Comparison of activity of OsDmc1A recombinase of rice (Oryza sativa) in presence of Ca2+ and Mg2+ ions.

Recombinases are known to play an important role in the homology search and strand exchange during meiosis as well as homologous recombination (HR)-mediated DNA repair specifically require Mg2+ ion for their activity. The Ca2+ has been shown to stimulate the strand exchange activity of hDmc1 and ScDmc1 by forming the extended filaments on DNA. Oryza sativa disrupted meiotic cDNA1A (OsDmc1A), a homologue of yeast and human Dmc1 from rice shows the hallmark functions of recombinase. Here, we report the effects of Ca2+ and Mg2+ on OsDmc1A activity from rice (Oryza sativa). OsDmc1A showed a concentration-dependent binding with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) substrates in presence of Mg2+ or Ca2+. The ssDNA and dsDNA binding activities, as well as renaturation activity of OsDmc1A were similar in the presence of Ca2+ or Mg2+. Increasing the Ca2+ or Mg2+ increased the DNA binding, renaturation and strand exchange of OsDmc1A. But, OsDmc1A showed only a slight stimulation of strand exchange activity in presence of Ca2+, when compared the activity in presence of Mg2+. Electron microscopy showed that OsDmc1A formed ring-like structures in presence of Mg2+ or Ca2+. However, OsDmc1A formed filament like structures with both ss and dsDNA in presence of Mg2+ or Ca2+. Taken together, Ca2+ did not affect OsDmc1A recombinase activity significantly.

Differential distribution of pigment-protein complexes in the Thylakoid membranes of Synechocystis 6803.

Thylakoids in Synechocystis 6803, though apparently uniform in appearance in ultrastructure, were found to consist of segments which were functionally dissimilar and had distinct proteomes. These thylakoid segments can be isolated from Synechocystis 6803 by successive ultracentrifugation of cell free extracts at 40,000×g (40 k segments), 90,000×g (90 k segments) and 150,000×g (150 k segments). Electron microscopy showed differences in their appearance. 40 k segments looked feathery and fluffy, whereas the 90 k and 150 k thylakoid membrane segments appeared tiny and less fluffy. The absorption spectra showed heterogeneous distribution of pigment-protein complexes in the three types of segments. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) showed unequal distributions in 40 k, 90 k and 150 k segments which were substantiated with low temperature fluorescence measurements. The ratio of PSII/PSI fluorescence emission at 77 K (λ(ex) = 435 nm) was highest in 150 k segments indicating higher PSII per unit PSI in these segments. The chlorophyll fluorescence lifetimes in the membranes, determined with a time-correlated single-photon counting technique, could be resolved in three components: τ(1) (=) <40 ps, τ(2) (=) 425-900 ps and τ(3) (=) 2.4-3.2 ns. The percentage contribution of the fastest component (τ(1)) decreased in the order 40 k > 90 k > 150 k segments whereas that of the other two components showed a reversed trend. These studies indicated differential distribution of pigment-protein complexes in the three membrane segments suggesting heterogeneity in the thylakoids of Synechocystis 6803.

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 (60)Co gamma 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.

Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions.

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000 x g, followed by 90,000 x g and finally at 150,000 x g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000 x g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000 x g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO2 fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.

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.