Sudhakar Bharti

Chlorophyll a fluorescence study revealing effects of high salt stress on Photosystem II in wheat leaves.

In order to study the effects of high salt stress on PS II in detached wheat (Triticum aestivum) leaves, the seedlings were grown in Knop solution and temperature was 20 +/- 2 degrees C. Detached leaves were exposed to high salt stress (0.1-0.5 M NaCl) for 1 h in dark and Chl a fluorescence induction kinetics was measured. Various parameters like Fv/Fm, ABS/RC, ETo/TRo, performance index and area over the florescence curve were measured and the energy pipeline model was deduced in response to salt stress. Our results show that the damage caused due to high salt stress is more prominent at the donor side rather than the acceptor side of PS II. Moreover the effects of high salt stress are largely reversible, as the acceptor side damage is completely recovered (approximately 100%) while the recovery of the donor side is less than 85%. Based on our results we suggest that in response to high salt stress, the donor side of PS II is affected more as compared to the acceptor side of PS II.

Phylogenetic relationships as in Ocimum revealed by RAPD markers

Genetic relationships were examined among thirty germplasm accessions belonging to five Ocimum species using RAPD markers. A very high degree of polymorphism (98.20%) was observed. UPGMA cluster analysis of genetic similarity indices grouped all the accessions into two major clusters corresponding to previously reported botanical sections. Intra-clustering within the two clusters precisely grouped the accessions belonging to one species in one sub-cluster as expected from their genetic background. Our results show that RAPD technique is a sensitive, precise and efficient tool for genomic analysis in Ocimum species, that may be useful in future studies, by assigning new unclassified germplasm accessions to specific taxonomic groups and reclassifying previously classified accessions of other Ocimum species by traditional criteria on a more objective basis.

Analysis of salt stress induced changes in Photosystem II heterogeneity by prompt fluorescence and delayed fluorescence in wheat (Triticum aestivum) leaves

In order to investigate changes in the heterogeneity of PSII, prompt fluorescence induction curves (PFIC) and delayed fluorescence induction curves (DFIC) were measured in wheat leaves after salt treatment. From these data, antenna heterogeneity and reducing side heterogeneity were estimated. Results show that antenna size, which is further differentiated into α, β and γ PSII centers, is changed under salt stress conditions. At higher salt concentration, there is a decrease in the number of α PSII centers with simultaneous increase in the amount of β and γ PSII centers. Another aspect of antenna heterogeneity is explained in terms of connectivity (or grouping) between PSII centers which did not change significantly under salt stress. Reducing side heterogeneity was assessed by both DFIC and PFIC and results show that a significant increase in the conversion of Q(B)-reducing centers to Q(B)-non-reducing centers is observed under salt stress.

INORGANIC ANIONS INDUCE STATE CHANGES IN SPINACH THYLAKOID MEMBRANES

The role of cations in excitation energy distribution between the two photosystems of photosynthesis is well established. This paper provides evidence, for the first time, for an important role of anions in the regulation of distribution of absorbed light energy between the two photosystems. Inorganic anions caused redistribution of energy more in favour of photosystem I, as judged from measurements of chlorophyll a fluorescence transients, rates of electron transport in low light and 77 K fluorescence emission spectra: the Fv/Fm ratio was decreased by inorganic anions even in the presence of DCMU, the PS II electron transport was decreased whereas PS I electron transport was increased and the F735 (77 K emission from PS I)/F685 (77 K emission from PS II) ratio was increased. Such changes were observed with inorganic anions having different valencies (Cl−, SO2− 4, PO3− 4): the higher the valency of the inorganic anion, the more the energy transferred towards PS I. Change in the valency of the inorganic anions thus regulates distribution of absorbed light energy between the two photosystems. However, organic anions like acetate, succinate, and citrate caused no significant changes in the Fv/Fm ratio, and in rates of PS I and PS II electron transport, showing their ineffectiveness in regulating light energy distribution.

Computational analysis of fluorescence induction curves in intact spinach leaves treated at different pH.

Effects of change in pH have been investigated on spinach leaf discs by measuring fluorescence induction kinetics using plant efficiency analyzer (PEA). On the basis of computational analysis of the results, we have reported that acidic pH causes a significant inhibition of the donor and the acceptor side of PS II. Energy flux models have been presented using the software Biolyzer HP 3. Effects of pH were investigated on the antenna size heterogeneity of PS II and a relative change in the proportions of α, β, and γ centers was observed.

Heat-induced changes in photosystem I activity as measured with different electron donors in isolated spinach thylakoid membranes.

Heat-induced changes in photosystem I (PSI) have been studied in terms of rates of oxygen consumption using various donors (DCPIPH2, TMPDred and DADred), formation of photo-oxidized P700 and changes in Chl a fluorescence emission at 77 K. Linear heating of thylakoid membranes from 35 degrees C to 70 degrees C caused an enhancement in PSI-mediated electron transfer rates (DCPIPH2-->MV) up to 55 degrees C. However, no change was observed in PSI rates when other electron donors were used (TMPDred and DADred). Similarly, Chl a fluorescence emission spectra at 77 K of heat-treated thylakoid membranes did not show any increase in peak at 735 nm, however, a significant decrease was observed as a function of temperature in the peaks at 685 and 694 nm. In DCMU-treated control thylakoid membranes maximum photo-oxidized P700 was generated at g = 2.0025. In heat-treated thylakoid membranes maximum intensity of photo-oxidized P700 signal was observed at approximately 50-55 degrees C without DCMU treatment. The steady-state signal of the photo-oxidized P700 was studied in the presence of DCPIPH2 and TMPDred as electron donors in DCMU-treated control and in 50 degrees C treated thylakoid membranes. We present here the first of such comparative study of PSI activity in terms of the rates of oxygen consumption and re-reduction kinetics of photo-oxidized P700 in the presence of different electron donors. It appears that the formation of the P700+ signal in heat-treated thylakoid membranes is due to an inhibited electron supply from PSII and not due to spillover or antenna migration.

Differential response of chloride binding sites to elevated temperature: a comparative study in spinach thylakoids and PSII-enriched membranes

A study of heat effects was performed in thylakoids and photosystem II (PSII)-enriched membranes isolated from spinach in relation to Cl−-induced activation of PSII catalyzed oxygen evolution and the retention of Cl− in the PSII complex. For this, Cl−-sufficient membranes and low-Cl− membranes were used. The presence of Cl− in the reaction medium did accelerate oxygen evolution, which remained unaffected by heat treatment up to 40°C in PSII membranes and up to 42.5°C in thylakoids. Heat resistance of Cl−-induced activation of oxygen evolution was found to be independent of the presence of ‘bound Cl−’ in the preparations. However, the functional stability of the PSII complex during heat treatment showed a marked dependence on the presence of bound Cl− in PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center/YD+ showed that there was little loss of Mn2+ up to 42°C in our preparations, although the PSII activity was significantly lowered. These observations together with data from steady state chlorophyll a fluorescence imply that the site of action of Cl− causing direct activation of oxygen evolution was different from the site of primary heat damage. A differential response of chloride binding sites to heat stress was observed. The high-affinity (tightly bound, slow exchanging) site of chloride is affected earlier (∼37°C) while low-affinity (loosely bound, fast exchanging) site gets affected at higher temperatures (42.5°C in thylakoids and 40°C in the case of PSII-enriched membranes).

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.

Evaluation of the specific roles of anions in electron transport and energy transfer reactions in photosynthesis

Ionic environment is important in regulating photosynthetic reactions. The roles of cations, Mn2+, Mg2+, Ca2+, Na+, and K+ as cofactors in electron transport, energy transfer, phosphorylation, and carbon assimilation are better known than the roles of anions, except for chloride and bicarbonate. Only a limited information exists on the roles and effects of nitri formate, sulphate, and phosphate. In this review, we evaluate and highlight the roles of some specific anions on electron transport as well as on excitation energy transfer processes in photosynthesis. Anions exert significant effects on thyla membrane conformation and membrane fluidity, possibly by redistributing the thylakoid membrane surface charges. The anion/cation induced phase transitions in the hydrophilic domains of the thylakoid membranes are probably responsible for the various structural and co-related functional changes under stress. Anions are also important in regulation of energy distribution between the two photosystems. Anions do not only divert more energy from photosystem (PS) 2 to PS1, but can also reverse the effect of cations on energy distribution in a valence-dependent manner. Anions affect also the structure of the photosynthetic apparatus and excitation energy distribution between the two photosystems.

Vacuole Cysteine Proteases and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Degradation during Monocarpic Senescence in Cowpea Leaves

Characterisation of proteases degrading ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC: 4.1.1.39) was studied in the cowpea leaf during monocarpic senescence 3 and 9 d after flowering (DAF), representing early and mid pod fill. The stage at 3 DAF coincided with decrease in the metabolic parameters characterising senescence, i.e., contents of total soluble proteins, RuBPCO, and leaf nitrogen. At 9 DAF, there was a decline in total soluble proteins and an appearance of a 48 kDa cysteine protease. Characterisation of the proteases was done using specific inhibitors. Subcellular localisation at 3 DAF was studied by following the degradation of RuBPCO large subunit (LSU) in the vacuole lysates using immunoblot analyses. Cysteine proteases played a predominant role in the degradation of RuBPCO LSU at the crude extract level. At 9 DAF, expression of cysteine protease isoforms was monitored using polyclonal antibodies against papain and two polypeptides of molecular masses 48 and 35 kDa were observed in the vacuole lysates. We confirmed thus the predominance of cysteine proteases in the vacuoles during different stages of pod development in cowpea leaf.