Functional Analysis Of A Tomato PSⅡ Light Harvesting Antenna Protein Gene LeLhcb2under Chilling Stress

Chilling is one of the most serious environmental stresses that affect the growth, yield and quality of crops severely. The combination of low temperature with low light also has the potential to enhance the amount of excess excitation energy, leading increased accumulation of reactive oxygen species (ROS) and inducing photoinhibition of photosystem II (PSII), even photooxidation. In the long term of evolvement, plants have evolved many kinds of defense mechanism to protect themselves. Light-harvesting chlorophyll-protein complex (LHC) had a function in adjusting the light energy distribution in the two photosystems, dissipating excess excitation energy and eliminating ROS to maintain the normal physiological function of cells. Therefore, it is of great theoretical significance and practical value to explore the mechanism of LHC proteins in plant chilling stress tolerance.In this study, a tomato LHCII (light-harvesting antenna system of PSII) protein gene LeLhcb2was cloned from tomato leaves. The roles of LeLhcb2in chilling stress tolerance were investigated here using sense transgenic tobacco. The major results are as follows:(1) We isolated the full length cDNA of the gene by RT-PCR using a pair of primers designed based on the chlorophyll a/b binding proteins sequence (GenBank accession number: AK246535.1). The full length of the gene is892bp and the ORF is798bp, encoding266amino residues. NCBI protein blast revealed that the isolated cDNA belonged to chlorophyll a/b binding protein superfamily, contained a chlorophyll a/b-binding domain. Amino acid sequence alignment between the gene and the12Arabidopsis chlorophyll a/b-binding proteins showed that this sequence shared the highest degree of sequence similarity to AtLhcb2(about88%). Moreover, amino acid sequence alignment between tomato LHCB2and LHCB2proteins from other plants (Thellungiella halophila, Chlamydomonas reinhardtii, Pisum sativum, Populus trichocarpa, Sedum alfredii, Selaginella moellendorffii) also showed a high degree of sequence similarity. Phylogenetic tree analysis by DNAman showed tomato LHCB2had the highest identities with Populus trichocarpa and Sedum alfredii. All the above results suggested that we have isolated the Lhcb2gene from tomato. We named it LeLhcb2.(2) p35S-LeLhcb2-GFP fusion protein was constructed and transiently expressed in Arabidopsis protoplasts derived from leaf tissue. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts, demonstrating the LeLhcb2subcellular localization in chloroplast.(3) Semi-quantitative PCR and Northern blot showed that the transcript of LeLhcb2was relatively most abundant in the leaves. qRT-PCR and Northern blot showed that the LeLhcb2expression was up-regulated by4℃and NaCl, but down-regulated by osmotic and oxidative stresses. Meanwhile, LeLhcb2was involved in the response to different semiochemicals.(4) Prokaryotic expression vector pET-LeLhcb2was constructed and transformed into E.coli BL21. The proteins induced by IPTG was purified and used to immunize the mice to obtain the antiserum, which was used in the following Western analysis. The titer of antibody was1:10000. The results of Western showed that LeLhcb2protein was induced by chilling and high-salinity.(5) The full-length ORF of LeLhcb2was subcloned into the expression vector pBI121under the control of the35S-CaMV promoter to form sense constructs. The Agrobacterium tumefaciens-mediated leaf disk method was used to generate transgenic tobacco plants. Kanamycin-resistant plants were detected by PCR, qRT-PCR and Western blot. The results suggested that over-expression lines were obtained.(6) The growth performance of WT and transgenic tobacco indicated no significant difference between the WT and transgenic plants under normal condition. However, under chilling stress, the transgenic young seedlings showed better survival and growth than WT. They showed higher fresh weight and chlorophyll content. The grown transgenic plants also displayed a more adaptive phenotype than WT under chilling stress. And they showed higher net photosynthetic rate (Pn) and less membrane damage. These results showed that overexpression of LeLhcb2enhanced transgenic tobacco tolerance to chilling stress.(7) Under chilling stress, the sense transgenic plants showed higher Fv/Fm, NPQ and D1protein content compared with WT, indicating that over-expression of LeLhcb2alleviated the photoinhibition of PSII.(8) Under chilling stress, the sense transgenic plants showed lower superoxide radical (02-) and hydrogen peroxide (H2O2) levels compared with WT. Furthermore, the relatively lower ROS levels were not considered to due to the higher activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD) in the transgenic plants under chilling condition.