UDP-glucose pyrophosphorylase: Isolation, purification and characterization from developing thermotolerant wheat (Triticum aestivum) grains.

UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9) activity was determined in four different thermotolerant varieties of wheat viz. WH-1021, PBW-373, Raj-3765 and DBW-16. The specific activity of UGPase was found to be highest at 21 days after anthesis (DAA) in the variety WH-1021 which has been developed by Haryana Agricultural University, Hisar (Haryana, India). Hence, crude extract prepared from immature grains (21 days after anthesis) of WH-1021 was used for purification of UGPase using standard protein purification techniques which exploit differences in protein properties viz. ammonium sulphate fractionation (based on solubility differences), DEAE-ion exchange chromatography (based on charge differences) and molecular sieving through Sephadex G-100 gel (based on molecular mass differences). Near homogeneous enzyme preparation with molecular mass of 82 kDa and subunit molecular weight of 39 kDa was obtained. The purified enzyme had thermostability upto 50 °C. Kinetic studies revealed that the enzyme followed Michaelis Menten kinetics with Km value of 0.9 mM and 1.66 mM for UDP and PPi, respectively. Physico-chemical and kinetic characterization suggested that the enzyme UGPase from WH-1021 is a homodimer which has adapted to high temperature stress and that lower availability of substrates and high Km values may be responsible for reduced starch synthesis/grain yield.

Characterization of seed storage proteins in high protein genotypes of cowpea [Vigna unguiculata (L.) Walp].

Twenty one genotypes and two check varieties viz. CS-88 and V-240 of cowpea [Vigna unguiculata (L.) Walp. ] were screened for total proteins. The total protein content ranged from 22.4 (HC-3) to 27.9 % (HC-98-64) in 21 genotypes whereas in check varieties it was 25.6 (V-240) and 26.0 % (CS-88). Seven genotypes viz. HC-6, HC-5, CP-21, LST-II-C-12, CP-16, COVU-702 and HC-98-64 having high protein content (26.7 to 27.9 %) were selected for further characterization of their seed storage proteins. Globulins were the major protein fraction ranging from 55.6 (LST-II-C-12) to 58.8 % (CP-16 and HC-6) of total protein. Glutelins was the second major fraction ranging from 14.4 to 15.6 % followed by albumins (8.2 to 11.9 %) and prolamins (2.3 to 5.0 %). Content of free amino acids also showed variations amongst genotypes with COVU-702 having maximum and LST-II-C-12 having minimum content. Essential amino acid analysis revealed that S-amino acids (cysteine and methionine) were the first limiting amino acids followed by tryptophan. From the results presented here it could be suggested that two genotypes viz. LST-II-C-12 and HC-5 be used in breeding programmes aimed at developing high protein moth bean varieties with good quality.

Amelioration of Chromium VI Toxicity in Sorghum (Sorghum bicolor L.) using Glycine Betaine.

The main objective of the present research work was to study the effect of Cr toxicity and its amelioration by glycine betaine (GB) in sorghum (HJ 541 and SSG 59-3). Chromium (Cr VI), 2 and 4 ppm led to a significant reduction in plant height, root length, chlorophyll content, antioxidant enzymes viz. catalase, peroxidase, ascorbate peroxidase, glutathione reductase, polyphenol oxidase, and superoxide dismutase; and metabolites viz. ascorbate, proline, and glutathione. The results of the present study supported the findings that the application of GB can minimize or reduce the toxic effects caused by Cr VI which reaches the plants via soil, water, and air pollution. It is concluded that GB at both 50, as well as 100 mM concentrations, successfully ameliorated Cr VI (up to 4 ppm) toxicity and its application may be recommended for crops affected by Cr VI toxicity to get better growth and yield.

Photosynthetic carbon reduction cycle metabolites and enzymes of sucrose and starch biosynthesis in developing Brassica pods.

Rate of net CO2 exchange and activities of the key enzymes of fru-2,6-P2, sucrose and starch synthesis and levels of certain intermediates of Calvin cycle were determined in Brassica pods at different stages of their development. The rate of net CO2 exchange, activities of FBPase, UDPG-pyrophosphorylase and SPS, and the contents of 3-PGA, DHAP, RuBP and UDPG increased up to day 21 after anthesis followed by a continuous decrease thereafter. However the content of fru-6-P started decreasing only after 28 days of anthesis. Changes in the levels of fru-2,6-P2 were closely associated with the changes in F6P 2-kinase activity rather than with F2,6-P2ase activity. Similarly, activities of ADPG-pyrophosphorylase and ADPG-starch synthetase closely followed the pattern of starch accumulation in pod tissues. These observations suggest that during the early phase of pod development (up to 21 days after anthesis), which is also the active phase for pod photosynthesis, carbon is mainly utilised for sucrose synthesis and that during the later phase of pod development (from day 21 to 42 after anthesis), there is shift in metabolic path of carbon from sucrose to starch.

Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.).

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg(2+) ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K(m) of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO(3) (-) with S(0.5) value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, alpha-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca(2+) and Mn(2+). Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.

Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L.

Activities of key enzymes of the Calvin cycle and C(4) metabolism, rates of CO(2) fixation, and the initial products of photosynthetic (14)CO(2) fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv ;Toria.' Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C(4) metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of (14)CO(2) assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO(2) during light. However, respiratory losses were very high during the dark period.

Effect of water stress on photosynthesis and in vitro activities of the PCR cycle enzymes in pigeonpea (Cajanus cajan L.).

Leaf water potential was decreased by withholding irrigation to provide three levels of stress described as mild ({ie69-1}) moderate ({ie69-2}) and severe ({ie69-3}). The specific activity of NADP linked glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, aldolase, phosphogluco-isomerase and RuBP carboxylase decreased under mild stress, but the activity of phosphoglucomutase showed an increase whilst ribulose-5-phosphate kinase was least affected. With further decrease in water potential, the activity of NADP linked glyceraldehyde-3-phosphate dehydrogenase and aldolase showed a decrease, whereas, the activities of fructose-1,6-bisphosphatase, phosphoglycerate kinase, phosphogulcomutase and RuBP carboxylase increased. Net CO2 fixation decreased sharply with stress, whereas, respiration and photorespiration increased in moderate stress, but decreased under severe stress. Stomatal resistance also increased with decrease in water potential. It seems that in vitro enzyme activities of PCR cycle are not responsible for decreased photosynthesis in pigeonpea under short term water stress.

Effect of cadmium and nickel on photosynthesis and the enzymes of the photosynthetic carbon reduction cycle in pigeonpea (Cajanus cajan L.).

The effect of Cd(2+) and Ni(2+) on the rate of photosynthesis and activities of key enzymes of the photosynthetic carbon reduction cycle was examined in leaves from pigeonpea (Cajanus cajan L., cv. UPAS-120) grown in nitrogen free sand culture. Two different concentrations of Cd(2+) and Ni(2+) were applied through the rooting medium at two growth stages. The application of Cd(2+) and Ni(2+) (0.5 and 1.0 mM) at an early vegetative stage (30 days after sowing) resulted in about 50% and 32% reduction in net photosynthesis, respectively. However, enzyme activities were decreased to different levels (2-61%) depending upon the enzyme and the concentration of the metal ion.These concentrations (0.5 and 1.0 mM of Cd(2+) and Ni(2+)) had no effect when applied at a later vegetative stage i.e. 70 days after sowing. However, when the concentration of Cd(2+) was increased to 10 mM, there was about an 86% reduction in the rate of photosynthesis but the enzyme activities were reduced by only about 40%. Although Ni(2+) reduced the photosynthetic rate by 65%, it had little effect on enzyme activities. The reduction in photosynthesis seems to occur indirectly through a decrease in chlorophyll content and stomatal conductance but not due to decreased enzyme activities. Oxygen evolution by leaf discs was inhibited by Cd(2+) and Ni(2+) in parallel with a reduction in photosynthesis. These data confirm the earlier reported effects of Cd(2+) and Ni(2+) on O2 evolution in isolated chloroplasts.