Study On Response Mechanism And Resistance-Related Genes Expression Analysis Of Two Apple Rootstocks To Drought Stress

Drought is one of the most serious problems in sustainable agriculture, influencing plant growth and crop yield in agricultural production in many regions of the world. However, the mechanism research on drought influence of fruit trees is behind other crops. Apple is one of the world’s major fruit trees of cultivated species and one of the mostly cultivated deciduous fruit trees in northern China. Apple trees are propogated by crafting, therefore the resistance of apple trees to environmental stress depends on the resistance ability of rootstocks. In China, little has been done about the research of apple rootstocks resistance to environmental stress, thus understanding the resistance ability of apple rootstocks to drought stress and the physiological and molecular mechanism of their response to drought stress are becoming more and more important for genetic modification of apple trees under abiotic stress.In present thesis, two apple rootstocks with significant differences under drought stress were used as plant materials. Two-year old pot grown plants were treated to drought stress by withholding water. The accumulation of reactive oxygen species （ROS）, the oxidation of membrane lipid, the activities of antioxidant enzymes, the contents of antioxidants, the change of plant hormone and leaf anatomical structure as well as subcellular ultrastructure were studied, we were about to reveal the physiological mechanism of injury and the adaptive metabolism of apple rootstocks seedlings under drought stress, and we discussed the relationship between these changes and drought tolerance. To investigate how M. prunifolia, an excellent apple rootstock with strong drought tolerance, adapts to drought stress and to identify genes responsible to this important trait, we constructed a cDNA library with drought-treated leaves of M. prunifolia, using suppression subtractive hybridization （SSH） technique. A PCR-based screening method of SSH cDNA library then was used for identification of resistance-related expression sequence tags （ESTs）. To get an insight into their behavior and regulation by drought stress, we have characterized the expression profile of mRNA from these apple leaves and roots during drought treatment using real-time RT-PCR. To better understand their function at the molecular level in stress responses, we isolated and identified several novel full-length cDNA, using in silico cloning and reverse-transcription PCR. We characterized the nucleotide sequence and deduced amino acid sequence of these novel genes, and multiple alignment and phylogenetic analyses were made. To further characterize these genes, we also applied a model for predicting their homology of protein structure with other species, and their putative roles were discussed. Both organ-specific and stress-related expression profile of the genes were detected by quantitative real-time PCR and semi-quantitative RT-PCR. The main results are included as follows:1. The general responses of the two apple rootstocks to drought are different, and we identified significant differences in the adaptive metabolisms employed to achieve tolerance. Drought-tolerant M. prunifolia showed a better protection mechanism against drought damage by maintaining higher constitutive and induced activities of antioxidant enzymes as well as contents of antioxidants than drought-sensitive M. hupehensis.In this study, we compared responses of anti-oxidant defence system in the leaves of two contrasting apple rootstocks. Two-year old pot-grown plants were subjected to drought for 12 days by withholding water, until plants began to wilt. Under well-watered conditions, there are significant differences between the lipid peroxidation and relevant antioxidant parameters in both apple species during the whole control experiment. Water stress increased lipid peroxidation, hydrogen peroxide （H₂O₂） as well as superoxide radicals （O₂ ） production in leaves of stressed plants. Compared with M. prunifolia, M. hupehensis was more sensitive to drought stress, resulting in larger increases in the levels of H₂O₂, O₂ and MDA. The activities of antioxidant enzymes superoxide dismutase （SOD）, catalase （CAT）, ascorbate peroxidase （APX） and glutathione reductase （GR） displayed an initial increase with gradual development of water deficit, reaching the highest level after 6 days of withholding water, and then dehydroascorbate reductase （DHAR） and monodehydroascorbate reductase （MDHAR） activities peaking on Day 6 and 8, respectively. But peroxidase （POD） activity was substantially enhanced, and peaked on Day 10, and then decreased at the end of the experiment （Day 12）. In addition, the contents of antioxidant ascorbic acid （ASA） as well as glutathione （GSH） content peaked on Day 4 and 6, respectively. In responses to drought stress, the activities of SOD, POD, APX, GR, and DHAR as well as contents of ASA and GSH in M. prunifolia were more increased than that of M. hupehensis, but CAT and MDHAR activities changed. These results demonstrated that both antioxidant enzymes activities and antioxidant contents in leaves of M. prunifolia and M. hupehensis are increased in response to soil drought stress to minimize oxidative damage, and the former exhibited higher antioxidant ability and protective action during drought stress.2. We compared changes in lamina anatomical structure and subcellular ultrastructure in leaves of two apple rootstocks during drought treatment. Under drought stress, observation through method of light microscope showed that the palisade tissue thickness, leaf lamina thickness, and the ratio of palisade tissue to leaf lamina thickness （CTR） of two apple rootstocks decreased, but their sponge tissue thickness, leaf cuticle thickness and the ratio of sponge tissue to leaf lamina thickness （SR） increased. Changes of lamina anatomical structure in M. prunifolia was less than that of M. hupehensis.Ultrastructural observations on mesophyll cells showed, that under soil drought stress, drought cells exhibited obvious ultrastructural changes over control, and cellular damage rised with the increasing drought stress. Main ultrastructural changes included nucleus condensation, cytoplasm degeneration, decreased organelles, and damaged membrane systems, decreased the number of chloroplast, greater starch grain disappeared, deformation and vacuolization of chloroplasts, much less grana stacking, loosening and distortion of thylakoid, mitochondria swelling and cristae appear vague, and increased stomatal density, and so on. Our results demonstrated that M. prunifolia was able to maintain structural cell integrity longer than M. hupehensis under water stress.3. A cDNA library was constructed from drought-stressed and well-watered leaves of M. prunifolia, using suppression subtractive hybridization technique, in which the rate of recombination was 95% and the size of inserts ranged from 250 bp to 600 bp. Based on their blue/white coloring, recombinant colonies were then selected using PCR-based method of screening the SSH cDNA library. To identify which M. prunifolia genes are differentially expressed under drought treatment, positive clones obtained from the subtraction libraries were subjected to sequencing. BLASTN and BLASTP alignment analyses were used to search against database of NCBI, whose related-stress genes encoding proteins were involved in energy and metabolism, signal transduction and defense, protein metabolism, and no hit or unknown ESTs. Functional annotation and classification of these ESTs were made by GO method.Analysis of sequences from the positive clones picked randomly revealed that many drought stress associated genes, including cysteine protease, metallothionein, apoptosis facilitator, glycine-rich RNA-binding protein, chlorophyll a/b-binding protein, oxygen evolving enhancer protein, et al., were obtained. The successful construction of the subtracted cDNA libraries enabled us to identify new differentially expressed genes involved in the resistance mechanism of Malus plants. Some related-stress genes in leaves and roots of M. prunifolia were selected for further expression characterization under drought stress using quantitative real-time RT-PCR analyses. These results indicated that expression patterns of the genes revealed by real-time RT-PCR analyses were induced by drought stress, with different expression levels, inducing that these genes were involved in the adaptive mechanism to drought.4. Two apple drought-related protein genes, which named MpGR-RBP1 （HM042682） and MhGR-RBP1 （HQ380209）, were successfully identified from the drought-treated leaves of M. prunifolia and M. hupehensis, respectively, using screening library, in silico cloning, or RT-PCR methods. This is the first report of this class of proteins in Malus plants. The encoded proteins of two homologous genes, MpGR-RBP1 and MhGR-RBP1, consisted of 171 and 164 amino acids, respectively. Their deduced amino acids contain an amino-terminal RNA recognition motif （RRM） and a carboxyl-terminal glycine-rich domain, with structural similarity to a class of stress-induced GR-RBP proteins found in other plants. Phylogenetic analysis confirmed that both MpGR-RBP1 and MhGR-RBP1 proteins belong to the plant GR-RBP family, members of which play important roles in posttranscriptional regulation of gene expression under various stress conditions. The expression profiles of the two apple GR-RBP transcripts were detected by quantitative real-time RT-PCR and semi-quantitative RT-PCR. MpGR-RBP1 and MhGR-RBP1 were expressed in roots shoots, and leaves, and the expression level of MpGR-RBP1 in leaves is higher than that of MhGR-RBP1 under drought stress. MhGR-RBP1 in leaves was expressed both under drought and high salt stresses, with higher expression levels in the former than the latter. Additionally, MhGR-RBP1 was sensitive to ABA and JA treatment and down-regulated with different concentrations of ABA and JA, and indicated MhGR-RBP1 might play a role in the signal pathway of independent-ABA or independent-JA. And the putative roles of MpGR-RBP1 and MhGR-RBP1 proteins are discussed. These results indicated that MpGR-RBP1 and MhGR-RBP1 might be involved in apple rootstocks to stresses response.