Chloroplast ultra structure, photosynthesis and enzyme activities in regenerated plants of Stevia rebaudiana (Bert.) Bertoni as influenced by copper sulphate in the medium.

Stevia rebaudiana (Bert.) Bertoni is an important medicinal plant used as noncaloric commercial sweetener. Plants regenerated with higher levels of copper sulphate in the medium exhibited enhanced activity of peroxidase and polyphenoloxidase (PPO) enzymes. Transmission electron microscopy (TEM) revealed increase in size and number of electron dense inclusions in the chloroplasts of plants regenerated at optimised level of copper sulphate (0.5µM) in the medium. There was decrease in chlorogenic acid (CGA) content. Chl-a-fluorescence transient pattern (OJIP) showed that the photosynthesis process was more efficient at 0.5µM CuSO4 in the medium. 

Carboxy terminal region of a chloroplast DNA polymerase accessory factor stimulates DNA polymerase activity.

p43, a glycoprotein of pea chloroplast (ct), acts as an accessory protein of pea chloroplast DNA polymerase. p43 binds to DNA, binds to ct-DNA polymerase and stimulates the ct-DNA polymerase activity. In the work presented here, the C-terminal domain of p43 (p22) has been overexpressed in E. coli. South Western analysis reveals that the recombinant p22 lacks in DNA binding activity. However, the recombinant p22 can form complex with the pea ct-DNA polymerase quite efficiently and stimulates the DNA polymerase activity to a greater extent than the native p43. Thus the DNA binding domain of p43 appears to be spatially separate from the domain responsible for the DNA polymerase accessory activity. The DNA binding domain is also highly O-glycosylated and loss of glycosylation of p43 leads to enhanced DNA binding as well as repression of ct-DNA polymerase activity. These findings allow us to propose a model to explain how glycosylation of p43 helps ct-DNA polymerase latch onto the DNA template for enhanced processivity. The predictive components of the model have been discussed.

Imaging oscillations in Gonyzaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.

Isolation of 34 kDa protein from spinach chloroplasts having ferrooxidase and H2O2-dependent dark O2 evolution activities.

A latent form of 'Ferrooxidase' exhibiting ferrocyanide-dependent O2 uptake was detected in the isolated spinach chloroplasts. Presence of a cationic detergent hexadecyl trimethyl ammonium bromide (CTAB) in the medium was essential to induce this activity. The association of this enzyme activity with photosystem II (PSII) particles as well as the ability of PSII particles to show oxidation of H2O2 (catalase like activity) indicated its possible relationship with water oxidation system. The protein catalysing this activity was purified to homogeneity and its molecular mass was found to be 34 kDa. The purified protein showed a complete dependence on an electron acceptor, namely ferricyanide, for the oxidation of H2O2. While with ferrocyanide in the presence of CTAB, the protein exhibits the ferrooxidase activity. For both activities, a sharp pH optima at 6.1 was observed. The km for H2O2 was 12.2 mM. The purified enzyme protein contained 4 atoms of calcium and 2 atoms of iron per mole of the enzyme. Unlike catalase, the enzyme reaction was insensitive to sodium azide even at 500 microM concentration. The enzyme was found to be sensitive to metal chelators like ethylene-glycol-bis-(beta-aminoethylether) N, N+ tetra acetic acid (EGTA) (2mM), alpha,alpha-dipyridyl (500 microM) and 1,10-orthophenanthroline (200 microM). The sensitivity of the reaction to alpha,alpha-dipyridyl and 1,10-orthophenanthroline suggested the involvement of Fe2+ in the reaction. Inhibition of enzyme activity by EGTA and restoration of activity by supplementation of CaCl2 to the EGTA-dialysed sample confirmed the absolute requirement for calcium for this activity. Calcium was absent in the EGTA-dialysed enzyme. Apart from these inhibitors, NaF and NH2OH were potent inhibitors of the enzyme reaction.(ABSTRACT TRUNCATED AT 250 WORDS)