Study On Photoprotection And Related Gene Function Of Phyllostachys Edulis

Photoinhibition is a common phenomenon in plants under high light. There are a variety of photoprotection mechanisms in plants, among which the xanthophyll cycle is one of the important protective mechanisms in response to photoinhibition. Zeaxanthin epoxidase(ZEP) and Violaxanthin de-epoxidase(VDE) are two key enzymes involved in xanthophyll cycle, which can catalyzed the inter-conversion of the violaxanthin(V), antheraxanthin(A) and zeaxanthin(Z). Based on understanding of the characteristics of photoinhibition under high light and the roles of xanthophylls cycle in protecting photosynthesis apparatus from the damage of excessive light in moso bamboo(Phyllostachys edulis) leaves, we isolated and characterized zeaxanthin epoxidase gene(Pe ZEP) and violaxanthin de-epoxidase gene(Pe VDE) of moso bamboo. In addition, functional analysis of Pe PsbS gene and their promoters were also conducted. The main results are as follows:1. The characteristics of photoinhibition of moso bamboo under high light and the roles of xanthophylls cycle in protecting photosynthetic apparatus from the damage of excessive light were investigatied by using chlorophyll fluorescence techniques. Our results showed that the value of maximum photochemical efficiency(Fv/Fm) decreased significantly(p<0.01) and that of non-photochemical quenching(NPQ) increased in leaves of moso bamboo exposed to high light or a natural high light condition at noon in summer. However, the Fv/Fm value could be restored in darkness or in the afternoon with the decreased intensity of sunlight. Dithiothreitol(DTT) is one of Xanthophyll cycle inhibitors which can suppress the activity of violaxanthin cyclooxygenase. The value of NPQ was inhibited in the leaves of moso bamboo treated with DTT under high light, while the values of Fv/Fm and qP were dropped significantly. These results suggested that xanthophyll cycle play an important role in photoprotection of moso bamboo.2. For the screening of related genes involved in photoprotection of moso bamboo, three samples of leaves treated with high light for 0 h(CK), 0.5 h(0.5H), and 8 h(8H) were respectively selected to perform further high-throughput RNA sequencing(RNA-Seq), on the basis of bamboo leaves Fv/Fm and trends of NPQ under high light stress as well as the light irradiation time of natural environment. As a result, 1293 differentially expressed genes were detected, including some up-regulated genes such as OHP, SEP, Elip and Psb S. ZEP and VDE are two key gene involved in xanthophyll cycle, which were also up-regulated. This result provided a basis for further study on the genes related to photoprotection of moso bamboo.3. A full length cDNA of Pe ZEP was isolated from moso bamboo by RT-PCR and RACE methods, and named as Pe ZEP(Gen Bank No. KP274885). The full length cDNA of Pe ZEP was 2532 bp, encoding a putative protein of 670 amino acids. Sequence analysis showed that PeZEP had similar characteristics of ZEPs in the other species, which had high homology with those ZEPs from gramineous plants such as Oryza sativa(more than 90%). The tissue-specific expression of Pe ZEP showed that its transcript was detected in roots, stems, leaves and leaf sheaths, with the highest level accumulated in leaves. The expression of Pe ZEP was induced by high light and inhibited by low temperature(4 ℃) and exogenous ABA. In addition, overexpressing Pe ZEP increased the sensitivity of transgenic Arabidopsis to high light besides the tolerant ability to drought.4. The expression of Pe VDE in the leaves of moso bamboo under different stress conditions were studied by using real-time quantitative PCR. The results indicated that Pe VDE in the leaves of moso bamboo was increased under the treatments of high light, high temperature(42℃) and drought stress. However, it was firstly induced by low temperature stress(4℃) and exogenous ABA in the short term, and thereafter significantly inhibited with prolonged treatment. The function of Pe VDE was studied using an Arabidopsis mutant npq1 with vde deletion. The functional heterologous complementation demonstrated that Pe VDE could rescue NPQ of Arabidopsis npq1 mutant. In addition, overexpressing Pe VDE could increase the value of NPQ and the defense capability of photoinhibition in transgenic Arabidopsis under high light and low temperature stresses.5. There are at least two homologous genes of Psb S in moso bamboo, such as Pe Psb S and Pe Psb S1. The ORF of the two genes were 810 bp and 807 bp, and encoded proteins with 269 and 268 amino acids respectively, which shares 91.8% sequence identity. Pe Psb S and Pe PsbS1 had the same structures with four exons and three introns. Expression analysis showed that Pe Psb S and Pe PsbS1 also had similar expression patterns, which were expressed mainly in green organs and were induced by high light. The function of Psb S was studied using an Arabidopsis mutant npq4. The functional heterologous complementation demonstrated that both Pe Psb S and Pe Psb S1 could rescue NPQ of Arabidopsis npq4 mutant. In addition, the overexpression of Pe Psb S or Pe Psb S1 could increase the values of NPQ and Fv/Fm in transgenic Arabidopsis under high light.6. The promoter activity of Pe PsbS and Pe PsbS1 was analyzed by transient expression method with the fusion of GUS reporter gene. On the basis of moso bamboo genome, primer pairs were designed for the sequences of Pe PsbS and Pe Psb S1 promoter, which were isolated respectively. The sequences were 2373 bp and 1500 bp separately upstream of translation initiation codon ATG, respectively. Sequence analysis showed many cis-acting elements involved in light responses were present in the two promoters. The two promoters were fused to the reporter gene GUS and transformed into tobacco for transient expression, respectively. GUS histochemical staining analysis showed that both promoters could drive the expression of GUS gene in tobacco leaves, indicating they both had biological activity.This study laid a foundation for us to better understand the photoprotection mechanisms in moso bamboo, and provided certain basis for us to explain why the environmental adaptability of bamboo widely distributed in nature, which also provided a good reference for the development and utilization of bamboo gene resource.