| Abiotic stress, particularly drought, is the primary cause of crop loss worldwide, and is responsible for an average yield loss of more than50%in the major crops. Drought tolerance mechanisms are typically under multigenic control, and thus difficult to select for in conventional breeding programmes. Thus the generation of crop varieties with improved salinity or drought tolerance has not proved to be very successful to date. An alternative approach, which avoids the prior need for gene identification and isolation, relies on the existence of naturally evolved stress tolerance in certain non-crop species. Although the majority of these species are typically sexually incompatible with even their related crop species, the technique of somatic hybridization can be exploited to transfer desirable traits. As it sidesteps the need for conventional crossing, somatic hybrids can then be used as parental material from which tolerance can be bred into crop varieties by conventional breeding and phenotypic selection.Improving the genetic adaptation of wheat (Triticum aestivum) to drought stress is one of the main objectives of regional breeding programmes in China. The feasibility of creating somatic hybrids between wheat and some of its wild relatives has been demonstrated for some years. Specifically, a number of introgression lines have been selected among the progeny of asymmetric somatic hybrids formed between the bread wheat cultivar Jinan177and the couch grass, salinity and drought tolerance were both evaluated in the field. One derivative in particular, named Shanrong No.3(SR3) proved, in addition, to be sufficiently improved with respect to drought tolerance that it has been successfully accepted as a commercial cultivar by the Shandong Crop Varieties Examining Committee (Lu-Nong-Shen-Zi No.[2004]030).The objectives of the present study were try to evaluate the osmotic-resistance mechanism of Shanrong No3under PEG-induced osmotic stress based on phynotype investigation, physiological indices determination, genome-wide transcriptional analysis, overexpression analysis of target gene in Arabidopsis and wheat. The main results are summarized as follows:1. Botanical traits surveyinga. More than80%of SR3plants survived completely from green dried state after rewatering and none was recovered for JN177in seedling stage. b. The results of coleoptiles and taproot elongation during germination stage treated with20%PEG-6000showed that, the coleoptiles length, classified as five grades in which the first grade behaved the mostly drought-resistant, presented normal distribution for SR3with the third grade the most, while it presented skew distribution for JN177with the fifth grade the most. The top root length of SR3was also significantly longer than that of JN177.c. The elongation of root in seedling stage treated with10%PEG-6000for one week showed that, the total root length, total root area and total root tips of SR3all significantly exceeded those of JN177.d. The water loss rate of excised leaf was significantly lower in SR3than in JN177at seedling stage.2. Physiological indices determinationMore suitable stress concentration screening of PEG-6000:Seedlings of SR3and JN177with three-leaf-stage were treated with15%,18%and20%w/v polyethylene glycol (PEG6000), respectively. Proline content and POD activity were determined after24h and48h of treatment. The results showed that the highest values for the two indices appeared under15%PEG treatment in Jinan177, while they appeared under18%PEG treatment in SR3. Thus18%of PEG was selected.Comprehensive physiological indices related to osmotic regulation and antioxidants were determined with and without18%of PEG stress. The results showed that SR3leaves were more effective in accumulating proline and soluble sugar than those of Jinan177, and their osmotic potential was significantly reduced in response to the applied osmotic stress. The mean increase in activities of SOD in the stressed root and leaf of SR3were higher than in Jinan177, as was the size of the AsA and GSH pool. Chlorophyll fluorescence parameters determined in flag leaf showed that Fv/Fm was higher in SR3than in JN177after drought stress. The ETR and Yield were always higher in SR3whether in normal or drought stressed condition.3. Comparative transcriptional profiling analysis both under control and PEG induced osmotic stressAffymetrix wheat genome array containing61127probe sets were firstly used to explore the transcription differences between SR3and JN177both under control and PEG-induced osmotic stressed at seedling stage. The results showed that, compared with JN177, a total of2195and 6390probe sets were differentially expressed in root and leaf of SR3respectively under control, while1187and1457probe sets were differentially expressed after PEG stress. GO molecular function analysis revealed that the differentially expressed probe sets mainly involved in the process related to catalytic activity, binding activity, molecular transducer activity and antioxidant activity etc. A few randomly selected genes were validated through RT-PCR. Among which the most interesting one was target Ta.23268.1, which was annotated as putative CEO protein and related to ROS signal pathway.4. The allelic variation of TaCEO1Four members of TaCEO1family were obtained from somatic hybrid SR3and named hTaCEO1-1, hTaCEO1-2, hTaCEO1-3and hTaCEO1-4. Homologous analysis of genome sequences of the genes from two parent lines and hybrid revealed that hTaCEO1-1, hTaCEO1-2, hTaCEO1-3came from the corresponding allelic variation in wheat parent JN177, but hTaCEO1-4arose from TaCEO1-1in parent wheat, which may be caused by replication, transfer or slippage of TaCEO1-1during somatic hybridization. Comparison between our cloned TaCEO1and deduced TaCEO1from reported ESTs in wheat cultivar Chinese Spring indicated that TaCEO1-1was not the homolog gene as reported, it was a new gene localized at chromosome4A. Possible modification site and functional motif in deduced protein of all TaCEO1homology protein deduced from nucleotide sequences were predicted online. The results showed that they all possess PARP-catalytic profile to the C-terminus. Besides, hTaCEO1-1still holds a WWE-domain and a nuclear localization signal profile.5. Functional characterization of h TaCEO1Two hTaCEO1with the significantly different sequences, hTaCEO1-1and hTaCEO1-2, were selected for functional characterization. The expression pattern of the two genes revealed that they were up-regulated upon PEG, salt and H2O2stress in SR3root. Function analysis in yeast mutant yap-1revealed that hTaCEO1-1could increase resistance of mutant yap-1toward oxidative-stress to a greater extent than hTaCEO1-2. hTaCEO1-1was then over-expressed in background of wild type Arabidopsis (Col-0). Functional analysis revealed that over-expression of hTaCEO1-1in Arabidopsis caused significant redox change based on the ratio of NAD+NADH and NADP+/NADPH. with the ratios higher in OE lines than in WT under control. The higher ratios of NAD+/NADH and NADP-/NADPH accelerated the substance and energy metabolism in OE lines, caused them grew more quickly and produced more ROS than WT. As signal molecules, the elevated ROS in OE lines caused down regulation of oxidative stress gene At2g21640and brought resistance of OE lines to a few abiotic stress elements such as H2O2, mannital, NaCl and MV.In summery, phenotypic and transcription profiles analysis revealed great differences between somatic hybrid SR3and its wheat parent line JN177. This could result from the introgression of Agropyron elongatum chromatin to wheat genome and gene shuffling in the process of somatic hybridization. Large numbers of differential expressed genes under PEG stress responsible for the constitutive difference between SR3and JN177, and only small part were responsive to PEG stress in SR3. Among these hTaCEO1-1studied in this work likely play an important role in the complex network controlling the osmotic tolerance of SR3. |
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