Purification and properties of antiviral proteins from the leaves of Bougainvillea xbuttiana.

A non-phytotoxic, resistance inducing, proteinaceous antiviral principle was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration from the leaves of Bougainvillea xbuttiana. It imparted resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in their respective test hosts viz. Nicotiana glutinosa, N. tabacum var. Samsun NN, and Cyamopsis tetragonoloba, respectively. The purified principle eluted as a single peak upon gel filtration, but exhibited two polypeptides on SDS-PAGE with Mr 28,000 and 24,000. The two polypeptides were found to be highly basic, rich in lysine with pI around 10.0 and 10.5, respectively. Since this principle effected local lesion inhibition in both treated and untreated top leaves of test host, it might be acting in the initial stages of virus infection as a systemic inducer.

A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves.

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process.

Expression of Rhizobium meliloti symbiotic promoters in Azorhizobium caulinodans.

Plasmids containing Rhizobium meliloti symbiotic promoters P1 (promoter of nifHDK) and P2 (promoter of fixABCX) when mobilized into the cells of Azorhizobium caulinodans strain IRBG 46 showed strong expression of these promoters under free-living microaerobic as well as symbiotic conditions. Under free-living conditions microaerobiosis (3% or less O2) was found to be sufficient to activate these promoters; expression being higher at 1% than at 3% O2 concentration. Under symbiotic conditions the expression was much more stronger-with bacteroids in stem nodules showing higher expression than those in root nodules. Under both the conditions expression of the promoters in the native R. meliloti strain Rm102F34 was lower than that in the A. caulinodans strain IRBG 46. The results suggest a functional homology of these promoters in the heterologous background of A. caulinodans.

Effect of combined nitrogen on the expression of nitrate reductase and nitrite reductase in Azorhizobium caulinodans.

In aerobically grown Azorhizobium caulinodans strain IRBG 46, in vivo expression of nitrate reductase (NR) and nitrite reductase (NiR) requires the presence of either nitrate or nitrite. On the contrary mere microaerobic conditions are sufficient for the expression of NR and NiR, however, addition of nitrate to the growth medium enhanced the activities of the enzymes. Optimum concentration of nitrate for maximum expression of NR and NiR activities was different in aerobic and microaerobic conditions. Nitrite was released into the medium both in aerobic and microaerobic conditions beyond a particular concentration of nitrate in the medium. Dissimilatory nitrate reduction was affected to a lesser extent by ammonium compared to assimilatory nitrate reduction.

Effect of nitrate on nitrogen fixation and nitrate assimilation in Azorhizobium-Sesbania rostrata symbiotic system.

Root nodule formation was inhibited by 30% and 50% respectively at low concentration of 1 mM and 2 mM nitrate, while stem nodule formation was enhanced by 50% only at 1 mM nitrate. The nodule specific nitrogenase activity decreased with the increasing concentration of nitrate. At 1 mM nitrate nitrogenase activity per plant stem nodule was not affected, but it was less than 50% in the root nodules as compared to control. Increasing concentration of nitrate increased in vivo activity of nitrate reductase (NR) significantly in stem, root nodules and leaves. Nodule cytosolic NR utilized both NADH and succinate as electron donor, but not reduced MV. However bacteroidal NR utilised reduced MV as reductant more efficiently than succinate.

Purification and characterization of proteolytic enzymes from normal and opaque-2 Zea mays L. developing endosperms.

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease II disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common—optimum pH and temperature were respectively, 5·7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-Ltyrosine ethyl ester and poly-L-glutamic acid. The Km values of day 15 and 25 normal maize endosperm proteases ranged from 2·73–3·30, while for opaque-2 maize endosperm protease I it was 3·33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein.