Nitric oxide: a common antipathogenic factor of plants.

In the present study, nitric oxide synthase/nitric oxide (NOS/NO) status was tested in the host plants infected with fungi, bacteria and virus. In each case cytosolic nitric oxide synthase (Cyt-NOS) of diseased plants was inhibited and inhibition was competitive in nature in respect to l-arginine, the substrate for the enzymic activity. Elevation of host nitric oxide (NO) level before infection using nitric oxide (NO) donor protected disease initiation significantly. The nature of enzyme kinetics and the manner of disease protection by nitric oxide donor (NO-donor) was similar in all the three cases of infection. It was concluded that nitric oxide was a common antipathogenic factor of plants.

Purification and characterization of cytosolic pyruvate kinase from developing seeds of Brassica campestris L.

Cytosolic pyruvate kinase (ATP: Pyruvate phosphotransferase, EC 2.7.1.40; PKc) was purified to apparent homogeneity with about 22% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive Blue Sepharose-CL-6B. The purified enzyme with molecular mass of about 214 kDa was a heterotetramer with subunit molecular mass of 55 and 57 kDa. The enzyme showed maximum activity at pH 6.8 and absolute requirement for a divalent (Mg2+) and a monovalent (K+) cation for activity. Typical Michaelis-Menten kinetics was obtained for both the substrates with Km values of 0.10 and 0.11 mM for PEP and ADP, respectively. The enzyme could also use UDP or GDP as alternative nucleotides, but with lower Vmax and lesser affinities. The enzyme was inhibited by glutamate, glutamine, fumarate, citrate, isocitrate, oxalate, 2-PGA, ATP, UTP and GTP and activated by glucose-6-phosphate, fructose-1,6-bisphosphate and Pi, suggesting its regulation mainly by TCA cycle intermediates and the cellular need for carbon skeletons for amino acid biosynthesis. ATP inhibition was of competitive type with respect to PEP and non-competitive with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. Initial velocity and product inhibition studies except for pyruvate inhibition were consistent for a compulsory-ordered tri-bi mechanism.

Enzymes of glycolytic and pentose phosphate pathways in cytosolic and leucoplastic fractions of developing seeds of Brassica campestris.

Distribution of the enzymes of glycolytic and pentose phosphate pathways were studied in cytosolic and leucoplastic fractions of the developing seeds of Brassica. Leucoplasts were isolated using a discontinuous percoll gradient. Intactness of leucoplasts was checked by ADP-glucose pyrophosphorylase assay in presence and absence of triton X-100. No contamination by microbodies, mitochondria and cytosol was observed as assessed by measuring the activities of marker enzymes. The recovery, latency and specific activity of each enzyme in different fractions were compared. The leucoplastic fraction contained complete set of the enzymes of glycolytic and pentose phosphate pathways, indicating that the two subcellular compartments metabolize carbon independently by these pathways. However, the enzymes showed higher activities in cytosolic fraction as compared to those in the leucoplasts, suggesting the need for exchange of metabolites in the two compartments through various translocators, for acting in cooperation to produce energy, reducing power and carbon skeletons for different biosynthetic activities in the non-photosynthetic plastids. Based on these compartmentation studies, a model for carbon flow for fatty acid synthesis in leucoplasts of developing Brassica seeds has been proposed.

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.