The Improvement Of Plant Acclimation To Abiotic Stress By Ameliorating Ub/26S System

Most of the plant growth and development are controlled by the selective removal of short-lived regulatory proteins. The Ub/26S proteasome proteolytic pathway is one of the most elaborate regulatory mechanisms in plants, which is very important in controlling and regulating different life courses. This fact was formally acknowledged by the awarding of the 2004 Nobel Prize in Chemistry to Aaron Ciechaover, Avram Hersko, and Irwin Rose for the discovery of ubiquitin-mediated protein degradation. In this pathway, Ub is attached to proteins destined for degradation; the resulting Ub-protein conjugates are then recognized and catabolized by the 26S proteasome. Increased expression of ubiquitin is evident during rapid growth and stress, consistent with the role of the Ub/26S proteasome pathway in removing short-lived regulatory proteins and abnormal polypeptides. Ubiquitin is present in all eukaryotic species examined, but little work has been done on genetic engineering of over-expressing ubiquitin in transgenic plants till now.In this study, the expression pattern of two wheat ubiquitin genes, Ta-Ub1 and Ta-Ub2, under water stress was detected; the sense vector with Ta-Ub2 (encoding monoubiquitin) and the antisense vector with Nt-Ub1 (tobacco ubiquitin gene) were constructed and transformed into tobaccos; the acclimation responses of sense and antisense transgenic tobaccos to abiotic stresses, including water desiccation, salt and low temperature stress, were investigated. The results showed that the expressions of Ta-Ub1 and Ta-Ub2 in wheat could respond to water stress; over-expression of sense Ta-Ub2 gene enhanced the tolerance of transgenic tobaccos to abiotic stresses, whereas antisense repression of Nt-Ub1 expression led programmed cell death and decreased the stress tolerance of the transgenic tobaccos. The main results are as follows:1. Transcript levels of Ta-Ub1 (accession number AY862401) mRNA in wheat coleoptiles increased gradually in response to water stress; that is, there was more Ta-Ub1 mRNA accumulation under severe water stress (30% PEG treatment) than that under moderate water stress (20% PEG treatment), and all of them were higher than that under normal water condition.2. The mRNA expression of Ta-Ub2 (accession number AY297059) was also regulated by water stress, which was increased in response to moderate water stress (20% PEG treatment), while decreased in response to severe water stress (30% PEG treatment), even less than that under well-watered condition.3. The sense vector with Ta-Ub2 was constructed and transformed into tobacco plants. In order to construct antisense vector of Ub gene, a partial ubiquitin gene in tobacco, Nt-Ub1 (accession number DQ830978) was cloned. The sense and antisense transgenic tobaccos were both confirmed by semi-quantitative RT-PCR, which indicating that transgenic lines with different Ub transgene levels were obtained.4. Immunology analysis with commercial ubiquitin antiserum indicated that neither over-expressing Ta-Ub2 (encoding a monoubiquitin) nor antisence repressing the expression of Nt-Ub1 (partial ubiquitin gene from tobacco) could significantly alter the level of ubiqutin pool (including free ubiquitin and higher molecular weight ubiquitin proteins) in transgenic tobaccos. But the acclimation of transgenic tobacoos to abiotic stress showed that both of sense and antisense transgenes were clearly biologically active, as judged by their effects on plant growth and stress tolerance, as shown below:(1) Sense transgenic seedlings over-expressing Ta-Ub2 gene grew better than controls under normal condition.(2) Under water stress and salt stress, over-expression of Ta-Ub2 resulted in enhanced germination speed and the growth rate of transgenic seedlings; meanwhile, in plant physiology characteristics, including water content and photosynthetic gas exchange parameters of transgenic plants were better than those of control plants under both drought and salt stress conditions. In a word, over-expression of sense Ta-Ub2 gene enhanced the tolerance of transgenic plants to multiple stresses (the tolerance to drought, salt and low temperature stress). Our results also indicated that over-expression of Ta-Ub2 gene enhanced the tolerance to stress combination (e.g. low temperature combined with drought stress, low temperature combined with salt stress) in transgenic tobaccos.(3) Antisense repression of tobacco Nt-Ub1 gene expression led to programmed cell death (PCD)-like Phenotype, e.g. leaf senescence early, more yellow leaves, lower pigment content and necrotic lesions in transgenic tobacco leaves. Under salt stress, growth status of antisense transgenic seedlings was worse than controls; water content and photosynthetic gas exchange of transgenic plants were worse than those of control plants, indicating that antisense repression of Nt-Ub1 expression decreased the tolerance to drought and salt stress in transgenic tobaccos.5. Staining of tissue samples with NBT to reveal superoxide anion (O2.-) level in tobacco plants showed that antisense transgenic tobaccos accumulate more O2.- than control seedlings; whereas, the sense transgenic tobaccos accumulated less O2.- than controls, suggesting that O2.- accumulation might be involved in the acclimation of sense and antisense transgenic plants to stresses. Perhaps ubiquitin/26S proteasome pathway was involved in removing ROS (reactive oxygen species).Farmers and breeders have long known that often it is the simultaneous occurrence of several abiotic stresses, rather than a particular stress condition in field, that is most lethal to crops. Researches have revealed that the response of plants to a combination of two more different abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the different stresses applied individually. Thus, besides multiple tolerance, tolerance to a combination of different stress conditions, particularly those that mimic the field environment, should also be the focus of future research programs aimed at developing transgenic crops and plants with enhanced tolerance to naturally occurring environmental conditions. This study suggests that over-expression of monoubiquitin enhanced not only the tolerance to drought, salt and cold stress individually but also to stress combination (the co-occurrence of different stresses) in transgenic plants, which proposing a new clue of developing plants with enhanced tolerance to diversiform adverse conditions as well as stress combination.