| Experiments were conducted in the experimental fields of Xianlin campus,Nanjing Normal University, Nanjing, China. A mutant of two-line sterile rice 812HS was isolated from the progeny of japonica rice 812S. At the seedling stage, the leaves of 812S and 812HS were uniform. There were no significant differences in agronomic characters between 812HS and 812S. After plum rain season, at the tillering and jointing stages, the tips of 812HS leaves showed etiolation, especially in leaf tips.However, the leaves of 812S were green. Genetic analysis and gene mapping showed that the trait was controlled by a new dominant gene, leaf photo-oxidation 1 (LPO1(t))on chromosome 4 between RMM22and M102. This study focused on the changes of chloroplast structure and function of rice mutant 812HS, paying particular attention to the dynamic changes of energy absorption, transformation and transmission in chloroplast, physiological and biochemical processes of the whole growth and proteomics and transcriptomics during the period of photooxidation. It is of great significance to further elucidate the molecular mechanism of photooxidation and rice breeding with high photosynthetic efficiency. The results showed:(1) Phenotype observation of 812HS and 812S. The Chl content in the leaves of 812HS decreased and the Chl a/b ratio appeared highest compared with that of 812S on August 4. The yellow coloration of 812HS leaves was evaluated by testing the response of 812HS to different light treatments. After the plum rain season, the tip of 812HS leaf shaded by the integument remained green, but the leaf exposed to natural light turned yellow. No significant difference was observed in 812S leaf exposed to similar conditions. This observation directly suggested that the yellowing of 812HS was light-induced and the mutation enhanced the photosensitivity of rice leaves.Detection of chlorophyll synthesis precursors revealed that chlorophyll content may be reduced by Urogen III to Proto IX phase arrest. Compared the ultrastructure of plastids in the 812HS mutant and wild-type plants at different developmental stages by using TEM, we found that the 812S chloroplasts exhibited an orderly arrangement of grana and stroma thylakoids on June 23. The number and volume of stoma were increased in 812S, while the chloroplast in leaves of 812HS appeared to be spherical on July 14. They displayed well-developed membrane systems with grana connected by stroma lamellae. The stacks of grana in the 812HS mutant appeared loose and lacking granal membranes and starch granules as compared to wild type on August 4.At senescence stage on September 4,the inner structure of the chloroplast was disorganized and the outer membrane of some chloroplasts was broken on.(2) The changes of photosynthetic performance of 812HS and 812S. The chlorophyll a fluorescence transient dynamics analysis indicated a timely and constant responsive pattern related to PSI and PSII. In the condition of strong light,the oxygen-evolving complex of 812HS was mainly damaged,thereby suppressed the electron transport chain. The energy dissipation efficiency (?D0) increased and the quantum yield of reduction of end electron acceptors of PSI (?R0) decreased. It was concluded that PSII in 812HS suffered serious damage in energy trapping and electron flow during photooxidation. The value of ABS/RC increased in 812HS on August 4 possibly because the apparent antenna size increased and a fraction of RCs was inactivated. The inactivation of RCs increased, which was considered to be a downregulation mechanism,to dissipate the excess of absorbed light. Dissipation indicators (?D0, DI0/RC, and DI0/CSM) increased so as to protect the leaves from photodamage, which resulted in a low level of electron transport per PS? RC(ETo/RC). The value of performance index (PIABS) and FV/FM showed that the photosynthesis of 812HS was inhibited. It was evidenced by changes of the photosynthetic parameters and primary photochemical properties.(3) The activities of RuBisCO were decreased, the activities of PEPCase and NADP-ME were increased in 812HS on Aug. 4, suggesting that it might be the compensation effect of C4 photosynthetic pathway on the degradation of RuBisCO activity or degraded prematurely. The ROS generation was mainly caused by abiotic stress, and the contents of O2·-, H2O2 and MDA in 812HS leaves increased gradually under strong light. The activities of SOD in 812HS increased on August 4, however,the rate of O2·- production in 812HS was much higher than that in 812S, probably due to the ability to remove O2·- by increased SOD activity exceeded O2·-. The activities of POD, CAT, APX, and GR increased slightly at senescence on September 15, which resulted in the accumulation of H2O2. The structure of the chloroplast membrane was damaged as well as the yellowing pathology confirmed these results. With the increase of light intensity, the xanthophyll cycle pool in 812HS gradually increased,indicating that the xanthophyll cycle library had been started. V decreased gradually,and the contents of A and Z increased, which indicated that 812HS could dissipate the excess excitation energy by thexanthophyll cycle to relieve the damage on the leaves.(4) Two-dimensional electrophoresis (2-DE) was used to compare all extractable rice leaf proteins from 812HS and 812S at the jointing stage on August 4. It was found that 53 differentially expressed protein spots were successfully identified,covering a wide range of biological functions and metabolic pathways. There were 32 proteins were upregulated and 21 proteins were downregulated in 812HS as compared to 812S (P < 0.05, and 1.5- to 4.9-fold). These differential proteins were classified into nine groups according to their function, and mainly related to photosynthesis(30.43%), respiration and energy metabolism (16.67%), resistance (17.39%), protein storage or degradation (13.04%) and some unknown proteins (13.04%). According to the subcellular localization analysis, the 53 proteins were mainly located in chloroplast (50%), mitochondria (5.76%), cytoplasm (11.54%), cell membrane(7.69%) and peroxisomes (1.92%), and 23.09% of proteins with unknown positional distributions. At the same time, in 812HS the RuBisCO large subunit, and RuBisCO large chain precursor were upregulation. RuBisCO activating enzyme was down-regulated. These results would lead to photosynthesis capacity decreased. While the activities of peroxidase and defense-related proteins, such as reactive oxygen species in 812HS were up-regulation, indicating that these proteins played an important role in rice photo-oxidation.(5) In the study of transcriptomics, we can select the differentially expressed genes between the samples by the two factors (|log2 (Fold change)|> 1) and significant level (q value <0.05). We found that there were 2540 differential genes in 812HS and 812S. Among these, 1342 genes were up-regulated and 1198 were down-regulated. A total of 546 Unigenes were involved in 21 KEGG metabolic pathways by Cuffdiff analysis. In these pathways, metabolic and genetic information processing were two enriched metabolic pathways, the pentose phosphate pathway, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, citric acid cycle glycolysis /sugar Carbon metabolism, amino acid biosynthesis, and metabolic pathways involved in metabolic pathways. Ribosome, transport, splicing, and processing of proteins in the endoplasmic reticulum were included in the genetic information processing class.In addition, some important pathways were also enriched, including organic systems,cell processes and environmental information processing. These results suggested that genes involved in these pathways played a key role in photooxidative conditions, and that these results provided valuable resources for studying the specific process,function and pathway of photooxidation. |
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