Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

Altered development of polysomal RNA activity in chick-pea (Cicer arietinum L.) embryonic axes. Effects of abscisic acid and temperature

The in vitro activity of polysomal polyadenylated RNA (poly(A)RNA) was studied using chick-pea (Cicer arietinum L.) embryonic axes subjected to treatments retarding germination (H₂O 30°C and abscisic acid [ABA] 30°C) or inducing a false germination (thiourea 30°C) in which normal protein synthesis and growth did not occur. All treatments induced a smaller proportion of poly(A)RNA compared with the control (H₂O 25°C). However, poly(A)RNA obtained in the presence of ABA had a similar in vitro activity to that of the control. The translation of mRNA from embryonic axes germinated at high temperatures was extensively blocked (70%) by methyl-7-guanosine-5-triphosphate, whereas mRNA translation from axes treated with H₂O-25°C and ABA was completely blocked (100%), indicating a greater cap dependence in the latter cases. Polyacrylamide gel electrophoresis showed that ABA and H₂O-30°C each induced the synthesis of a polypeptide with an approximate M_r of 32 kDa, probably a germination regulator. It is suggested that ABA and high temperatures could regulate germination at the translational level as well as affecting ionic-exchange properties, as has been previously demonstrated (Hernández-Nistal et al. 1983, Physiol. Plant. 57, 273–278).Abbreviations ABA abscisic acid - Poly (A) RNA polyadenylated RNA - TU thiourea     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Partial purification and properties of a 36-kDa 1-aminocyclopropane-1-carboxylate N-malonyltransferase from mung bean

A 36-kDa 1-aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase, which converts the ethylene precursor ACC into the conjugated derivative malonyl-ACC (MACC), has been isolated from etiolated mung bean ( Vigna radiata ) hypocotyls, and partially purified in a four-step procedure. The enzyme is stimulated about 7-fold by 100 m M K⁺ salts or 0.5 m M Co²⁺ salts, and is inhibited competitively by D-phenylalanine (K_i= 1.3 m M ) and non competitively by CoASH (0.3 m M ). Beside malonyl-CoA, it is capable to use succinyl-CoA as an acyl donor. The 36-kDa enzyme described here exhibits a lower optimum temperature (40°C) and a 7- or 3-fold lower apparent K_m for ACC (68 μ M ) and malonyl-CoA (74 μ M ), respectively, when compared with its 55 kDa isoform already isolated from the same plant material. This data support the idea that several isoforms of ACC N-malonyltransferase exist in plants. These isoforms may play a differential role in regulating the availability of ACC, and consequently the rate of ethylene production, as well as detoxifying cells from D-amino acids.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate N-malonyltransferase from etiolated mung bean hypocotyls

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mung bean (Vigna radiata) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg⁻¹ protein h⁻¹. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent K_m of 0.5 mm for ACC and 0.2 mm for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol⁻¹ degree⁻¹.     

Partial purification and some biochemical properties of nonspecific alkaline phosphatase in germinating chick-pea (Cicer arietinum) seeds

A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the K_m for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (K_m= 2.4 m M ), NADP⁺ (K_m= 4.0 m M ), 5'-AMP (K_m= 4.5 m M ), 5'-ADP (K_m= 6.1 m M ) and ribose-5P (K_m= 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn²⁺, K⁺, Hg²⁺ and Mo⁶⁺ were strong inhibitors, whereas F⁻ and Ca²⁺ inhibited weakly; Co²⁺ and Ni²⁺ were activators.     

Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.)

Plants of chick-pea (Cicer arietinum L. cv. ILC1919) inoculated with Mesorhizobium ciceri strain ch-191 were grown in a controlled environmental chamber, and were administered salt (0, 50, 75, and 100 mM NaCl) during the vegetative period. Four harvests (4, 7, 11, and 14d after treatment) were analysed. The aim was to ascertain whether the negative effect of saline stress on nitrogen fixation is due to a limitation on the photosynthate supply to the nodule or a limitation on the nodular metabolism which sustains nitrogenase activity.Plant growth was affected only by the highest NaCl concentration, whereas nitrogenase activity was affected from 50 mM. At the first harvest, Rubisco, PEPC and MDH activities in leaves rose with salt, but fell during the following harvests. The increase of PEPC and MDH in nodules at the two first samplings was clearly related to salt concentration. While 50 mM NaCl increased GS and GOGAT in nodules at some harvests, 100 mM strongly inhibited these activities at all the harvests. The accumulation of proline, amino acids and carbohydrates was clearly related to salt especially in the leaves, whereas in the nodules the protein content was boosted by salt. Although photosynthesis declined with NaCl, the response of nitrogen fixation to salt was more pronounced. This situation, together with carbohydrate accumulation, suggests that the lack of photosynthate does not cause the inhibition of nitrogenase activity under this type of stress. The similar trend observed for the PEPC-MDH pathway and the ARA support the hypothesis concerning the limitation in the supply of energy substrate, mainly malate, to the bacteroids. The accumulation of compatible solutes is more a consequence of damage produced by salt stress than of a protective strategy.     

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.     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Comparative study of nitrogen fixation and carbon metabolism in two chick-pea (Cicer arietinum L.) cultivars under salt stress

Two cultivars of Cicer arietinum with differential tolerance to salinity have been compared by analysing growth, photosynthesis, nodulation, nitrogenase activity, and carbon metabolism in the nodule cytosol. The aim was to help elucidate the relationships between, on the one hand, sucrose and malate metabolism in nodules and, on the other, the inhibition of nitrogen fixation under salt stress. Chick-pea cultivars Pedrosillano (sensitive) and ILC1919 (tolerant) inoculated with Mesorhizobium ciceri strain Ch-191 were grown in a controlled environmental chamber and were treated with salt (0, 50, 75, and 100 mM NaCl) from sowing to harvest time (28 d). Plant growth and photosynthesis were more affected by salt in Pedrosillano than in ILC1919. Also the effect of salt on nodulation and nitrogen fixation was much more pronounced in Pedrosillano. The increase in nodular mass in ILC1919 can partially counteract the inhibition of nitrogenase activity. The enzymes of sucrose breakdown were inhibited by NaCl, but in ILC1919 a rise in alkaline invertase was observed with salinity, which could compensate for the lack of the sucrose synthase hydrolytic activity. The activity of PEPC was stimulated by salt in ILC1919. Also, this cultivar showed higher malate concentrations in root nodules.     

Exposure of seeds to static magnetic field enhances germination and early growth characteristics in chickpea (Cicer arietinum L.)

Seeds of chickpea (Cicer arietinum L.) were exposed in batches to static magnetic fields of strength from 0 to 250 mT in steps of 50 mT for 1-4 h in steps of 1 h for all fields. Results showed that magnetic field application enhanced seed performance in terms of laboratory germination, speed of germination, seedling length and seedling dry weight significantly compared to unexposed control. However, the response varied with field strength and duration of exposure without any particular trend. Among the various combinations of field strength and duration, 50 mT for 2 h, 100 mT for 1 h and 150 mT for 2 h exposures gave best results. Exposure of seeds to these three magnetic fields improved seed coat membrane integrity as it reduced the electrical conductivity of seed leachate. In soil, seeds exposed to these three treatments produced significantly increased seedling dry weights of 1-month-old plants. The root characteristics of the plants showed dramatic increase in root length, root surface area and root volume. The improved functional root parameters suggest that magnetically treated chickpea seeds may perform better under rainfed (un-irrigated) conditions where there is a restrictive soil moisture regime.     

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 M zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 M zeatin plus 0.2 M indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 M BA or 4.6 M kinetin. This regeneration system may be useful for genetic transformation of chickpea.     

Genetics of nodulation in Chickpea (Cicer arietinum L.)

Combining ability, components of genetic variance and graphic analysis revealed that nodulation in the cultivars of Chickpea (Cicer arietinum L.) under study, was predominantly under the control of non-additive gene action although substantial additive effect was also present. The crosses giving high specific combining ability effects also manifested highly significant positive heterosis. The parents F-61, Giza and Annegiri possessed mostly dominant alleles while Phule G-5, NEC-249 and N-31 possessed mostly recessive alleles having positive effect on nodule weight.     

Factors influencing growth of embryonic axes from dormant seeds of hazel (Corylus avellana L.)

Chilling (after-ripening) of hazel seeds stimulates subsequent growth of excised embryonic axes on sucrose and leads to growth which is less susceptible to inhibition by abscisic acid. Chilling also obviates the need for inorganic salts in the culture medium. Axes from dormant seeds grow normally when only one-third of a cotyledon is left attached.Abbreviation ABA abscisic acid     

Colchicine induced polyploidy in chickpeas (Cicer arietinum L.)

Summary The most successful induction of tetraploidy was obtained with 2 hour treatment by 0.25% aqueous colchicine solution of 18-hour watersoaked desi chickpeas material. However, kabuli types needed only 1 hour treatment under similar conditions. Gigantism accompanied induction of polyploidy in desi as well as kabuli types but yield and fertility were greatly reduced. The meiotic abnormalities accompanying polyploidy were multivalent association of chromosomes followed by unequal disjunction, chromosome bridges, laggards etc. The percentage of stainable pollen, however, was at par between diploids and tetraploids. Gene control of percentage seed setting was observed in both levels of ploidy. A striking feature of the studies was the high seed setting percentage in 4n F ₁ material resulting from diverse crosses, viz., desi×kabuli. A probable reduction in multivalent association coupled with yield increases in segregants from the later generation of tetraploids indicates the possibility of selection for higher yield and fertility from polyploids, particularly from some hybrid material.     

Quantitative trait loci governing carotenoid concentration and weight in seeds of chickpea (Cicer arietinum L.)

Chickpea is a staple protein source in many Asian and Middle Eastern countries. The seeds contain carotenoids such as beta-carotene, cryptoxanthin, lutein and zeaxanthin in amounts above the engineered beta-carotene-containing golden rice level. Thus, breeding for high carotenoid concentration in seeds is of nutritional, socio-economic, and economic importance. To study the genetics governing seed carotenoids in chickpea, we studied the relationship between seed weight and concentrations of beta-carotene and lutein by means of high-performance liquid chromatography in segregating progeny from a cross between an Israeli cultivar and wild Cicer reticulatum Ladiz. Seeds of the cross progeny varied with respect to their carotenoid concentration (heritability estimates ranged from 0.5 to 0.9), and a negative genetic correlation was found between mean seed weight and carotenoid concentration in the F₃. To determine the loci responsible for the genetic variation observed, the population was genotyped using 91 sequence tagged microsatellite site markers and two CytP450 markers to generate a genetic map consisting of nine linkage groups and a total length of 344.6 cM. Using quantitative data collected for beta-carotene and lutein concentration and seed weight of the seeds of the F₂ population, we were able to identify quantitative trait loci (QTLs) by interval mapping. At a LOD score of 2, four QTLs for beta-carotene concentration, a single QTL for lutein concentration and three QTLs for seed weight were detected. The results of this investigation may assist in improving the nutritional quality of chickpea.     

Purification and characterization of a membrane-bound ATP diphosphohydrolase from Cicer arietinum (chick-pea)roots.

Microsomal membranes from Cicer arietinum (chick-pea) roots contained an ATP phosphohydrolase activity that could be solubilized by high-ionic-strength media. The enzyme has been purified to homogeneity by affinity and ion-exchange chromatography. It has the properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) that hydrolyses different nucleoside di- and tri-phosphates but has no activity towards monophosphoric esters and pyrophosphate. No stimulation by K+ could be demonstrated for either the membrane-bound or the purified enzyme, and therefore it would seem not to be related to the K+ -dependent ATPase postulated to mediate K+ transport in plants.