion. Sucrose infusion partly rescued reproductive failure in chickpea by increasing vegetative growth enabling more flower production and by providing sucrose for pod and seed growth. We conclude that insufficient assimilate availability limits yield in salt-stressed chickpea.

Evaluation of Extraction and Degradation Methods to Obtain Chickpeasaponin B1 from Chickpea (Cicer arietinum L.).

The objective of this research is to implement extraction and degradation methods for the obtainment of 3-O-[α-l-rhamnopyranosyl-(1→2)-ß-d-galactopyranosyl] soyasapogenol B (chickpeasaponin B1) from chickpea. The effects of microwave-assisted extraction (MAE) processing parameters-such as ethanol concentration, solvent/solid ratio, extraction temperature, microwave irradiation power, and irradiation time-were evaluated. Using 1g of material with 8 mL of 70% aqueous ethanol and an extraction time of 10 min at 70 °C under irradiation power 400W provided optimal extraction conditions. Compared with the conventional extraction techniques, including heat reflux extraction (HRE), Soxhlet extraction (SE), and ultrasonic extraction (UE), MAE produced higher extraction efficiency under a lower extraction time. DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) saponin can be degraded to structurally stable saponin B by the loss of its DDMP group. The influence of pH and the concentration of potassium hydroxide on transformation efficiency of the target compound was investigated. A solution of 0.25 M potassium hydroxide in 75% aqueous ethanol was suitable for converting the corresponding DDMP saponins of chickpeasaponin B1. The implementation by the combining MAE technique and alkaline hydrolysis method for preparing chickpeasaponin B1 provides a convenient technology for future applications.

Salt sensitivity in chickpea is determined by sodium toxicity.

MAIN CONCLUSION: Salt sensitivity in chickpea is determined by Na(+) toxicity, whereas relatively high leaf tissue concentrations of Cl(-) were tolerated, and the osmotic component of 60-mM NaCl was not detrimental. Chickpea (Cicer arietinum L.) is sensitive to salinity. This study dissected the responses of chickpea to osmotic and ionic components (Na(+) and/or Cl(-)) of salt stress. Two genotypes with contrasting salt tolerances were exposed to osmotic treatments (-0.16 and -0.29 MPa), Na(+)-salts, Cl(-)-salts, or NaCl at 0, 30, or 60 mM for 42 days and growth, tissue ion concentrations and leaf gas-exchange were assessed. The osmotic treatments and Cl(-)-salts did not affect growth, whereas Na(+)-salts and NaCl treatments equally impaired growth in either genotype. Shoot Na(+) and Cl(-) concentrations had markedly increased, whereas shoot K(+) had declined in the NaCl treatments, but both genotypes had similar shoot concentrations of each of these individual ions after 14 and 28 days of treatments. Genesis836 achieved higher net photosynthetic rate (64-84 % of control) compared with Rupali (35-56 % of control) at equivalent leaf Na(+) concentrations. We conclude that (1) salt sensitivity in chickpea is determined by Na(+) toxicity, and (2) the two contrasting genotypes appear to differ in 'tissue tolerance' of high Na(+). This study provides a basis for focus on Na(+) tolerance traits for future varietal improvement programs for salinity tolerance in chickpea.

Evaluation of antagonistic and plant growth promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against Sclerotium rolfsii in chickpea.

AIMS: To evaluate the potential of chitinolytic endophytic Actinomycetes isolated from medicinal plants in order to diminish the collar rot infestation induced by Sclerotium rolfsii in chickpea. METHODS AND RESULTS: Sixty-eight chitinolytic endophytic Actinomycetes were recovered from various medicinal plants and evaluated for their chitinase activity. Among these isolates, 12 were screened for their plant growth promoting abilities and antagonistic potential against Sc. rolfsii. Further, these isolates were validated in vivo for their ability to protect chickpea against Sc. rolfsii infestation under greenhouse conditions. The isolates significantly (P < 0·05) increased the biomass (1·2-2·0 fold) and reduced plant mortality (42-75%) of chickpea. On the basis of 16S rDNA profiling, the selected antagonistic strains were identified as Streptomyces diastaticus, Streptomyces fradiae, Streptomyces olivochromogenes, Streptomyces collinus, Streptomyces ossamyceticus and Streptomyces griseus. CONCLUSION: This study is the first report of the isolation of endophytic Actinomycetes from various medicinal plants having antagonistic and plant growth promoting abilities. The isolated species showed potential for controlling collar rot disease on chickpea and could be useful in integrated control against diverse soil borne plant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our investigation suggests that endophytic Actinomycetes associated with medicinal plants can be used as bioinoculants for developing safe, efficacious and environment-friendly biocontrol strategies in the near future.

Isoflavones extracted from chickpea Cicer arietinum L. sprouts induce mitochondria-dependent apoptosis in human breast cancer cells.

Isoflavones are important chemical components of the seeds and sprouts of chickpeas. We systematically investigated the effects of isoflavones extracted from chickpea sprouts (ICS) on the human breast cancer cell lines SKBr3 and Michigan Cancer Foundation-7 (MCF-7). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that ICS (10-60 µg/mL) significantly inhibited the proliferation of both cell lines in a time-dependent and dose-dependent fashion. Wright-Giemsa staining as well as annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V/PI) staining showed that ICS significantly increased cytoclasis and apoptotic body formation. Quantitative Annexin V/PI assays further showed that the number of apoptotic cells increased in a dose-dependent manner following ICS treatment. Semiquantitative reverse transcription PCR showed that ICS increased the expression of the apoptosis-promoting gene Bcl-2-associated X protein and decreased the expression of the antiapoptotic gene Bcl-2. Western blot analysis showed that treatment of SKBr3 and MCF-7 cells with ICS increased the expression of caspase 7, caspase 9, P53, and P21 in a dose-dependent manner. Flow cytometry assays using the fluorescent probe 3,3'-dihexyloxacarbocyanine iodide showed a dose-dependent decrease in mitochondrial membrane potential following ICS treatment. Treatment using ICS also induced a dose-dependent increase in reactive oxygen species production. This is the first study to demonstrate that ICS may be a chemopreventive or therapeutic agent against breast cancer.