Study On The Enhancement Of Abiotic Stress Tolerance Of Wheat Plants By Introduction Of Glycinebetaine-synthesis Pathway

Wheat (Triticum aestivum L.) is one of the most widely distributed and planted food crops in the world. With the continual increase of population, the deterioration of natural environment, the increasing soil salinization and water shortage, the contradictions between the increased equirement and decreased supply of wheat come into prominence. It is urgent to enhance the yields and abiotic stress tolerance of wheat nowadays.The traditional wheat breeding depends on the hybridization between cultivars, distant hybridization and mutation breeding to create new wheat varieties and germ plasm. With long years of hard work, the yields and quality of wheat have been improved significantly. However, the development of traditional wheat breeding has been limited due to the narrow germ plasm sources in the recent years. Although there are a large number of materials produced by distant hybridization, these materials usually have some disadvantages and need many generations of selection to be directly used for wheat breeding. The development and application of genetic engineering technology break the restriction of gene flow between species and could transfer exogenous genes into wheat and produced transgenic plants, which could be used to breed new varieties and develop the wheat breeding approaches. It is very important to enhance wheat salt and drought tolerance by using biotechnology.Due to the great difficulty of wheat genetic transformation, the huge genome and long experimental cycle, the progress of wheat genetic engineering was far slower than other crops such as rice, maize and cotton etc. It is an important subject to create salt-and drought-tolerant cultivars in wheat genetic engineering. In the study, we introduced genes of glycinebetaine (GB) biosynthesis into elite wheat varieties and carried out drought and salt tolerance studies based on the techniques of genetic transformation for wheat shoot apical meristem in our laboratory. The genes used in this study are betA [encoding choline dehydrogenase (CDH)] from E.coli, ApGSMT2 and ApDMT2 [encoding glycine sarcosine methyltransferase (ApGSMT) and dimethylglycine methyltransferase (ApDMT) respectively] from a halotolerant cyanobacterium(Aphanothece halophytica). Generation of transgenic wheat plants and expression analysis of the transgenesIn the study, three genes of betA, ApGSMT2 and ApDMT2 were introduced into three wheat varieties (Jinan 17, Jimai 19 and Jimai 22) by Agrobacterium mediated shoot apical meristem transformation. After selection of herbicide (the selectable marker genes are bar or epsp, which encode glufosinate-ammonium or glyphosate resistant enzyme respectively) and molecular identification (PCR, Southern blotting etc.),15 lines of betA transgenic plants from Jinan 17,27 lines from Jimai 19 and 27 lines from Jimai 22 of ApGSMT2 and ApDMT2 transgenic plants were obtained. The transformation frequency was 3.67-5.35%. Southern blotting analysis showed the foreign genes had been integrated into wheat genome with one or two copies and transmitted to the progenies stably. RT-PCR analysis indicated that the foreign genes in transgenic lines had different expression levels.Three homozygous transgenic lines (BL1, BL2 and BL3) in T2 generation and WT (wild-type, Jinan 17) were selected for betA gene expression analysis in seedlings under salt treatment. Real-time RT-PCR assay indicated there was no expression in the WT. Before salt treatment, betA transcripts abundance of different transgenic lines were about 1% of actin transcripts abundance, although the expression of betA was controlled by the constitutive Ubiquitin promoter which is a strong promoter in monocotyledon plants. After 12 days of 200mM NaCl treatment, the amounts of transcripts from betA in the lines BL1, BL2 and BL3 increased by 6.3,8.8 and 10.3 folds respectively comparing with themselves before the salt treatment. The GB contents of transgenic lines BL1, BL2 and BL3 were 116.5-134.9μmol g-1DW before salt stress, which were significantly higher than that of WT (106.02μmol g'DW). After 12 days of 200mM NaCl treatment, the GB contents of transgenic lines BL1, BL2 and BL3 were up to 179.7,281.2 and 363.0μmolg-1DW respectively, which were 1.1,1.8 and 2.3 fold of the WT (157.4μmolg-1DW). This suggested the increment of half-life of betA transcripts heightened the transcript amounts which led to more GB synthesis in the cells.Three T3 generation of homozygous transgenic lines (BL6, BL7 and BL9) and WT (Jinan 17) were selected for betA expression analysis under drought treatment at the stage of seedlings. The expression amounts of betA were also only about 1% of actin gene abundance before drought treatment. After 7 days of drought treatment, the expression levels of betA increased by 4.3,8.3, and 5.2 folds comparing with those before drought treatment respectively. The GB content measurement indicated that the GB contents of transgenic lines BL6, BL7 and BL9 were 82.6-97.1μmol g-1DW before drought stress, which were obviously higher than that of WT (72.1μmol g-1DW). After 7 days of drought stress, the GB contents of transgenic lines BL6, BL7 and BL9 were up to 194.1,281.9 and 250.0μmol g-1DW, which were 1.4,2.0 and 1.7 folds of WT (143.7μmol g-1DW). This indicated, as cellular osmoregulation substance and protective substance of macromolecular structure, the increment of GB contents was beneficial to maintain the normal function of cells under drought stress condition.The expression levels of ApGSMT2 in T1 wheat plants were analyzed by RT-PCR analysis. The results indicated the expression levels of ApGSMT2 were different between the transgenic lines. The expression level of A-19L4 was the lowest among the 5 transgenic lines from Jimai 19 while A-19L5 was the highest. The expression level of A-22L1 was the lowest among the 5 transgenic lines from Jimai 22 while A-22L4 and A-22L5 were the higher ones. GB contents of transgenic lines were significantly higher than that of WT (P<0.01, n=4). The GB contents of the 5 transgenic lines from Jimai 19 were 84.8-115.0μmol g-1DW, which were 17%-56% higher than WT (Jimai 19,72.2μmol g-1DW). The GB contents of the 5 transgenic lines from Jimai 22 were 27%-55% higher than WT (Jimai 22,74.31μmol g-1DW). It could be concluded that after introduction of ApGSMT2 and ApDMT2 from lower eukaryote into wheat, the GB accumulation levels could be increased by the prolonged half-life of betA transcripts. Analysis of salt tolerance of betA-expressing wheat plantsIn order to determine whether the expression of betA could improve the salt tolerance of wheat plants and investigate the mechanism of salt tolerance improvement, three homozygous transgenic lines (BL1, BL2 and BL3) in T2 generation and WT (Jinan 17) were selected for salt tolerance analysis. The emergence, seedlings and green up stages in early spring were sensitive stages of winter wheat to salt stress, and the salt tolerance was ultimately evaluated by the growth and economic output of the plants in saline soil. The study was focused on the salt tolerance at the emergence and seedlings stages of transgenic wheat plants and also investigated their salt tolerance in the coastal saline land.Seeds of homozygous transgenic lines (BL1, BL2 and BL3) and WT (Jinan 17) were sowed in sand pots, watered respectively with different concentrations of NaCl solution (0,100,200 and 300mM) to determine the germination rate and growth potential. The results indicated that the germination rates of transgenic lines were significantly higher than that of WT and the growth of transgenic lines were also better under salt stress conditions. Under 200mM NaCl conditions, the emergence rate of BL3 was 94%, which was 1.45 folds of WT, the salt injury of BL3 seedlings was the least among the 3 transgenic lines, and showed much better growth comparing to WT.The seedlings in sand pots from the three transgenic lines (BL1, BL2 and BL3) and WT (Jinan 17) were watered with Hoagland solution after emergence. When the seedlings reached the 5-leaf stage, the salt stress treatments commenced with Hoagland nutrient solution supplemented with NaCl. The concentration of NaCl was increased by 50 mM each day to a final concentration of 200 mM. The transgenic lines exhibited better development of shoot and root system compared with WT under salt stress conditions. After 12 days of 200 mM NaCl treatment, the shoot biomass of transgenic lines BL2 and BL3 were 23.7% and 33.8% higher than that of WT, and the root biomass of lines BL2 and BL3 were 18.9% and 34.1% higher than WT, respectively.Under 200 mM NaCl stress, the Na+contents of the root and leaves of transgenic lines and WT showed a increasing tendency, while the K+contents increased slightly in the first few days and then decreased. However, the transgenic lines accumulated less Na+ and more K+ compared with the WT under salt stress conditions. As a result, the K+/Na+ ratio was significantly higher especially in BL2 and BL3 than WT after 12 days of 200 mM NaCl treatment. During 12 days of 200 mM NaCl stress, the chlorophyll contents of all lines showed a little increase initially and then decreased, however, in the later days of salt treatment of 12 days, the chlorophyll contents of the transgenic lines were significantly higher than that of WT. The chlorophyll contents of BL2 and BL3 were 2 and 2.1 fold higher than WT respectively after 12 days of salt stress. The photosynthesis rate, stomatal conductance, intercellular oncentration and Fv/Fm ratio indicated that photosynthesis rate decreased evidently in the short time of 200 mM NaCl stress, which may be due to the decrease of stomatal conductance. In the later days of salt stress, the decrease of photosynthesis rate may be co-effected by of the decrease of stomatal conductance and the inhibition of the PS II complex activity. However, the photosynthesis rate, stomatal conductance, intercellular CO2 concentration and Fv/Fm of the transgenic lines always exhibited higher than those of WT, which lead to more synthesis of carbohydrate. This was the reason for the better growth of transgenic wheat plants under salt stress conditions.During 200 mM NaCl treatment, the solute potential (Ψs) of wheat leaves decreased with the prolonged time of salt stress. Moreover,Ψs of BL2 and BL3 decreased significantly than that of WT, which would be benefit to water retention and water uptake in the transgenic plants. There were higher soluble sugar and proline contents in transgenic wheat leaves compared with that of WT. These results indicated that transgenic lines BL2 and BL3 accumulated more soluble substance (including proline, soluble sugar, GB etc.) under salt stress conditions, which help to keep a lower cellularΨs and maintain cell normal metabolism such as photosynthesis and plant growth.Under salt stress, the MDA contents and the electrolyte leakage were lower in transgenic lines than in WT which showed that the degree of membrane damage in transgenic lines was lower, which displayed a negative correlation with the higher accumulation level of GB in the transgenic lines. This was also coincident with the higher PSⅡcomplex activity of the transgenic lines. These results suggested that the higher level of GB in transgenic wheat lines can enhance the protection to the biomembrane and the activity of biological macromolecule complex.In addition, it was noticeable that the transgenic lines exhibited better in the field evaluation in the saline land. The economic traits such as tillering number per plant, flag leaf area, grain yield per plant and yields of square meter of transgenic lines, especially BL2 and BL3, were significantly higher than those of WT. The transgenic wheat lines exhibited remarkably improvement of salt tolerance, which created new salt tolerant materials for wheat breedings.Analysis of drought resistance of betA-expressing wheat plantsThe emergence and young seedling of wheat plants are sensitive to water deficit. Drought could greatly decrease the germination rate, tillering number per plant and grain yields of wheat. The drought stress tolerance of three transgenic lines from Jinan 17 was investigated at germination and young seedlings stages.Seeds of three homozygous transgenic lines (BL6, BL7, BL9) from Jinan 17 in T3 generation and WT (Jinan 17) were germinated in petri dishes containing two filter papers moistened with MS salt solution supplemented with 0,10%,15%,20% and 25%(w/v) PEG-6000 respectively. The germination percentage was recorded daily and the maximum shoot and root length were measured. The results showed that the transgenic lines BL7 and BL9 germinated earlier than WT under osmotic stress of 25%(w/v) PEG-6000, and the maximum shoot and root length were significantly longer than that of WT.Wheat young seedlings suffered from sustaining drought treatment and their morphological changes were observed, finally the aerial part and root of the plants were weighed for biomass. The transgenic wheat plants grew better under drought treatment, with much better developed root system and significantly higher biomass compared with the WT. Different transgenic lines showed discrepant drought resistance, and the best one was BL7, after 28 days of drought treatment the biomass of aerial part and root from line BL7 were 79.8% and 38.5% higher compared with the WT respectively. Wheat seedlings at 5-leaf stage were subject to drought stress in soil pots for seven days, the WT plants showed disastrous water deficit and leaves wilted severely, whereas most of the transgenic plants grew feebly without serious symptoms from water stress, and the relative water contents (RWC) of transgenic lines were significantly higher than that of WT. After 7 days of drought treatment, the chlorophyll content, net photosynthesis, stomatal conductance and transpiration rate of BL7 and BL9 were 45.5% and 28.5%,44.7% and 28.2%,71.0% and 35.8%,46.9% and 40.0% higher than that of WT respectively. Under drought stress condition, the solute potential of wheat seedlings decreased greatly, and soluble sugar and proline increased, however, the solute potential of the transgenic lines decreased more than that of WT and the contents of soluble sugar and proline were higher in the transgenic lines. These results suggested that more solutes were accumulated in transgenic cells compared with WT, which would be beneficial to retain and uptake more water to maintain cell turgor in the transgenic plants than in WT under osmotic stress.After 7 days of drought stress, except transgenic line BL6, the cell electrolyte leakage and MDA level in the other two lines were significantly lower than those of WT (P<0.05, n=3), which suggested less membrane damage and lipid peroxidation occurred in these transgenic lines. In addition, the SOD and POD activity in these transgenic lines were also significantly higher than those of WT after drought stress for 7 days which demonstrated better anti-peroxidation ability in transgenic wheat under drought stress. Those results implied that the elevated accumulation of GB in vivo might help to maintain the stability of cell membranes and the activity of enzymes.To investigate the mechanism of the enhanced drought resistance in the transgenic wheat lines with more accumulation of GB, the contribution of total soluble sugars, proline contents and GB to osmotic potential were calculated in transgenic line BL7. The results indicated that the contribution of GB was the first, and soluble sugars was second, proline content played minor role for its low concentration although it rose greatly in drought stress treatment. Among the lines BL7 had maximal GB content and showed lowest solute potential under drought stress. These results indicated that GB might play more important role in osmotic adjustment in wheat than the soluble sugar and the proline. After re-watering for 7 days, the transgenic lines recovered more rapidly than WT, this suggested that the higher accumulation of GB provided more protection to the transgenic lines.Above all, this study has created some valuable materials for wheat drought and salt resistance breeding, and made a little contribution to wheat production and utilization of large area of arid and saline soils in China. Moreover, the study has provided some important information to better understand the molecular mechanisms that wheat plants resist drought and salt stress.The innovative points in the study:1) It was the first time to introduce heterologous GB biosynthetic pathway into wheat cultivars and obtained transgenic wheat lines with significantly increased GB contents. Genetic stability of transgenes expression was analyzed systematically and created new materials with enhanced salt and drought resistance.2) The growth, development and physiological changes of betA-expressing wheat plants were analyzed systematically under salt and drought treatment, the results confirmed that massive accumulation of GB in wheat could enhance stress tolerance under stress conditions. The massive accumulation of GB in wheat also could promote the root system development and enhance the capacity of cellular water-retaining, which help plant to maintain cell turgor and enhance photosynthesis along with plant growth.3) The results showed that the introduction of ApGSMT2 and ApDMT2 from Aphanothece halophytica into monocotyledon wheat could efficiently synthesize GB in wheat and produced good materials for wheat breeding. The materials had greatly improved GB content and normal growth.4) A series of ApGSMT2 and ApDMT2 transgenic wheat lines with glyphosate herbicide resistance under the control of different promoter combinations were obtained. And these lines could be used to investigate influences of different promoters on target gene expression and GB accumulation, and also be used in anti-herbicide wheat breeding.The biggest shortcoming in this study:there were no systematical genetic and physiological analysis to ApGSMT2 and ApDMT2 transgenic wheat lines, which greatly weakened the innovation and systematization of the study.