Surendra Chandra Sabat

Analysis of chlorophyll a fluoresence changes in weak light in heat treated Amaranthus chloroplasts.

After preheating of Amaranthus chloroplasts at elevated temperatures (up to 45°C), the chlorophyll a fluorescence level under low excitation light rises as compared to control (unheated) as observed earlier in other chloroplasts (Schreiber U and Armond PA (1978) Biochim Biophys Acta 502: 138–151). This elevation of heat induced fluorescence yield is quenched by addition of 0.1 mM potassium ferricyanide, suggesting that with mild heat stress the primary electron acceptor of photosystem II is more easily reduced than the unheated samples. Furthermore, the level of fluorescence attained after illumination of dithionite-treated samples is independent of preheating (up to 45°C). Thus, these experiments indicate that the heat induced rise of fluorescence level at low light can not be due to changes in the elevation in the true constant F0 level, that must by definition, be independent of the concentration of QA. It is supposed that the increase in the fluorescence level by weak modulated light is either partly associated with dark reduction of QA due to exposure of chloroplasts to elevated temperature or due to temperature induced fluorescence rise in the so called inactive photosystem II centre where QA are not connected to plastoquinone pool. In the presence of dichlorophenyldimethylurea the fluorescence level triggered by weak modulated light increases at alkaline pH, both in control and heat stressed chloroplasts. This result suggests that the alkaline pH accelerates electron donation from secondary electron donor of photosystem II to QA both in control and heat stressed samples. Thus the increase in fluorescence level probed by weak modulated light due to preheating is not solely linked to increase in true F0 level, but largely associated with the shift in the redox state of QA, the primary stable electron acceptor of photosystem II.

Superoxide dismutase—mentor of abiotic stress tolerance in crop plants

Abiotic stresses impact growth, development, and productivity, and significantly limit the global agricultural productivity mainly by impairing cellular physiology/biochemistry via elevating reactive oxygen species (ROS) generation. If not metabolized, ROS (such as O2•−, OH•, H2O2, or 1O2) exceeds the status of antioxidants and cause damage to DNA, proteins, lipids, and other macromolecules, and finally cellular metabolism arrest. Plants are endowed with a family of enzymes called superoxide dismutases (SODs) that protects cells against potential consequences caused by cytotoxic O2•− by catalyzing its conversion to O2 and H2O2. Hence, SODs constitute the first line of defense against abiotic stress-accrued enhanced ROS and its reaction products. In the light of recent reports, the present effort: (a) overviews abiotic stresses, ROS, and their metabolism; (b) introduces and discusses SODs and their types, significance, and appraises abiotic stress-mediated modulation in plants; (c) analyzes major reports available on genetic engineering of SODs in plants; and finally, (d) highlights major aspects so far least studied in the current context. Literature appraised herein reflects clear information paucity in context with the molecular/genetic insights into the major functions (and underlying mechanisms) performed by SODs, and also with the regulation of SODs by post-translational modifications. If the previous aspects are considered in the future works, the outcome can be significant in sustainably improving plant abiotic stress tolerance and efficiently managing agricultural challenges under changing climatic conditions.

Expression and purification of soluble bio-active rice plant catalase-A from recombinant Escherichia coli

Catalase in plants is a heme-coordinated tetrameric protein that primarily disproportionates hydrogen peroxide into water and oxygen. It plays an important role in maintaining cellular concentration of hydrogen peroxide to a level, necessary for all aspects of normal plant growth and development. Except for its recombinant expression in transgenic plants and insect cell line, the protein is yet to be synthesized in its bio-active form in prokaryotic expression system. Attempts made in past for recombinant expression of plant catalase in Escherichia coli consistently resulted in formation of insoluble and inactive aggregates of inclusion body. Here we have shown the specific requirement of a thioredoxin fusion partner, the involvement of trigger factor protein and the low temperature treatment during induction period for synthesis of completely solubilized rice plant catalase-A in recombinant E. coli. Furthermore, the bacteria required the supplementation of δ-aminolevulinic acid to produce bio-active recombinant rice catalase-A. The molecular and biochemical properties of the purified recombinant protein showed the characteristic features of a typical mono-functional plant catalase. These results attest to the usefulness of the present protocol for production of plant catalase using E. coli as heterologous expression system.

Senescence induced alteration in the electron transport in wheat leaf chloroplasts

Abstract Aging in vivo of primary wheat leaves not only induces loss in chloroplast O 2 evolution capacity but also alters the accessibility of the electron transport chain for exogenous electron acceptors and donors. The pH profile of ferricyanide Hill reaction in the presence of uncoupler methylamine shifts to the acidic side, upon leaf aging, close to the optimum pH of oxidized para -phenylenediamine-mediated photosystem-II-catalysed electron transport activity. This suggests that ferricyanide, which normally accepts electrons at more than one site in the electron transport chain, accepts electrons preferentially at a site close to that of electron acceptance by oxidized para -phenylenediamine in aged leaf chloroplasts. Leaf aging enhances the extent of inhibition by dibromothymoquinone, which suggests changes in the acceptor side of photosystem II. Leaf aging also enhances the rate of the photosystem-I-catalysed electron transport reaction supported by reduced dichlorophenol-indophenol and reduced tetramethyl- para -phenylenediamine. Furthermore, the fact that inhibition by KCN of reduced dichlorophenol-indophenol supported photosystem I activity in aged leaf chloroplasts is greater than the activity supported by reduced tetramethyl- para -phenylenediamine suggests an alteration in the site of feeding of electrons by reduced dichlorophenol-indophenol. Thus leaf aging appears to induce alterations in specific segments of the electron transport chain.

Artemisinin inhibits chloroplast electron transport activity: mode of action.

Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo), behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the QB; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth.

Characterization of Heat-stress Induced Stimulation of Photosystem I Electron Transport Activity in Amaranthus Chloroplasts: Effect of Cations

Summary Exposure of isolated Amaranthus chloroplasts to elevated temperatures (>25 °C for 5 min) induced a stimulation of photosystem I catalyzed electron transport rates only with reduced-dichlorophenolindophenol but not with -tetramethylparaphenylenediamine or -diaminodurene as electron donors. Uncoupler mediated stimulation in dichlorophenolindophenol supported photosystem I catalyzed electron transport was retained in 30 to 45 °C heated chloroplasts, but at 50 °C the sensitivity to uncouplers was lost. The heat induced enhancement of photosystem I activity was, however, found to depend on the concentration of dichlorophenolindophenol, with greater stimulation at low than at high concentration. Furthermore, the stimulation was enhanced both by monovalent and divalent cations. These results suggest that heat treatment of chloroplasts not only uncouples chloroplast electron transport activity but also induces some specific alterations in the site of electron donation by reduced dichlorophenolindophenol, resulting in the enhancement of photosystem I rates.

Heat induced alterations in the photosynthetic electron transport and emission properties of the cyanobacterium Spirulina platensis

Abstract Heat-stress-induced photosynthetic electron transport and emission properties were studied in the cyanobacterium Spirulina platensis. Heat treatment of intact cells up to 50 °C did not cause major changes in the absorption and emission properties of both chlorophyll a and phycocyanin. However, above 50 °C, there was a specific bleaching of phycobiliproteins and an uncoupling of energy transfer in phycobilisomes. Heat stress also reduced the extent and slowed down the decay kinetics of light-induced quenching of the long wavelength emission band which has been shown to be associated with the redox state of P 700, the primary donor of photosystem I. Electron transport activities measured in intact cells showed a decline in the photosystem II mediated Hill activity and an increase in the photosystem I activity with increasing temperatures. However, isolated thylakoid membranes did not exhibit heat-induced stimulation in photosystem I activity. This indicates that the enhancement of photosystem I activity in intact cells is mostly due to increased permeability of cells for the entry of acceptors and donors upon heat treatment. However, mild heat treatments induced damage at the plastoquinone pool, as indicated by the inhibition in the durohydroquinone to methylviologen intersystem electron flow. These results suggest that unlike higher plants, the thylakoid membranes of the cyanobacterium Spirulina platensis do not show heat-induced stimulation in photosystem I activity. We argue that the lack of heat-induced stimulation in photosystem I activity in this cyanobacterium may arise as a result of the absence of light harvesting chlorophyll a/b complex and also variations in the membrane lipid organizations.

Relative sensitivity of various spectral forms of photosynthetic pigments to leaf senescence in wheat (Triticum aestivum L.)

The change in the characteristics of the absorption spectrum of chloroplasts which were isolated from the mature and senescing primary wheat leaves, was examined at various wavelengths in which the photosynthetic pigments mostly absorb. Chlorophyll (Chl) a was observed to be relatively more sensitive to leaf senescence than Chl b and carotenoids. Furthermore, the various spectral invivo forms of Chl a, did not degrade to a similar extent; the far red absorbing forms of Chl a including species that absorb maximally at 692 nm (Chl a-692), 700 nm (Chl a-700) and 708 nm (Chl a 708) were found to be extremely sensitive to senescence induced losses. Both attached and detached senscing primary wheat leaves exhibited nearly similar pattern in the loss of photosynthetic pigments which suggests that the loss in long wavelength absorbing forms of Chl a is a selective indicator of leaf senescence.

Alteration in phosphate uptake potential of wheat plants co-cultivated with salicylic acid.

The phosphate uptake potential was significantly stimulated with low concentration of salicylic acid (SA). In one of the wheat cultivars (Triticum aestivum Var. Sonalika), 50 microM SA stimulated phosphate (PO(4)(3-)) uptake. At higher concentrations of SA (500 and 1000 microM), the rate of uptake was reduced. These findings underscore the light and dose-dependent biphasic action of SA: a low 50 microM stimulated, and high concentrations 500 and 1000 microM inhibited, PO(4)(3-) uptake.

Copper Ion Inhibition of Electron Transport Activity in Sodium Chloride Washed Photosystem II Particle Is Partially Prevented by Calcium Ion

Abstract The inhibitory effects of copper ion (Cu2+) on the photosynthetic electron transport function was investigated both in NaCl washed (depleted in 17 and 23 kDa polypeptides) and native (unwashed) photosystem II membrane preparations from spinach (Beta vulgaris) chlo-roplasts. Copper in the range of 2.0 to 15 μᴍ strongly inhibited the electron flow from water to 2,6-dichlorobenzoquinone in NaCl washed particles in a concentration dependent manner. Com plete inhibition was noticed at 15 μᴍ Cu2+. Oppositely in native membranes, 15 μᴍ C u2+ inhibited only 10-12% of control activity. It was found that calcium ion (Ca2+) significantly reduced the Cu2+ inhibition of electron transport activity. The Ca2+ supported prevention of Cu2+ toxicity was specific to Ca2+. Further analysis indicated that both Cu2+ and Ca2+ act competitively. Since Ca2+ is known to have stimulating/stabilizing effect at the donor side of photosystem II, it is therefore suggested that Cu2+ in NaCl washed particles exerts its inhibitory effect(s) at the oxidizing side of photosystem stimulates/stabilizes the oxygen evolution.