Exploration of biochemical and molecular diversity in chickpea seeds to categorize cold stress-tolerant and susceptible genotypes

Chickpea (Cicer arietinum L.) genotypes are sensitive to low temperature (<10°C) during its reproductive stage suffer from abortion of flowers, infertile pods and small shriveled seeds that resulted in a significant decrease in crop yield. In the present investigation seeds of a number of cold stress-tolerant and susceptible genotypes were evaluated for biochemical and molecular diversity with the purpose to categorize them. The activities of various antioxidative enzymes (superoxide dismutase, glutathione reductase, ascorbate peroxidase and catalase), content of H₂O₂ and malondialdehyde, enzymes involved in phosphate metabolism (acid and alkaline phosphatases), and content of phytic acid and proline were determined in seeds of 20 cold stress tolerant and seven cold stress susceptible genotypes. Higher activities of superoxide dismutase, ascorbate peroxidase, catalase and acid phosphatase and low content of malondialdehyde and phytic acid were observed in cold stress-tolerant genotypes as compared to cold stress susceptible genotypes. Seventeen chickpea genotypes comprising both cold stress-tolerant and susceptible ones were evaluated through 20 randomly amplified polymorphic DNA (RAPD) primers. The results of cluster analysis revealed two major groups. In the first group five tolerant (group 1a) and six susceptible genotypes (group 1b) clustered together whereas in second group all the tolerant genotypes clustered together (group 2). Out of 20 RAPD primers, 4 primers (Opa-13, Opa-14, Opa-15 and Opa-16) have been identified as markers for cold stress tolerance. In general high SOD activity, and H₂O₂ content and low MDA and phytic acid content are related with cold stress tolerance. The status of these markers was more pronounced in genotypes clustered in group 2 after RAPD analysis than in group 1a of cold stress-tolerant genotypes as compared to susceptible genotypes. The observed biochemical and molecular diversity could be useful for identifying and developing cold stress-tolerant genotypes of chickpea.     

Low temperature induced floral abortion in chickpea: relationship to abscisic acid and cryoprotectants in reproductive organs

Cold stress (temperature <8 °C) during reproductive growth of chickpea is detrimental to flowering and pod set. Cold-sensitive genotypes either delay their flowering or abort their flowers produced during intermittent warm spells upon exposure to cold stress. To find out the mechanism of floral abortion in chickpea, a cold-sensitive genotype (PBG1) was grown under controlled (28 °C/12 °C) and cold-stressed conditions (12–15 °C/4–6 °C). Pod set was 95% in controlled conditions versus 20% under cold stress. The relative leaf water content (RLWC) of stressed plants was decreased by 26% over controls. The retained and aborted flowers harvested from the plants growing under cold stress along with flowers from control plants were examined for functioning of gametes, endogenous levels of ABA, sucrose, glucose, fructose, trehalose and proline in whole flowers, and their male (pollen, anthers) as well as female (stigma–style, ovary) parts. Flowers from stressed plants showed a decrease in functioning of gametes in comparison to those from control plants. The pollen germination (in vivo and in vitro) and viability did not differ significantly between the retained and aborted flowers of the stressed plants but stigma receptivity and ovary viability were significantly lower in aborted flowers. The ABA content in aborted flowers was 23% higher than in retained flowers. The content of sucrose, glucose and fructose in aborted flowers was 40, 25 and 23% lower, respectively, and the content of trehalose and proline was 50 and 73% lower, respectively, than in flowers on the plant. Male parts of aborted flowers contained 7–12% greater concentration of ABA and solutes than those retained on the mother plant but the female parts of the former, especially ovary contained 25% more ABA and 23–26% lower solute levels. Since the male parts of aborted flowers were relatively little affected by cold stress, the events in female parts appeared to be primarily responsible for floral abortion in chickpea. Reciprocal pollination between plants growing in controlled and stressed conditions indicated greater impairment of female parts than male parts under cold stress. Proline (5 mM) application to stressed plants improved the floral retention and pod set by 33%.     

Analysis of emergence stage facilitates the evaluation of chickpea (Cicer arietinum L.) genotypes for salinity tolerance imparted by mycorrhizal colonization

Salinity is an ever-increasing constraint limiting crop production in arid and semi-arid regions. Arbuscular mycorrhiza (AM) helps host plant to cope with detrimental effects of salinity. Experiments were aimed to examine the hypothesis that emergence is a better stage to determine salt tolerance of chickpea genotypes than germination and genotypic variability in their tolerance ability at emergence and subsequent vegetative growth is the manifestation of differential benefits imparted by mycorrhiza. Investigations were carried out at germination and emergence stage of genotypes (PBG 5, GPF 2, PBG 1, BG 1053, L 550) at 0, 40, 60, 80 mM NaCl. Significant genotypic variations in salt tolerance were observed at emergence rather than germination because of greater inhibitory effects on seedling emergence. Percent mycorrhizal colonization (MC) and its resulting impact on respiration rate (RR) and salt tolerance index (STI) at emergence indicated that PBG 5, with lowest RR, highest STI and mycorrhiza benefit percentage was the most tolerant whereas, L 550 the most sensitive genotype. Genotypic variability recorded at 30 days was consistent with that at emergence stage. Superior salt tolerance of PBG 5 than L 550 could be attributed to higher correlation between MC and physio-biochemical traits (RWC, chlorophyll a/b, proline accumulation, antioxidant activities). The study supported the hypothesis that both emergence stage and mycorrhizal effectiveness are important determinants of salt tolerance in chickpea genotypes. Evaluation of genotypes for relative adaptation to salinity should include estimation of their differential salt tolerance at different growth stages and symbiotic effectiveness of AM .     

Differential proline metabolism in vegetative and reproductive tissues determine drought tolerance in chickpea

Proline is emerging as a critical component of drought tolerance and fine tuning of its metabolism under stress affects the plants sensitivity and response to stress. Thus the study was carried out to analyse the effect of water deficit on the proline content and principal enzymes involved in its synthesis (Δ¹-pyrolline-carboxylate synthetase) and catabolism (proline dehydrogenase) at different developmental stages and in different organs (roots, nodules, leaves, pod wall, and seeds) of two chickpea (Cicer arietinum L.) cultivars differing in drought tolerance (drought tolerant ICC4958 and drought sensitive ILC3279). It was observed that increased Δ¹-pyrolline-carboxylate synthetase activity under moderate stress in roots and nodules of ICC4958 caused an increase in proline content during initiation of reproductive development whereas increased proline dehydrogenase activity in nodules and leaves at this period helped to maintain reducing power and energy supply in tissues and proper seed development as seed biomass increased consistently up to maturity. On the other hand, roots and nodules of ILC3279 responded to stress by increasing proline content after the developmental phase of reproductive organs was over (near maturity) which negatively affected the response of pod wall to stress. Concurrent increase in activities of Δ¹-pyrolline-carboxylate synthetase and proline dehydrogenase in pod wall of ILC3279 aggravated the oxidative stress and affected seed development as seed biomass initially increased rapidly under stress but was unaffected near maturity.     

Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

Indigenous salt-tolerant rhizobacterium <Emphasis Type="Italic">Pantoea dispersa</Emphasis> (PSB3) reduces sodium uptake and mitigates the effects of salt stress on growth and yield of chickpea

Salt stress has multiple damaging effects on plants including physiological damage, reduced growth, and productivity. Plant growth-promoting rhizobacteria (PGPR) are one of the valuable options to mitigate the negative effects of this stress. In the present study, native bacteria from chickpea’s rhizosphere were isolated, and checked for their salt tolerance and plant growth-promoting attributes (phosphate (P) solubilization, siderophores, indole-3-acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production). One isolate, subsequently identified as Pantoea dispersa, showed appreciable production of IAA (218.3 µg/ml) and siderophores (60.33% SU), P-solubilization (3.64 µg/ml) and ACC deaminase activity (207.45 nmol/mg/h) in the presence of 150 mM NaCl, under laboratory conditions. Salt stress in uninoculated chickpea (GPF2 cultivar) plants induced high accumulation of Na⁺ ions (3.86 mg g^?1 dw) in the leaves, along with significant reduction in K⁺ uptake, membrane integrity, chlorophyll concentration, and leaf water content, thus resulting in impaired growth of the plant and yield (pods and seeds) in a salt concentration-dependent manner. The damage due to salt stress was restored significantly in plants inoculated with P. dispersa. A significant improvement in biomass (32–34%), pods number (31–34.5%), seeds number (32–35.7%), pods weight (30–32.6%), and seeds weight (27–35%) per plant occurred in salt stress-affected plants, which was associated with significant reduction in Na⁺ uptake, reduced membrane damage, significantly improved leaf water content, chlorophyll content, and K⁺ uptake. This study suggests for the first time that native P. dispersa strain PSB3 can be used to alleviate the negative effects of salt stress on chickpea plants and holds the potential to be used as a biofertilizer.     

Freezing tolerance of chickpea: biochemical and molecular changes at vegetative stage

The aim of this study was to find a correlation between the freezing tolerance of three chickpea (Cicer arietinum L.) cultivars (?nci, I??k-05, and Sar?-98) and their wild relative C. echinospermum and physiological responses. Chickpea plants (15-d-old) were subjected to cold acclimation (CA) (10 °C for 7 d), freezing (-3 or -5 °C for 2 h), and subsequent rewarming (25 °C for 7 d). In two separate experiments with three replications, we determined growth, water status, photosystem 2 photochemical activity, photosynthetic pigments, H₂O₂, malondialdehyde, and proline content, relative leakage ratio, antioxidant enzyme activities, and gene expressions in cultivars different in freezing tolerance. Freezing temperatures adversely affected all the physiological parameters of all cultivars. Rewarming did not lead to complete recovery. The cultivar ?nci was more tolerant to the freezing temperatures than others.     

Effect of drought stress implemented at pre- or post-anthesis stage on some physiological parameters as screening criteria in chickpea cultivars

Drought is one of the most important factors limiting chickpea production in arid and semi-arid regions. There is little information regarding genotypic variation for drought tolerance in chickpea cultivars. Screening for drought tolerance is very important. It is essential to identify the physiological mechanisms of drought tolerance to complete conventional breeding program. Glasshouse experiment was carried out to study the genotypic variation among 11 chickpea (Cicer arietinum L.) cultivars. Plants were grown either under optimum conditions or drought stress was implemented at pre-or post-anthesis stages. The drought susceptibility index (DSI) was used as the measure of drought tolerance. Relationships between DSI and excised-leaf water loss (RWL), relative water content (RWC), membrane permeability, ascorbic acid, proline, and chlorophyll contents, lipid peroxidation, and hydrogen peroxide concentrations were determined in order to find out whether these physiological parameters could be used as the genotypic selection criteria for drought tolerance. The results of this study indicated that there was a wide variation in tolerance to drought stress among the chickpea cultivars, which could be exploited in breeding new chickpea cultivars with high drought tolerance. The results also demonstrated that drought-tolerant cultivars had a higher RWC, ascorbic acid and proline concentrations, but lower RWL and membrane permeability in comparison to drought-sensitive cultivars. The significant and a well defined relationships between DSI and RWC, RWL, ascorbic acid, proline, and membrane permeability were found. It was concluded that these parameters could be instrumental in predicting the drought tolerance of chickpea cultivars. This text was submitted by the authors in English.     

Exploration of Distinct Physiological and Biochemical Alterations in Chickpea (<i>Cicer arietinum</i> L.) under Varying Organic Materials,Drought and Proline Application

Chickpea yield is decreasing day by day due to drought stress, which could be an immense risk for future food security in developing countries. Management practices could be the most excellent approach to diminish loss due to this abiotic factor. The current research work was designed to explore the tolerance reaction of chickpea genotypes against management practices, through morphological and biochemical parameters and evaluate yield performance across drought prone location of Bangladesh. Four genotypes BD-6048, BD-6045, BD-6090, BD-6092 and eight management practices, e.g., severe water stress (SWS), i.e., without irrigation, 10 cm thick mulching with rice straw (MRS), 10 cm thick mulching with water hyacinth (MWH), organic amendment through compost (OAC) @ 3 t ha⁻¹, organic amendment through cow dung @ 5 t ha⁻¹ (OACD), organic amendment through poultry manure @ 2 t ha⁻¹ (OAPM), inorganic amendment through proline application (IAPA) as foliar spray and 16 h hydro-priming (HP). The study revealed that the genotypes BD-6048 showed excellent performance because of the highest chlorophyll, carotenoids, phosphorus, potassium, proline and protein content. The highest pod number plant⁻¹ also increased seed yield in BD-6048. Considering management practices, IAPA increased relative water content, carotenoids, leaf phosphorus and potassium compared to other management practices and severe water stress. Finally, BD-6084 was selected as best genotype because of a significant increase in chlorophyll a and b, carotenoids, and relative water content with IAPA. Identified top performing genotypes can be used for releasing variety and cultivated for sustainable production in drought prone area of Bangladesh.     

Genetics and characterization of an open flower mutant in chickpea

The chickpea (Cicer arietinum L.) is a self-pollinated grain legume with cleistogamous flowers. A spontaneous open-flower mutant, designated OFM-3, was identified in which reproductive organs were not enclosed by the keel petals and thus remained exposed. All 10 stamens in this mutant were free, whereas these are in diadelphous (9 fused + 1 free) condition in normal chickpea flowers. A large number of pods (73%) remained unfilled (empty) in OFM-3, though its pollen fertility was as high as the standard cultivars. The open-flower trait was found to be recessive and controlled by a single gene. OFM-3 was crossed with earlier reported open-flower mutants, ICC 16341 and ICC 16129, to establish trait relationships of genes controlling open flower traits in these mutants. It was found that each of these mutants has a unique gene for open flower trait. The genes controlling open flower trait in ICC 16341, ICC 16129, and OFM-3 were designated ofl-1, ofl-2, and ofl-3, respectively. Breeding lines with open flower trait and higher percentage of filled pods have been developed from the progenies of the crosses of OFM-3 with normal-flowered lines. The open flower trait offers opportunity for exploring hybrid technology in the chickpea.     

Exploration of the antioxidative defense system to characterize chickpea genotypes showing differential response towards water deficit conditions

The present investigation was carried out to characterize genotypic variability in chickpea for water deficit tolerance by exploring the antioxidative defense system and seedling growth. Twenty nine chickpea genotypes including cultivars and advanced lines were grown under control and water deficit conditions induced by adding 3 % mannitol. The genotypes showed differential response in seedling growth under water deficit conditions. The activities of catalase (CAT) and superoxide dismutase (SOD) were observed to be differentially expressed in the roots of various genotypes, under control and water deficit conditions. The contents of H₂O₂, malondialdehyde (MDA) and proline were also observed to be variable in the roots of all the genotypes, under control and water deficit conditions. Stress tolerance index for the various parameters, viz, CAT and SOD activity, H₂O₂, MDA and proline content, root length, shoot length and their biomass was determined and the level of stress resistance calculated. The genotypes which showed increased activities of CAT and SOD, decreased contents of H₂O₂ and MDA together with least affected seedling growth under water deficit conditions exhibited higher stress resistance capacity. Multivariate principal component analysis for all the parameters affected under water deficit conditions, grouped the genotypes into three clusters having different (high, moderate and low) levels of stress resistance. Complete linkage clustering grouped these genotypes into two major clusters-I and II. The genotypes present in sub–sub cluster ‘A1’ and sub cluster ‘B’ of major cluster-I have been observed to possess high stress resistance levels for respective parameters. It can thus be concluded that chickpea genotypes exhibiting increased stress resistance levels in relation to SOD and CAT activities, H₂O₂ and MDA contents and seedling growth would have higher stress tolerance under water deficit conditions.     

Influence of Several Sources and Amounts of Iron on DNA, Lipid and Protein Oxidative Damage During Anaemia Recovery

Nanoparticles (NPs) in agricultural systems can potentially be used as appropriate candidate for change in growth, development, productivity, and quality of plants. In the present study, we assessed the effect of TiO₂ NP concentrations (0, 2, 5, and 10 ppm) on changes of membrane damage indexes like electrolyte leakage index (ELI) and malondialdehyde (MDA) during cold stress (CS) 4 °C in sensitive (ILC 533) and tolerant (Sel 11439) chickpea (Cicer arietinum L.) genotypes. Aggregation of NPs within the vacuole and chloroplast indicated absorbed NPs in seedlings. Bioaccumulation of NPs showed that, under thermal treatments, the sensitive genotype had more permeability to NPs compared to the tolerant one, and TiO₂ content was higher during CS compared to optimum temperature. Physiological indexes were positively affected by NP treatments during thermal treatments. TiO₂ NP treatments (especially 5 ppm) caused a decrease in ELI during thermal treatments, whereas ELI content under CS treatment increased at 0 ppm TiO₂ in both genotypes. Under thermal treatments, although the genotype 11439 showed lower accumulation of MDA than ILC 533 genotype, a significant decrease was observed in MDA content at 5 ppm TiO₂. Results showed that TiO₂ treatments not only did not induce oxidative damage in sensitive and tolerant chickpea genotypes but also alleviated membrane damage indexes under CS treatment. It was suggested for the first time that TiO₂ NPs improved redox status of the genotypes under thermal treatments. New findings possibly would reveal the use of NPs generally or TiO₂ NPs especially for increase of cold tolerance in crops.     

What are the possible benefits of small size for energy‐constrained ectotherms in cold stress conditions?

In stressful environments, two main hypotheses have been proposed to explain the consequences of body size: (1) the absolute energy demand hypothesis (AED), which predicts that larger individuals are at a disadvantage under stressful conditions; (2) the relative efficiency hypothesis (RE), which predicts the reverse. We compared the effects of cold stress on different fitness traits of large and small individuals of the parasitoid wasp Aphidius ervi (Hymenoptera: Aphidiinae). For that, we exposed nymphs of this wasp to 5 treatment conditions as follows (control at 20°C; 7C1 and 7C2: constant cold temperature of 7°C for 1 and 2 weeks respectively; 4C1 and 4C2: constant cold temperature of 4°C for 1 and 2 weeks respectively). After cold stress, only the large females that emerged in the 7C2 and 4C2 treatments displayed a reduction in the fitness traits studied (longevity, egg load at emergence, life‐time fecundity). The decrease in lipid content in large adults may have been responsible for their lower fitness. Our results thereby supported the AED hypothesis. Furthermore, the small females in these treatments produced more eggs at emergence than the control females. This highlights the fact that in stressful environments, small females switch their reproductive strategy from a synovigenic strategy (in which females mature new eggs after emergence) to a more pro‐ovigenic one (in which females emerge with more mature eggs).     

First insights into the biochemical and molecular response to cold stress in <Emphasis Type="Italic">Cicer microphyllum</Emphasis>, a crop wild relative of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Identifying a potential crop wild relative (CWR) of legumes, especially one with high abiotic stress tolerance, has been a priority of plant breeders for many decades. Traditionally CWRs have been selected based on biometrical traits observed in the field, however this methodology is insufficient for research into nonmorphological traits such as stress tolerance. Biochemical and molecular analysis of potential CWRs allows for more informed selection. Specifically, we focus on Cicer microphyllum Benth, a CWR of cultivated chickpea Cicer arietinum L., which is distributed in Trans Himalayan ranges adjacent to glaciers of India and Pakistan at the alpine altitude gradient between 2700 to 6000 m. The objective of this study is to begin characterization of the biochemical and molecular bases of adaptation of C. microphyllum to cold stress and compare it to its cultivated relative (Cold susceptible genotype ILC533). Significant differences were recorded in terms of malondialdehyde (MDA) concentration, electrolyte leakage and proline accumulation in C. microphyllum, as compared to C. arietinum, upon cold exposure (4°C/24h). C. microphyllum exhibits more membrane stability under cold stress. Furthermore, proline overaccumulation and an increase in the enzymatic activities of antioxidants including superoxide dismutase, catalase, and ascorbate peroxidase were also observed in C. microphyllum under cold stress treatment. Expression of pyrroline-5-carboxylate synthetase, chalcone reductase, flavonoid 3',5'-hydroxylase and flavonoid 3'-monooxygenase are all upregulated under cold treatment in C. microphyllum. The characteristics recommend C. microphyllum both as a model for plant response to cold stress and as a potential source for abiotic stress resistant germplasm for chickpea breeding programs.     

Phenotypic evaluation of genetic variability and selection of yield contributing traits in chickpea recombinant inbred line population under high temperature stress

Heat is a major abiotic stress that drastically reduces chickpea yield. This study aimed to identify heat-responsive traits to sustain crop productivity by screening a recombinant inbred line (RILs) population at two locations in India (Ludhiana and Faridkot). The RIL population was derived from an inter-specific cross between heat-tolerant genotype GPF 2 (C. arietinum L.) and heat sensitive accession ILWC 292 (C. reticulatum). The pooled analysis of variance showed highly significant differences for all the traits in RILs and most of the traits were significantly affected by heat stress at both locations. High values of genotypic coefficient of variation (19.52–38.53%), phenotypic coefficient of variation (20.29–39.85%), heritability (92.50–93.90%), and genetic advance as a percentage of mean (38.68–76.74%) have been observed for plant height, number of pods per plant, biomass, yield, and hundred seed weight across the heat stress environments. Association studies and principal component analysis showed a significant positive correlation of plant height, number of pods per plant, biomass, hundred seed weight, harvest index, relative leaf water content, and pollen viability with yield under both timely-sown and late-sown conditions. Path analysis revealed that biomass followed by harvest index was the major contributor to yield among the environments. Both step-wise and multiple regression analyses concluded that number of pods per plant, biomass and harvest index consistently showed high level of contribution to the total variation in yield under both timely-sown and late-sown conditions. Thus, the holistic approach of these analyses illustrated that the promising traits provide a framework for developing heat-tolerant cultivars in chickpea.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00977-5.     

Response of transgenic rape plants bearing the <Emphasis Type="Italic">Osmyb4</Emphasis> gene from rice encoding a trans-factor to low above-zero temperature

Accumulation of soluble sugars (sucrose, fructose, and glucose), proline, phenols (total phenols and flavonoids), and antocyanins during adaptation to low-temperature stress (4°C) of two lines of spring rape (Brassica napus L., cv. Westar) characterized by weak (Bn-1) and strong (Bn-3) expression of the Osmyb4 transgene was studied. Vegetatively propagated transgenic and wild-type plants were grown in the hydroponic culture at 24°C; at the stage of 5–6 leaves, plants were exposed to 4°C for 5 days and then returned to the optimum temperature of 24°C for recovery. Transgenic plants were established to manifest improved cold and frost tolerance, which was evident from more active biomass accumulation at 4°C as compared with wild-type plants and from sustaining their viability after 2-day-long exposure to −6°C. Determination of MDA content showed that one of the reasons of their improved cold tolerance was their capability of maintaining oxidative homeostasis under low-temperature stress. This suggestion is supported by intense accumulation of phenols and antocyanins, manifesting pronounced antioxidant effects, by transgenic plants during their cold adaptation. Thus, during 2–5 days of plant exposure to 4°C, in transgenic plants the total content of phenols increased by 2.6–3.7 times, flavonoids — by 3.7–4.7 times, and antocyanins — by 3.5–5.3 times as compared with control plants growing at 24°C. Transgenic Bn-3 plants with strong expression of the Osmyb4 gene accumulated phenols and antocyanins at 4°C more actively than Bn-1 plants characterized by weak expression of this gene. Transgenic rape plants subjected to cold stress accumulated more proline, manifesting stress-protection effects, and lesser accumulation of soluble sugars. Before the beginning of experiment, the content of soluble sugars was approximately similar in wild-type plants and transgenic lines; at 4°C their level in transgenic plants was substantially lower than in control plants. As distinct from the process of cold adaptation, during recovery, the content of all tested stress-protection compounds dropped sharply. The results obtained indicate that active expression of the Osmyb4 gene from rice in the rape plants was accompanied not only by accumulation of compatible osmolytes but also by biosynthesis of antioxidants of phenolic nature.     

Proline improves copper tolerance in chickpea (Cicer arietinum)

The present study suggests the involvement of proline in copper tolerance of four genotypes of Cicer arietinum (chickpea). Based on the data of tolerance index and lipid peroxidation, the order for copper tolerance was as follows: RSG 888?>?CSG 144?>?CSG 104?>?RSG 44 in the selected genotypes. The basis of differential copper tolerance in chickpea genotypes was characterized by analyzing, antioxidant enzymes (superoxide dismutase, ascorbated peroxidase and catalase), phytochelatins, copper uptake, and proline accumulation. Chickpea genotypes showed stimulated superoxide dismutase activity at all tested concentrations of copper, but H₂O₂ decomposing enzymes especially; ascorbate peroxidase did not increase with 25 and 50 μM copper treatments. Catalase activity, however, increased at lower copper concentrations but failed to stimulate at 50 μM copper. Such divergence in responses of these enzymes minimizes their importance in protecting chickpea against copper stress. The sensitive genotypes showed greater enhancement of phytochelatins than that of tolerant genotypes. Hence, the possibility of phytochelatins in improving copper tolerance in the test plant is also excluded. Interestingly, the order of proline accumulation in the chickpea genotypes (RSG 888?>?CSG 144?>?CSG 104?>?RSG 44) was exactly similar to the order of copper tolerance. Based on hyperaccumulation of proline in tolerant genotype (RSG 44) and the reduction and improvement of lipid peroxidation and tolerance index, respectively, by proline pretreatment, we conclude that hyperaccumulation of proline improves the copper tolerance in chickpea.