Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

Phosphofructokinase from the host fraction of chickpea nodules

Two phosphofructokinases (EC 2.7.1.11; PFK) have been isolated from the host fraction of nitrogen–fixing chickpea ( Cicer arietinum L.) root nodules that were formed with Rhizobium sp. (Cicer) CC1192. The predominant enzyme, which is suggested to be cytosolic, was isolated as a large aggregate of molecular mass near 2 000 kDa and was purified to a high degree. The less abundant enzyme, suggested to be of plastid origin, had a native molecular mass of 186 kDa. Both forms of PFK displayed typical hyperbolic kinetics with MgATP and fructose-6-phosphate. The major form of PFK was strongly inhibited by phosphoenolpyruvate, 2-phosphoglycerate and 3-phos-phoglycerate, and to a lesser extent by ADP, pyrophosphate, glucose-6-phosphate and 6-phosphogluconate. KCl and phosphate were activators and relieved the effect of inhibitors. The major PFK was disaggregated in the presence of ATP into a species which had a molecular mass of approximately 550 kDa and a lower affinity for fructose-6-phosphate.     

Effect of plant growth promoting rhizobacteria,nematode parasitic fungi and root-nodule bacterium on root-knot nematodes Meloidogyne javanica and growth of chickpea

Effects of plant growth promoting rhizobacteria (Pseudomonas putida MTCC No. 3604 and Pseudomonas alcaligenes MTCC No. 493) and parasitic fungi (Pochonia chlamydosporia KIA and Paecilomyces lilacinus KIA) were studied, alone and together with Rhizobium sp. (charcoal commercial culture) on the growth of chickpea and multiplication of Meloidogyne javanica. Individually, P. putida 3604, P. alcaligenes 493 and Rhizobium caused a significant increase in the growth of chickpea in both nematode inoculated and uninoculated plants. Inoculation of Rhizobium with a parasitic fungus or with plant growth promoting rhizobaterium caused a greater increase in the growth of plants inoculated with nematodes than caused by either of them singly. Individually, P. lilacinus KIA caused a greater increase in the growth of nematode inoculated plants than caused by P. putida 3604 or P. alcaligenes 493. P. lilacinus KIA caused a greater reduction in galling and nematode multiplication followed by P. chlamydosporia KIA, P. putida 3604 and P. alcaligenes 493. Combined use of P. lilacinus KIA with Rhizobium was better in reducing galling and nematode multiplication than any other treatment. P. putida 3604 caused a greater colonization of root than P. alcaligenes 493 while P. lilacinus KIA was isolated from more nematodes than P. chlamydosporia KIA.     

Studies on the expression of marker genes in chickpea

Conditions were established for the optimum transient expression of beta-glucuronidase and neomycin phosphotransferase II genes introduced into zygotic embryos of chickpea (Cicer arietinum L. 6153 and CM72) by accelerated tungsten particles. Plasmid DNA at a concentration of 12 microgram per milligram of tungsten particles when accelerated with an inflow of helium gas at 60 kilogram per square centimeter through a distance of 24 centimeter in a chamber maintained at a negative pressure of 71.12 centimeter of mercury, resulted in optimal transient expression of the beta-glucuronidase gene in chickpea embryos. However, the expression of the marker genes was 20-40% higher under a cauliflower mosaic virus promoter in comparison to the Win6 and actin promoters. When Agrobacterium tumefaciens was used to transfer marker genes into zygotic embryos and the resultant plants were analysed for activity of the beta-glucuronidase and neomycin phosphotransferase II genes, both of these genes were expressed in tumorous tissues. When a disarmed strain of Agrobacterium was used, normal shoots were regenerated in which the lower parts showed expression of both genes at a frequency of 20%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of plant growth regulators on evolution of ethylene and methane by different explants of chickpea

Shoot tips, cotyledonary nodes and hypocotyls of chickpea (Cicer arietinum L.) were grown on 3 media: plant induction medium (PIM), callus induction medium (CIM), and shoot induction medium (SIM). Maximum growth and differentiation was seen in PIM, whereas minimum was observed in CIM. Shoot tips which differentiated to multiple shoots evolved negligible amounts of ethylene. Maximum ethylene evolution was recorded by hypocotyls in PIM. Ethylene appears to have stimulatory effect on shoot bud differentiation in cotyledonary nodes. But in hypocotyls, increased ethylene inhibited growth and differentiation. Calli on media containing only auxin (PIM) evolved significantly more ethylene, whereas those on media with cytokinin (SIM) evolved more methane. Callus forming explants like cotyledonary nodes and hypocotyls evolve more ethylene than shoot tips. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of salinity and sodicity upon nodulation and nitrogen fixation in chickpea (Cicer arietinum)

Production of grain legumes is severely reduced in salt-affected soils because their ability to form and maintain nitrogen-fixing nodules is impaired by both salinity and sodicity (alkalinity). Genotypes of chickpea, Cicer arietinum, with high nodulation capacity under stress were identified by field screening in a sodic soil in India and subsequently evaluated quantitatively for nitrogen fixation in a glasshouse study in a saline but neutral soil in the UK. In the field, pH 8.9 was the critical upper limit for most genotypes studied but genotypes with high nodulation outperformed all others at pH 9.0-9.2. The threshold limit of soil salinity for shoot growth was at ECe 3 dS m(-1), except for the high-nodulation selection for which it was ECe 6. Nodulation was reduced in all genotypes at salinities above 3 dS m(-1) but to a lesser extent in the high-nodulation selection, which proved inherently superior under both non-saline and stress conditions. Nitrogen fixation was also much more tolerant of salinity in this selection than in the other genotypes studied. The results show that chickpea genotypes tolerant of salt-affected soil have better nodulation and support higher rates of symbiotic nitrogen fixation than sensitive genotypes.     

Current status of wilt/root rot diseases in major chickpea growing areas of Ethiopia

Wilt/root rot diseases are a major chickpea production constraint in Ethiopia causing yield losses by reducing the number of plants. To determine the current status of disease incidence and distribution, surveys were conducted in the 2013/2014 and 2014/2015 cropping seasons in major chickpea growing zones of Ethiopia. Despite recent efforts in dissemination of improved varieties, low to high incidence of 0.0–83.4, 0.0–27.6, 1.3–19.8 and 0.0–16.3% and 1.0–81.9, 0.0–25.5, 3.0–13.9 and 1.0–21.5% in East Gojjam, Southwest Shewa, North Shewa and West Shewa in 2013/2014 and 2014/2015, respectively. Therefore, integrated pest management strategies should be developed and availed to farmers.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Active extrusion of protons and exudation of carboxylic acids in response to iron deficiency by roots of chickpea (Cicer arietinum L.)

A chickpea cultivar, K-850, acidified the nutrient solution in response to iron deficiency, with subsequent re-greening of chlorotic leaves. No recovery of chlorosis was observed when the nutrient solution was buffered at a pH 6.3. During the period of acidification induced by iron deficiency, the roots of K-850 exuded more carboxylic acids than when supplied with sufficient iron. However, the rate of extrusion of protons was much higher than the rate of exudation of carboxylic acids during the acidification period. The extrusion of protons was inhibited by the addition of vanadate at the beginning of the decrease in pH. It appeared that acidification of the solution in response to iron deficiency was mediated by a proton-pumping ATPase, located at the plasma membrane. The presence of cations in the solution was essential for the extrusion of protons under iron deficiency, but the species of cation made no significant difference to the rate of extrusion of protons from roots. Therefore, we concluded that non-specific H+/cation antiport was involved in the acidification process.     

Ascochyta blight of chickpea reduced 38% by application of Aureobasidium pullulans (anamorphic Dothioraceae,Dothideales) to post-harvest debris

In 2004–2005, application of non-amended suspensions of Aureobasidium pullulans conidia to post-harvest chickpea debris resulted in 37.9% fewer Ascochyta blight lesions on chickpea test plants relative to controls. Analogous tests in 2006–2007 resulted in 38.4% fewer lesions. Ascospores released from debris were predominantly Davidiella sp. (anamorph, Cladosporium sp.), followed by Didymella rabiei (anamorph, Ascochyta rabiei, agent of Ascochyta blight).     

Characterization of an rpoN mutant of Mesorhizobium ciceri

AIMS: To study the genetic basis of C(4)-dicarboxylate transport (Dct) in relation to symbiotic nitrogen fixation in Mesorhizobium ciceri. METHODS AND RESULTS: A Tn5-induced mutant strain (TL16) of M. ciceri, unable to grow on C(4)-dicarboxylates, was isolated from the wild-type strain TAL 620. The mutant lacked activities of the enzymes, which use C(4)-dicarboxylates as substrate. The sequencing of the 3.2kb EcoRI fragment, which was the site of Tn5 insertion, revealed three complete and two partial open reading frames. In the mutant, Tn5 interrupted the rpoN gene, of which only one copy was there. Complementation and biochemical studies suggest that the M. ciceri rpoN activity is required for C(4)-Dct, maturation of bacteroids and symbiotic nitrogen fixation. The fine structure of the ineffective nodules produced by TL16 on Cicer arietinum L changed in comparison with those produced by the wild type. CONCLUSIONS: The mutant strain TL16 suffered a disruption in the rpoN gene. Only one copy of rpoN gene is present in M. ciceri. The mutation abolishes Dct activity. It additionally abolishes the symbiotic nitrogen fixation activity of the bacteroids in the nodules. SIGNIFICANCE AND IMPACT OF THE STUDY: This first document in M. ciceri shows that a functional rpoN gene is essential for the transport of dicarboxylic acids and symbiotic nitrogen fixation.     

Genetic analysis of seed size, yield and days to flowering in a chickpea recombinant inbred line population derived from a Kabuli × Desi cross

Quantitative traits, seed size, yield and days to flowering were studied in a chickpea intraspecific recombinant inbred line (RIL) population (F_6:7) derived from a Kabuli × Desi cross. The population was evaluated in two locations over 2 years. Days to flowering was also evaluated in the greenhouse under short-day conditions. Seed size was the most heritable trait (0.90), followed by days to flowering (0.36) and yield (0.14). Negative and significant correlation was found between yield and seed size in the second year where environmental homogeneity was tested by analysing the controls included in each assay. During the first year, the environment was not considered homogeneous for yield in either location. Quantitative trait loci (QTLs) for the three characters were detected in linkage group (LG) 4. In relation to seed size, two QTLs were located in LG4 (QTL_SW1) and LG8 (QTL_SW2). QTL_SW1 accounted 20.3% of the total phenotypic variation and QTL_SW2 explained 10.1%. A QTL for yield (QTL_YD) was located in LG4 explaining around 13% of variation. QTL_YD might be pleiotropic with QTL_SW1. For days to flowering, a QTL (QTL_DF1) was located in LG4 for all environments analysed explaining around 20% of variation. QTL_DF1 was closely linked to QTL_SW1 and QTL_YD in LG4.     

Cobalt stress affects nitrogen metabolism,photosynthesis and antioxidant system in chickpea (Cicer arietinum L.)

Abstract

Plants of chickpea were exposed to varied levels of cobalt (Co) and sampled at the 60-day stage. Cobalt at concentration <100 µM significantly increased the number of nodules, their dry mass, leghemoglobin concentration and the activity of nitrogenase. Similarly, the activities of glutamate dehydrogenase, glutamine synthetase and glutamate synthase also exhibited an increase in the presence of Co <100 µM, in nodules and leaves, respectively. The various photosynthetic attributes in leaves and the activity of antioxidative enzymes both in nodules and leaves were inhibited by Co in a concentration-dependent manner. However, the lipid peroxidation and the content of proline exhibited a significant increase in response to Co and were at a maximum in the plants exposed to 250 µM concentration of cobalt. Since most of the parameters showed a significant increase in response to 50 µM cobalt, this concentration may be regarded as a threshold concentration.     

Genotypic alteration and competitive nodulation of Mesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils

Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium ciceri are chickpea (Cicer arietinum L.) rhizobia that share a high similarity of the symbiotic genes nodC and nifH, but they have different geographic distributions. M. muleiense has been isolated and found only in alkaline soils of Xinjiang, China, whereas the other two strains have been found in the Mediterranean and India. To investigate the species stability of M. muleiense during natural evolution and its capability of competitive nodulation against the other two exotic species, re-sampling of nodules in the field and competition experiments between the three species were conducted. The results showed that the predominant microsymbiont associated with chickpea grown in Xinjiang was still M. muleiense, but the predominant genotypes of M. muleiense had changed significantly during the four years since a previous survey. The data also showed that M. mediterraneum and M. ciceri were more competitive than the residential strain of M. muleiense CCBAU 83963^T in sterilized vermiculite or soils from Xinjiang. However, in non-sterilized soils, M. muleiense was the predominant nodule occupier. These results indicated that natural or adapting evolution of M. muleiense was occurring in fields subjected to changing environmental factors. In addition, the biogeography and symbiotic associations of rhizobia with their host legumes were also influenced by biological factors in the soil, such as indigenous rhizobia and other organisms.     

Morphological and pathogenic variability of Indian isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt

Wilt of chickpea (Cicer arietinum) caused by Fusarium oxysporum f. sp. ciceris is prevalent in almost all chickpea growing areas of the world and its incidence varied from 14.1 to 32.0% in the different states of India surveyed. The isolates were highly variable in their colony growth pattern, size of colony and pigmentations. The size of microconidia varied from 5.1–12.8 × 2.5–5.0 μm, whereas macroconidia ranged from 16.5–37.9 × 4.0–5.9 μm with 1–5 septations. One hundred and twelve isolates were grouped into 12 categories on the basis of their radial growth, size of macroconidia and growth pattern. Majority of the isolates were highly pathogenic causing more than 50% wilt in chickpea cultivar JG 62. Virulence analysis of 56 representative isolates on a set of 18 cultivars of chickpea, including 10 international differentials, grouped them into three categories. Chickpea cultivar KWR 108 differentiated all isolates of Punjab, Haryana and Delhi states and a few isolates of Rajasthan from others by showing resistant reactions and were placed in the first group. The rest of the isolates of Rajasthan state showed susceptible reactions on KWR 108 placed in a second group. Cultivar CPS 1 distinguished the isolates of Jharkhand state and placed them into a third group. An international set of cultivars recommended for race differentiation were not able to distinguish all the isolates into known races of the pathogen, therefore cultivar KWR 108 should be included in the existing differential set of cultivars.     

Molecular structure and chromosomal localization of major repetitive DNA families in the chickpea (Cicer arietinum L.) genome

Three major repetitive DNA sequences were isolated from a genomic library of chickpea (Cicer arietinum L.) and characterized with respect to their genomic organization and chromosomal localization. All repetitive elements are genus-specific and mostly located in the AT-rich pericentric heterochromatin. Two families are organized as satellite DNAs with repeat lengths of 162–168 bp (CaSat1) and 100 bp (CaSat2). CaSat1 is mainly located adjacent to the 18S rDNA clusters on chromosomes A and B, whereas CaSat2 is a major component of the pericentric heterochromatin on all chromosomes. The high abundance of these sequences in closely related species of the genus Cicer as well as their variation in structure and copy number among the annual species provide useful tools for taxonomic studies. The retrotransposon-like sequences of the third family (CaRep) display a more complex organization and are represented by two independent sets of clones (CaRep1 and CaRep2) with homology to different regions of Ty3-gypsy-like retrotransposons. They are distributed over the pericentric heterochromatin block on all chromosomes with extensions into euchromatic regions. Conserved structures within different crossability groups of related Cicer species suggest independent amplification or transposition events during the evolution of the annual species of the genus.     

Potential of DNA markers in detecting divergence and in analysing heterosis in Indian elite chickpea cultivars

 Molecular markers such as RAPDs and microsatellites were used to study genetic diversity in 29 elite Indian chickpea genotypes. In general, microsatellites were more efficient than the RAPD markers in detecting polymorphism in these genotypes. Among the various microsatellites, (AAC)₅, (ACT)_5, (AAG)₅ and (GATA)₄ were able to differentiate all 29 chickpea cultivars. The mean value of probability of identical match by chance was 2.32×10^-25 using DraI-(ACT)₅, TaqI-(AAC)₅, TaqI-(AAG)₅ and TaqI-(GATA)₄ enzyme-probe combinations. The dendrogram, constructed on the basis of similarity index values, grouped the chickpea genotypes into five main clusters with 8 cultivars genetically distant and outgrouped from the main clusters. To investigate if DNA markers are useful in predicting F₁ performance and heterosis in chickpea, we crossed 8 genotypes having important agronomic characters in a diallel set. The F₁s and their parents in the diallel set were analysed for agronomic traits for better parent and midparent heterosis. Heterosis was found to be much higher for yield than for yield components that fit a multiplicative model. The analysis of genetic divergence using D² statistics clustered the 8 cultivars into two groups. Although molecular marker-based genetic distance did not linearly correlate to heterosis, two heterotic groups could be identified on the basis of the general marker heterozygosity. Received: 1 August 1998 / Accepted: 29 September 1998