| As sessile organisms, plants suffer from divergent stress conditions continually, which may impair the growth, development or yields of plants. Rosa chinensis is one of the most important horticultural and ornamental plants in the world. In addition to the its commercial value, it also has some medicinal value. However, the growth of most R. chinensis varieties is limited by unfavorable environmental conditions, such as high temperatures and drought. Under stress conditions. these varieties do not blossom, but enter the dormancy condition directly. As the global warming becoming more severe today, research on enhancing the plant’s resistance to high temperature and other abiotic stresses is of significance.To isolate high temperature stress-related genes from R.chinensis, we compared the protein expression profile of two R.chinensis varieties, a heat-resistant variety. SM and a heat-sensitive variety, KP with or without 38 ℃ treatment for 3 h through two-dimensional (2D) electrophoresis gel. We chose three proteins which accumulated higher level in SM after heat shock. Based on mass spectrum, the three proteins were characterized as heat shock protein (HSP), eukaryotic translation initiation factor 5A (eIF5A) and late embryogenesis abundant (LEA) protein.In this study, we isolated the LEA gene from SM using homology cloning, inverse-PCR and 3’-RACE. The open reading frame (ORF) is 981bp, encoding 326 amino acid, which shared the most similarity with D95/Leal4 proteins according to the alignment with other LEA proteins. The hydropathy analysis of RcLEA suggested that it was a neutral protein, not a typical hydrophilic LEA protein. RcLEA was cytoplasm-localized and it was induced by high temperature. The expression of RcLEA was strongly induced after 38 ℃ treatment for 3 h in the SM variety, but it could not be detected with er without the treatment in KP.To reveal the function of RcLEA, it was transformed into Escherichia coli and Arabidopsis thaliana. The survival rate of E. coli in which RcLEA was expressed was higher than that transformed with the empty vector under high or low temperature treatment. Overexpression of RcLEA also improved its growth performance compared with the control strains when grown on medium containing NaCl or H2O2. These results indicated that the tolerance of E. coli with the overexpression of RcLEA, to high temperature, low temperature, NaCl or oxidative stress was improved.The transgenic Arabidopsis with overexpression of RcLEA showed better growth after high and low temperature treatment, produced more siliques and exhibited less peroxide according to 3,3-diaminobenzidine (DAB) staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in transgenic Arabidopsis. There were no differences of phenotype or electrolyte leakage between the transgenic and wild type Arabidopsis, no matter where they were cultured, on MS or in soil. RcLEA overexpressed in Arabidopsis increased resistance to high temperature or low temperature, but had no effect on resistance to NaCl or osmotic stresses.In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase (LDH) and citrate synthase (CS). It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation (BiFC) assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers.The data obtained from RcLEA expression in E. coli, Arabidopsis and the protective roles RcLEA played in vitro experiments suggested that RcLEA was mainly involved in high and low temperature stresses. Hence, there is a great chance that RcLEA could function in improving the adaptability of crops or flowers to the environment through genetic engineering.Another gene identified by our lab through 2D gel was a heat shock protein(HSP) coded gene. The ORF was cloned and it was turned out to be a small HSP, named RcHspl7.8. It was induced by abiotic stresses, such as high temperature and osmotic stress and confered resistance to a variety of stresses to E. coli, yeast and Arabidopsis. To further analyze the expression and regulation of RcHspl7.8, a 1,910 bp fragment of the upstream sequence of the RcHspl7.8 translation initiation codon were fused to a β-glucuronidase (GUS) report gene. The plasmid was transferred to Arabidopsis. The GUS could not be detected in any tissues without heat shock treatment. GUS staining was seen in all the organs of young seedlings after heat treatment. In floral, high level of heat-induced GUS staining was observed in filaments and the upper portion of the gynoecium. GUS activity was not induced in petals. Under heat treatment, both the upper and basal portion of the young siliques showed strong GUS staining. However, in mature siliques, no GUS staining was detected even after the treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected under other abiotic stresses.To identify the stress-related cis-acting elements in the promoter, some cis-acting elements of the RcHsp17.8 promoter were predicted using PlantCARE. Based on the prediction, five promoter deletion fragments which were lack of 5’sequences of the promoter were designed and fused to GUS. These constructs were transformed to Atabidopsis for analysis. Deletion experiments indicated that the region from-178 to-771 was essential the promoter’s response to high temperature, though there was no heat shock element (HSE) in the region, which indicated that except the HSE, the region around it was important for the promoter’s response to high temperature as well.GIGANTEA (GI) is a part of the photoperiod pathway to regulate flowering. It also has some effects on circadian rhythms. Besides, the tolerance of gi mutant to low emperature, oxidative stress or drought was different from that of WT. It has not been proved that how GI works in abiotic stress, thus there are needs to carry on further study to reveal the mechanism.gi-4 mutant was treated with various abiotic stresses and the mutant seedlings were sensitive to drought, high temperature, salt and osmotic stresses under long or short day conditions.The digital gene expression (DGE) was used to find out the differential gene expressed between WT and gi-4. Most of downstream genes of C-REPEAT-BINDING FACTOR 3 (CBF3), which is an important transcription factor functioning in abiotic stress response, were downregulated in gi-4. However, according to the quantitative RT-PCR, the expression level of CBF1, CBF2 or CBF3 was not lower in gi-4 than in WT. which implied that GI might regulate the downstream genes independent of CBFs, or GI had some effect on CBF proteins.What is more, the transcription factor AtWRKY44 was significantly downregulated in gi-4. It was proved to be directly regulated by GI through the GI-GR induction system and ChIP analysis. The phenotype that wrky44 was sensitive to NaCl indicated that GI might play roles in NaCl stress through WRKY44. |
