Jayashree Krishna Sainis

Shape analysis of grains of Indian wheat varieties

Shape variation, based on grain morphology, was quantified in 15 Indian wheat varieties by digital image analysis using custom-built software. Gray images of the grains of different varieties were captured in crease-down position using a HP scan jet IICX/T scanner in transparency mode. The software rotates each grain in the captured image for normalization of orientation. These rotated images were used for further analysis. Geometric features such as area, perimeter, compactness, major and minor axis length and their ratios, slenderness and spread were computed on the binary image. Five other shape factors were derived from these basic geometric features. Moment analysis for calculating standard, central, normalized central and invariant moments of grains was done using the gray images of grains. The data for each parameter for every grain in the image as well as mean, standard deviation (S.D.) and standard error (S.E.) for that parameter were stored for further use. The wheat varieties used in this study showed differences in geometric and shape related parameters. It was concluded that Euclidean distance calculated on the basis of these differences could serve as a basis of distinguishing between samples and to their identification.

Heterogeneity in thylakoid membrane proteome of Synechocystis 6803

Cell-free extracts of Synechocystis 6803 were fractionated by successive ultracentrifugation at 40,000 x g, 90,000 x g and 150,000 x g to obtain the three thylakoid fractions designated as 40k, 90k and 150k fractions respectively. These fractions showed differences in absorption and emission spectra. Nano-LC-ESI-Q-TOF MS analysis identified 123 proteins belonging to membrane as well as cytosolic fraction. Out of these proteins, there were 22 proteins with transmembrane helices and 12 proteins with signal peptide. There were 77 proteins common across all the three fractions. Most of these proteins were subunits of photosynthetic complexes, CF(0)-CF(1) ATP synthase or ribosomal proteins. Among the rest of the proteins, 8 were exclusive to 40k fraction, 3 were exclusive to 90k fraction and 13 were exclusive to 150k fraction. There were 10 proteins common between 40k and 90k fractions and 12 proteins common between 90k and 150k fractions. There were no common proteins detected between 40k and 150 fractions. The results suggested existence of heterogeneity in thylakoids of Synechocystis 6803, which may lead to micro-compartmentation and functional heterogeneity in the thylakoids of this organism as seen previously.

Immunoelectron microscopy for locating calvin cycle enzymes in the thylakoids of synechocystis 6803.

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing (HPF) and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle (phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase) and the catalytic portion of the chloroplast H+-ATP synthase (CF1) are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40,000, 90,000, and 150,000 g. Among these, the 150,000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate (R-5-P) +ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.

Effects of 60Co γ radiation on thylakoid membrane functions in Anacystis nidulans

In photosynthetic organisms oxidative stress is known to result in photoinactivation of photosynthetic machinery. We investigated effects of (60)Co gamma radiation, which generates oxidative stress, on thylakoid structure and function in cyanobacteria. Cells of unicellular, non-nitrogen fixing cyanobacterium Anacystis nidulans (Synechococcus sp.) showed D(10) value of 257 Gy of (60)Co gamma radiation. When measured immediately after exposure, cells irradiated with 1500 Gy (lethal dose) of (60)Co gamma radiation did not show any differences in photosynthetic functions such as CO(2) fixation, O(2) evolution and partial reactions of photosynthetic electron transport in comparison to unirradiated cells. Incubation of irradiated cells for 24h in light or dark resulted in decline in photosynthesis. The decline in photosynthesis was higher in the cells incubated in light as compared to the cells incubated in dark. Among the partial reactions of electron transport, only PSII activity declined drastically after incubation of irradiated samples. This was also supported by the analysis of membrane functions using thermoluminescence. Exposure of cyanobacteria to high doses of (60)Co gamma radiation did not affect the thylakoid membrane ultrastructure immediately after exposure as shown by electron microscopy. The level of reactive oxygen species (ROS) in irradiated cells was 20 times higher as compared to control. In irradiated cells de novo protein synthesis was reduced considerably immediately after irradiation. Treatment of cells with tetracycline also affected photosynthesis as in irradiated cells. The results showed that photoinhibition of photosynthetic apparatus after incubation of irradiated cells was probably augmented due to reduced protein synthesis. Active photosynthesis is known to require uninterrupted replenishment of some of the proteins involved in electron transport chain. The defective thylakoid membrane biogenesis may be leading to photosynthetic decline post-irradiation.

DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis.

Rad51 and disrupted meiotic cDNA1 (Dmc1) are the two eukaryotic DNA recombinases that participate in homology search and strand exchange reactions during homologous recombination mediated DNA repair. Rad51 expresses in both mitotic and meiotic tissues whereas Dmc1 is confined to meiosis. DNA binding and pairing activities of Oryza sativa disrupted meiotic cDNA1 (OsDmc1) from rice have been reported earlier. In the present study, DNA renaturation and strand exchange activities of OsDmc1 have been studied, in real time and without the steps of deproteinization, using fluorescence resonance energy transfer (FRET). The extent as well as the rate of renaturation is the highest in conditions that contain ATP, but significantly less when ATP is replaced by slowly hydrolysable analogues of ATP, namely adenosine 5′‐(β,γ‐imido) triphosphate (AMP‐PNP) or adenosine 5′‐O‐(3‐thio triphosphate) (ATP‐γ‐S), where the former was substantially poorer than the latter in facilitating the renaturation function. FRET assay results also revealed OsDmc1 protein concentration dependent strand exchange function, where the activity was the fastest in the presence of ATP, whereas in the absence of a nucleotide cofactor it was several fold (≈ 15‐fold) slower. Interestingly, strand exchange, in reactions where ATP was replaced with AMP‐PNP or ATP‐γ‐S, was somewhat slower than that of even minus nucleotide cofactor control. Notwithstanding the slow rates, the reactions with no nucleotide cofactor or with ATP‐analogues did reach the same steady state level as seen in ATP reaction. FRET changes were unaffected by the steps of deproteinization following OsDmc1 reaction, suggesting that the assay results reflected stable events involving exchanges of homologous DNA strands. All these results, put together, suggest that OsDmc1 catalyses homologous renaturation as well as strand exchange events where ATP hydrolysis seems to critically decide the rates of the reaction system. These studies open up new facets of a plant recombinase function in relation to the role of ATP hydrolysis.

Involvement of thylakoid membranes in supramolecular organisation of Calvin cycle enzymes in Anacystis nidulans.

The cells of unicellular photosynthetic cyanobacterium Anacystis nidulans were permeated with lysozyme, toluene, toluene-triton, toluene-triton-lysozyme. Transmission electron microscopy of semi-thin sections (500 nm) using TEM at 160 kV showed that cells permeated with only lysozyme or toluene showed the typical concentric arrangement of thylakoid membranes. However, when toluene-treated cells were further treated with triton and lysozyme the thylakoid membranes were disrupted. Sequential reactions of Calvin cycle were studied in the differentially permeated cells in vivo, using various intermediates such as 3-PGA, GA-3-P, FDP, SDP, R-5-P, RuBP and cofactors like ATP, NADPH depending on the requirement. RuBP and R-5-P + ATP dependent activities could be observed in all types of permeated cells. Sequential reactions of the entire Calvin cycle using 3-PGA could be detected in the cells that had retained the internal organisation of the thylakoid membranes after permeation and were lost on disruption of this organisation. Light dependent CO2 fixation could be detected only in the cells permeated with lysozyme. This activity was abolished in the cells after treatment with toluene. The results suggested that the integrity of thylakoid membranes may be essential for the organisation of sequential enzymes of the Calvin cycle in vivo and facilitate their functioning.

Hydroxylamine inhibition of the nitrate reductase complex from Amaranthus

Abstract Nitrate reductase from Amaranthus viridis is similar to nitrate reductase from other plant sources. NH2OH inhibits nitrate reduction from NADH by the nitrate reductase complex, but it does not inhibit either the NADH-dehydrogenase activity or nitrate reduction from reduced flavin mononucleotides. The inhibition observed was non-competitive with nitrate when the enzyme was pre-incubated with NH2OH and NADH, and competitive with nitrate without pre-incubation. The Ki values for NH2OH were 5 μM and 30 μM with or without pre-incubation respectively.

Employing a Photosynthetic Antenna Complex to Interfacial Electron Transfer on ZnO Quantum Dot.

Photosynthetic antenna complexes exhibit unidirectional energy-transport phenomena, which make them potential photosensitizers in interfacial electron-transfer processes. In the present study, we show the antenna function of phycocyanin-allophycocyanin (PC-APC) complex using transient emission and absorption spectroscopy. Interfacial electron-transfer dynamics in the PC-APC complex sensitized ZnO semiconductor quantum dot material is compared in native and denatured conditions. The downhill sequential energy transfer from a peripheral phycocyanin disk to a core allophycocyanin disk opens a new electron injection pathway from the allophycocyanin disk in addition to primary electron injection from directly photoexcited phycocyanin disk. Further, the large association of phycocayanobilin chromophores in PC-APC conjugates stabilizes the positive charge within the sensitizer, which leads to slower charge recombination in comparison to that in denatured condition. This study displays the antenna function of energy-efficient biomolecules in favor of better charge separation across the semiconductor interface.

Image analysis of wheat grains developed in different environments and its implications for identification

Varietal identification is an important aspect of crop research and utilization. Identification using computer-based image analysis could be an alternative to visual identification. However, the effectiveness of image analysis systems needs to be established under various real conditions. Three wheat varieties were sown on three different dates. Variation in the grain size and shape of these varieties, brought about by changes in the environmental conditions, was measured using Comprehensive Image Processing Software (CIPS). Some parameters showed considerable grain-to-grain variation, which was either inherent or due to environmental changes during grain filling. Euclidean distances were calculated using either means of all the parameters (ED1), or using only those parameters that did not show a high coefficient of variation (ED2). For samples of the same variety sown at different times, Euclidean distances were smaller compared with samples of different varieties, indicating that grains of the same variety resembled one another. By using the criterion of minimum Euclidean distance it was possible to distinguish between varieties, in spite of variation in grain shape and size due to environmental conditions. It was possible to identify correctly an unknown sample, taken as a test case.

Effect of the state of water as studied by pulsed NMR on the function of RUBISCO in a multienzyme complex

Summary Calvin cycle enzymes function in situ in stroma where protein concentration is extremely high and water is remarkably limited. The state of water molecules in spinach leaves, chloroplasts and aqueous solutions of hydrophilic macromolecules like bovine serum albumin (BSA) and polyethylene glycol (PEG) was studied by pulsed nuclear magnetic resonance (NMR). The results showed that water existed in two states in spinach leaves and chloroplasts. The relaxation times of the two components of water in leaves were 243 ms and 51 ms. In the chloroplasts, a further reduction in the mobility with relaxation times of 78 ms and 24 ms was observed, suggesting that most of the water molecules in situ may be bound to a variety of macromolecules and hence highly immobile. The aqueous solutions of hydrophilic macromolecules demonstrated a reduction in the mobility, in a concentration dependent manner. The solutions of BSA above 50 % showed the state of water to be similar to that in vivo . The binding of water molecules in vivo would reduce the aqueous potential of the cellular water affecting solvation capacity as well as diffusion and also influence the activities of the enzymes. In order to study the consequence of the reduced mobility of water on the function of enzymes, R-5-P + ATP and RuBP dependent CO 2 fixation activities of RUBISCO in the multienzyme complex of isomerase, kinase and RUBISCO were examined at different concentrations of BSA and PEG. Both the activities were inhibited to a similar extent at high concentrations of polymers due to a severely water restrained condition. The identical inhibition of R-5-P + ATP dependent coupled reaction support the hypothesis that RuBP may be channeled to RUBISCO in this multienzyme complex and channeling of intermediates might be offering a functional advantage in a crowded condition.