| As a monocot model plant, rice is one of the most important crops in the world. In this study, we mainly elaborate the function of OsLIR1 in maintaining the stability of chloroplast and the detailed mechanism, and elucidate the roles of OsEXPB2 in rice root development.Chloroplasts are the main organelle fulfilling the photosynthesis in the green plants. Light-harvesting complexes bound to the chlorophyll molecules form the pigment-protein complexes in the thylakoid membranes, which absorb the sunlight and transform it into the form of ATP. At the aging period of leaf, light-harvesting complexes separate from chlorophyll and then chloroplasts are degraded. Finally the leaf blade turns yellow gradually and nutrients in leaf transfer from the old leaves to fresh leaves and seeds. Premature leaf senescence severely affects the photosynthetic efficiency and nutrients transformation, which ultimately lead to the repression of plant growth. Therefore, the mechanism analysis of the role of light induced genes in maintaining the stability of chloroplast in rice is significant for further understanding the function of chloroplast. Meanwhile, this study will provide valuable gene sources for rice breeding in the future.OsLIR1 is a light-induced gene, which encoding a 218-amino acid protein, with a molecular weight of 13.317- kDa. The function of OsLIR1 was systematically investigated through the RNA interference(RNAi) and protein-protein interactions.The main resulted achieved were as follows:① OsLIR1 is a chloroplast protein and may locate in thylakoid lumenWe constructed the 35s::OsLIR1-GFP fusion expression vector, and transformed it into the tobacco protoplasts. The transient expression of GFP was observed by using the laser scanning confocal microscope. The result showed that Os LIR1 is a chloroplast protein. Analyzed by plant plasid online database(PPDB), the OsLIR1 sequence has 59% similarity to the light regulated protein AT3g26740.1 from Arabidopsis thaliana that existes in the thylakoid lumen. Furthermore, analyzed by ChloroP combined with TMHMM server 2.0 online software showed that the N-terminal of OsLIR1 protein contains 35 aa chloroplast transit peptide(cTP) and it does not belong to transmembrane proteins. Therefore, we speculate that Os LIR1 may be a thylakoid lumen protein. In addition, online analysis revealed that Os LIR1 protein interacts with the typical proteins which contain stress-response domains. These results suggest that OsLIR1 play crucial roles in growth and development of rice plants.② OsLIR1 is photoperiodic and rhythmic, specifically expressed in green tissues and responses to stress treatmentsWe measured the expression levels of OsLIR1 by quantitive real-time PCR(qRT-PCR) during the 3 d period: 12 h constant light followed by 12 h constant dark or, 24 h of light followed by 24 h dark. The results showed that expression of OsLIR1 is under photoperiod and circadian rhythm in rice. Furthermore, qRT-PCR was used to analyze the expression levels of OsLIR1 in the various tissues of Nipponbare(japonica), including the leaf blade, leaf sheath, stem and root at tillering stage. The results indicated that OsLIR1 mainly expressed in green tissues, of which, most in the leaf and few transcripts in the root. Under abiotic stress treatments, the mRNA levels of OsLIR1 were significantly increased under low temperature and UV irradiation, and greatly suppressed under condition of high concentration NaCl.③ OsLIR1 knockdown inhibited plant growth and changed the structure of the chloroplastOsLIR1- knockdown transgenic rice lines exhibited a variety of phenotypes, such as repression of plant growth, premature leaf senescence, abnormal spike development and low seed-setting rate. Compared with wild type, the chlorophyll content and photosynthetic efficiency of OsLIR1- knockdown transgenic lines were decreased significantly. Interestingly, the photochemical efficiency in terms of the values of maximal photochemical efficiency of photosystem II(PSII)(Fv/Fm) and effective quantum yield of PSII(ΦPSII) was not changed significantly compared with that of wild type. The chloroplast ultrastructure showed that the distribution of thylakoid membrane was in disorder and the thickness of grana became thinner in the OsLIR1-knockdown transgenic lines. The transcription levels of the key genes involved in the chlorophyll degradation pathway were significantly up-regulated, while the expression levels of the relative genes of chloroplast were decreased. Simultaneously, the thylakoid membrane complexes were analyzed by using BN-PAGE. The results showed that the super-complex of photosystem II was dissociation and disappeared in OsLIR1-knockdown transgenic lines compared with the counterparts of wild type. These results demonstrated that Os LIR1 might directly affect the stability of photosystem II supercomplexes and change the chlorophyll content, which finally result in the degradation of the chloroplast.④ OsLIR1 is necessary to maintain the stability of thylakoid membrane during dark-induced leaf senescenceIn the dark-induced senescent leaves of OsLIR1- knockdown transgenic rice lines, compared to those of wild type plants, the chlorophyll content, maximum efficiency of PSII(Fv?Fm) and quantum yield of PSII(ΦPSII) significantly decreased, and the relative mRNA levels of the major enzyme genes involved in chlorophyll degradation pathway were up-regulated. Furthermore, the array of grana stacks and inter grana in the ultrastructure of chloroplast became naturally disordered and were dramatically reduced. Meanwhile, thylakoid membranes complexes and photosynthesis-related proteins were acceleratedly degraded, respectively. These results showed that OsLIR1 is required for maintaining the function of chloroplast under dark-induced leaf senescence.⑤ OsLIR1 interacted with CP29(photosystem II light-harvesting complex protein, Lhcb4)We constructed the yeast two hybrid cDNA library of leaves from Nipponbare(japonica) at different growth stages. Eight candidate proteins including CP29 were obained from yeast two-hybrid cDNA library by using Os LIR1 as bait. Co-transformation of yeast strain Y2 H verified the OsLIR1 interacted with CP29(O. sativa, light-harvesting complex Lhcb4) of photosystem II. Furthermore, BiFC plasmids were constructed and transformed into the epidermis cell of tobacco. The YFP yellow fluorescence existed in the chloroplast of epidermis cell containing the OsLIR1 and CP29 plasmids. In addition, pull down experiment also confirmed that Os LIR1 interacted with CP29. These results of OsLIR1 interacting with CP29 elaborate that the OsLIR1 plays an important role in maintaining the ability of the photosystem II supercomplex stability and explain, to a degree, the reason of leaf premature senescence in OsLIR1-knockdown transgenic plant.In brief, these data showed that Os LIR1, as a light induced protein, affects the structure and function of chloroplast.As the endogenous regulators, expansins generally play important roles in pant cell wall loosening. Nevertheless, little is known about the function of expansin genes in rice. In this study, the function of a β-expansin gene was analyzed through transgenic technology in rice, and these results were as follows:① Multiple sequence alignment revealed that the N-terminal of OsEXPB2 shares six discontinuous Cysteine(Cys) residues and and the C-terminal shares four intermittent Tryptophan(Trp) residues. Phylogenetic analysis showed that OsEXPB2 falls into a branch with other β-expansin genes from different species.② The 35S::OsEXPB2-GFP fusion expression vector was constructed and transformed into the onion epidermal cell. The confocal microscope revealed the signal of GFP fluorescence under plasmolysis. Subcellular localization assay revealed that OsEXPB2 was localized in cell wall.③ Analysis of spatial- and temporal-expression patterns demonstrated that OsEXPB2 was predominately expressed in rice root, and it was up-regulated by abiotic stresses, such as phosphate(Pi) or Fe deficiency, while was repressed by ABA treatment.④ The clear difference was observed between OsEXPB2 RNAi transgenic lines and WT in the root system architecture and plant height. The suppression of OsEXPB2 resulted in a visible alteration of the width of the leaf blade. Anatomy analysis found that the cell size of root cortical cell in OsEXPB2 suppressed lines was significantly smaller than those of wild-type plants. Furthermore, Cryo-SEM analysis showed that the quantity of root hair seems to be suppressed in RNAi lines.All these results indicate that OsEXPB2 is a root-dominant gene with a key role in root-hair formation and has the potential to be utilized in transgenic root breeding to improve abiotic stress tolerance. |
PDF Full Text Request