Studies On Physiological Mechanisms In Response To Low Temperature And Drought In Regenerated Seedlings Of Chorispora Bungeana

Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) is a representative alpine subnival plant species that can survive and flourish under frequent temperature fluctuations and even freezing temperatures. It has proved to retain some stable variations of physiological and genetic characteristics in response to low temperature stress, and is an ideal plant species for research the mechanisms of cold-hardiness and stress-responsive genes. NADPH:protochlorophyllide oxidoreductase (POR) plays a pivotal regulatory role in Chl synthesis and chloroplast development. In order to examine whether C. bungeana POR is a main factor in this system, a POR gene was isolated and termed CbPORB (Accession No. FJ390503), and its expression patterns were investigated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot under cold stress. In addition, we also investigated changes in the antioxidant system of C. bungeana seedlings under the influence of EBR and subsequently exposed to drought stress, and a relationship between antioxidant system changes and the degree of stress resistance. All the results were as following:1. The full-length cDNA sequence of C. bungeana PORB (GenBank Accession No. FJ390503), obtained by 5'- and 3'-RACE (rapid amplification of cDNA ends) PCR, showed high homology to POR cDNA sequences from related species. The full-length cDNA sequence of CbPORB is 1444 bp containing a 1209-bp ORF, which encoded a predicted protein of 402 amino acid residues with a theoretical molecular weight of 43.37 kDa, and the isoelectric point was 9.43. In comparison with other available plant POR sequences at the protein level, C. bungeana PORB protein precursor is highly homologous to Arabidopsis PORB and we thus referred to it as CbPORB (GenBank Accession No. ACJ12925). The deduced CbPORB protein sequence showed high homology with the sequences of AtPORB, PsPOR, CsPOR and HvPORB.2. Sequence analysis showed that CbPORB protein contained all the characteristic domains found in other POR proteins, such as plastid transit peptides, the IRAQ tetrapeptide; the essential amino acid residues Tyr-277 and Lys-281 for POR catalytic activity and four highly conserved Cys residues. The alpha helix and random coil constituted the interlaced domain of the main part of the secondary structure. POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids.3. CbPORB expression was detected in leaves and stems, but not in roots, thereby demonstrating that this gene is organ-specific expression patterns. The expression pattern of the CbPORB transcript and protein level in response to low temperatures was studied by semi-quantitative RT-PCR and Western blot. When C. bungeana was exposed to 4℃, the transcript and protein levels of CbPORB maintained at approximately 85% of the control level. After exposure to-4℃, the transcript levels of CbPORB remained at approximately 70% of the control level, and the CbPORB protein abundance decreased substantially and reached the minimum value at 72 h. The changes of transcript level were not similar to that of previous researches, which demonstrated that CbPORB possesses certain resistant characteristic and is a major player in Ch1 biosynthesis process involved in plant growth and development in C. bungeana. Meanwhile, a correlation between the Ch1 content and the levels of CbPORB transcript and protein was found in chilling-stressed and freezing-stressed C. bungeana seedlings. In addition, CbPORB was inhibited by PEG, however, induced by EBR.4. Chlorophyll fluoreseenee analysis showed that values of Fv/Fm and FPSⅡdecreased significantly upon C. bungeana exposure to-4℃, which indicated that the active reaction centers of PSII were reduced, PSII itself was damaged and the eleetron transport chain was damaged, too. On the contrary, Fv/Fm and FPSⅡvalues did not show significant reductions at 4℃during the experiment. There were no significant responses of qP or NPQ to chilling and freezing treatments in C. bungeana seedlings. qP and NPQ showed no significant responses to cold treatment, which demonstrated that cold didn't markedly reduce the proportion of open reaction centres, and cause an increase in thermal dissipation in PSⅡantennae respectively.5. Brassinosteroids have been proposed to act as exogenous signal molecules responsible for inducing tolerance for environmental stresses in plants. The foliar application of 0.1μM EBR was investigated in drought-stressed C. bungeana plants. The results showed that exogenous EBR preserved PSII photochemical activity, reduced lipid peroxidation and membrane permeability. In addition, EBR application reduced the proline content under drought stress conditions, significantly increased the RWC and antioxidative enzyme activities, and ascorbate and glutathione concentrations. These results suggested that EBR enhanced the photoinhibition tolerance of PSII through protecting the structure and function of the oxygen-evolving complex of PSII, and considerably alleviated oxidative damage by maintaining higher antioxidative enzyme activities and antioxidant concentrations.