Studies On Structural And Functional Characterization Of Two Circadian Gene Rubisco Activase And Manganese Stabilizing Protein

In higher plants, circadian clock plays a crucial role in regulation of different physiological processes. In this study cDNA array containing 5837 rice unique genes was used to explore gene expression patterns at 4 h intervals throughout a day to isolate diurnal and circadian candidate genes in rice. Structural and functional characterization of two circadian gene Rubisco activase(RCA) and manganese stabilizing protein(MSP) were also studied in this paper and main results includes following four parts:1. cDNA array analysis of diurnal and circadian candidate genes in ricecDNA array containing 5837 rice unique genes was used to explore gene expression patterns at 4 h intervals throughout a day. It was found that there were 190 genes with diurnal changes, of which 170 genes could change in circadian manner. Analysis of those genes showed that circadian clock played a key role in coordination of many physiological processes such as glycolysis, water and mineral elements transport. By clustering diurnal change genes from cDNA data, we found 10 clusters with different expression patterns.2. Diural Change of Rubisco Activase Expression in Rice LeavesIn order to illuminate the regulation of Rubisco activase (RCA) expression, we investigated the changes of rca mRNA accumulation in leaves of rice seedlings grown under 12-h light/12-h darkness (LD), constant darkness (DD) and constant light (LL) by Northern blotting analysis. The changes in RCA contents in leaves of rice seedlings grown under these three treated conditions were also studied with single radial immune diffusion by rabbit polyclonal antibodies against rice Rubisco activase.The results showed that the accumulation of rca mRNA displayed a typical oscillating pattern under LD treatment, in which the levels of the maximum and minimum were detected at 8:00 a.m. and 8:00 p.m. respectively. The rhythmic accumulation pattern of rca mRNA level continued through two cycles after being transferred to DD and LL. Under the LL treatments the peak of rca mRNA accumulation started earlier and the amplitude of the fluctuation for the second cycle was greatly reduced to approximately 20% under both DD and LL treatments. Theobvious diurnal changes of RCA protein contents from the minimum levels before dawn to the maximum level in the afternoon had been observed under LD treatment. It was different from that of DD and LL treatments, indicating that the transcription of RCA was regulated by an oscillating circadian rhythm and also by light but the translation of RCA was regulated by light to a large extent.3. Structural and functional differentiation of three groups of tyrosine residues by acetylation of 7V-acetylimidozole in manganese stabilizingIn order to study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidozole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually -5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried) and 1 -2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of -6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further red shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.4. Identification of candidate lysine residues involved in maintaining the functional conformation of manganese stabilizing protein in solutionTo clarify structure-function relationships of lysine residues in MSP, lysine residues were specifically modified with N-succinimidyl propionate (NSP). When MSP was treated with 0.5 mM NSP, the modified MSP did not affect its reconstitution or oxygen evolving activities. Analysis with far UV circular dichroism (CD) or intrinsic fluorescence spectra showed that 0.5 mM NSP-modified MSP retained most of its native secondary structure. Increasing the NSP concentration to 4mM, the modified protein cannot rebind to PSII and completely lost the reactivating capability. The results from both far UV-CD and intrinsic fluorescence spectra analysis showed a clearly conformational changes upon 4 mM NSP-modified MSP, suggesting a loss of its rebinding to PSII was primarily caused by conformational change. Mapping of the sites of NSP modification was performed by staphylococcus Vs protease digestion of the modified protein and analysis by MALDI-TOF MS. These studies indicated that six lysine residues including Lys20, LyslOI, Lysl96, Lys207, Lysl30 (or 137) and Lys66 (or 76) were modified only with 4mM NSP. These candidate lysine residues seem to play a crucial role in maintaining functional conformation of MSP in solution.