Study On The Enhancement Of Drought Tolerance Of Ryegrass By Introduction Of The Glycine-methylation Biosynthetic Pathway Of Glycinebetaine

Ryegrass is one of the widely grown cool-season grasses in the world and used for forage or turf purposes. The two most important ryegrass species are annual ryegrass (Lolium multiflorum Lam.) and perennial ryegrass (Lolium perenne L.). Genetic improvement of ryegrass through traditional breeding has proven difficult because of its highly self-incompatible nature. Genetic transformation is a powerful tool to overcome these problems and contribute to plant improvement for certain traits. As the world is running out of water supply, drought stress is a severe environmental constraint to plant productivity in the world and ryegrass is drought-susceptible, improvement of drought tolerance is thus an important breeding objective for ryegrass. However, the study of the ryegrass drought tolerance genetic engineering is just at the beginning. Therefore, cultivating ryegrass with enhanced drought tolerance through biotechnology would have important economic and scientific benefits.The stress-resistance genes with self-owned intellectual property rights are crucial for improving plant stress tolerance by genetic engineering techniques. The transgenes for the glycinebetaine (GB)-biosynthetic enzymes were mostly from the pathway of dehydrogenation/oxidation of choline, although there have been many studies on GB-accumulating transgenic plants with increased tolerance to abiotic stress. Recently GB biosynthetic pathway from glycine was elucidated. But there are only a few reports on cloning and application of genes in this biosynthetic pathway. In this study two methyltransferase genes named ApGSMT2 and ApDMT2, which catalyze the glycine-methylation biosynthetic pathway of GB, had been cloned, broadening the gene resources for breeding GB-accumulating transgenic plants with enhanced stress tolerance. The functional analysis of the genes was carried out in the model plant tobacco. Then the genes were introduced into perennial ryegrass and annual ryegrass. The drought tolerance of the transgenic ryegrass plants was further studied.Cloning and functional analysis of ApGSMT2 and ApDMT2 genesBased on the sequences of ApGSMT (GenBank accession number:AB094497) and ApDMT (GenBank accession number:AB094498), two genomic DNA fragments were amplified from Aphanothece halophytica GR02 by PCR. The two DNA sequence are 798-bp and 834-bp long and contain an open reading frame (ORF), respectively. The sequence analysis and the in silico structure predictions suggested that one of the deduced amino acid sequences coded a glycine sarcosine methyltransferase homologue named ApGSMT2, and the other coded a dimethylglycine methyltransferase homologue named ApDMT2. Respective alignment of the deduced amino acid sequences with related proteins from other species revealed that ApGSMT2 shared 93% identity with ApGSMT,64% with EcGSMT,64% with AcGSDMT. ApDMT2 was 91% similar to ApDMT,50% to EcSDMT and 48% to AcGSDMT. At the nucleotide level, ApGSMT2 had only 83% sequence identity with ApGSMT and ApDMT2 had only 84% with ApDMT. ApGSMT2 and ApDMT2 proteins both contained the putative motifs (Motif I, Post I, Motif II and Motif III) that were referred to as methyltransferase signature motifs. The in silico structure predictions indicated that they both contained a mixed seven-stranded P-sheet (6↓7↑5↓4↓1↓2↓3↓) referred to as an "AdoMet-dependent MTase fold", which is used for identifying hypothetical Mtases.ApGSMT2 and ApDMT2 genes were introduced into E. coli BL21 (DE3). The E. coli cells co-expressing ApGSMT2 and ApDMT2 proteins could accumulate more GB, 1.3-2 folds higher than in the control cells. The increased level of GB in transgenic E. coli cells suggested that ApGSMT2 and ApDMT2 catalyzed GB biosynthesis from glycine. Tobacco is a GB non-accumulating plant. Heterologous expression of ApGSMT2 and ApDMT2 genes in transgenic tobacco resulted in GB accumulation (up to 0.4436μmol g-1 FW) and enhanced drought tolerance. Compared with the betA gene transgenic tobacco (named betA line), the ApGSMT2 and ApDMT2 genes transgenic plants (named GSD line) accumulated more GB (up to about 4.3-fold) and performed better under drought stress. The different levels of GB accumulation might be due to the different substrates, glycine and choline. Free glycine was abundant as a major amino acid in plant cells and free choline was limiting. Under 20% PEG, the seed germination rate of GSD lines was up to 65.2%, higher than that of betA line (50.9%), while the seed germination rate of the wild type were reduced to below 50%. The GSD lines tobacco seedlings also performed better than the betA line and wild type grown in vermiculite without water. The GB accumulation level (0.4436μmol g'FW) was still much lower than in natural GB accumulating species such as spinach (30-40μmol g"'FW), and physiological analysis of the tobacco in pots showed that under drought stress the GSD lines had higher relative water content, less cell membrane damage and better photosynthetic capacity than betA line and wild type. Therefore, in the GSD lines tobacco GB might maintain the integrity of membranes and stabilized PS II core complexes under drought stress, although GB could serve as osmoprotectants.In summary, two useful genes, ApGSMT2 and ApDMT2, which catalyze the glycine-methylation biosynthetic pathway of GB, had been cloned, broadening the gene resources for breeding GB-accumulating transgenic plants. It was also confirmed that when these two genes were transferred to tobacco, it accumulated more GB and had better drought tolerance than betA line, which catalyzes GB biosynthesis from choline. These results suggested that co-expressing ApGSMT2 and ApDMT2 for GB accumulation appeared to be a better choice for enhancing crop resistance to drought stress.Generation of transgenic ryegrass plants heterologously expressing ApGSMT2 and ApDMT2 genesFor genetic transformation of the monocotyledon, ryegrass, the ApGSMT2 and ApDMT2 gene were constructed into a plant expression vector. In this vector, the ApGSMT2 and ApDMT2 genes were under the control of maize ubiquitin 1 promoter, which has been widely used to drive constitutive transgene expression, respectively. And 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was the resistance gene for selection in plants. The ApGSMT2 and ApDMT2 genes which catalyzed the glycine-methylation biosynthetic pathway of GB, were introduced into perennial ryegrass (Lolium perenne L.) cv. Derby Xtreme and annual ryegrass (Lolium multiflorum Lam.) cv. Gulf by Agrobacterium-mediated shoot apical meristem transformation method. It was confirmed that ApGSMT2 and ApDMT2 genes had been integrated into the ryegrass genome and had expressed to different extent in different transgenic lines by southern blotting and RT-PCR analysis. For annual ryegrass, the progenies of the transgenic lines were be analyzed further and it was confirmed that the foreign genes had been transmitted to the progenies.Analysis of drought tolerance of transgenic ryegrass plantsIn this part three TO transgenic perennial ryegrass lines and three T2 transgenic annual ryegrass lines were used for drought tolerance analysis. For perennial ryegrass, eight tillers of each transgenic line and wild type (WT) were transplanted into pots filled with equiponderant homogenous soil. The tillers were hand clipped to leave a 5 cm stubble. After the transplanted tillers were established for 4 weeks, drought stress was imposed to them by withholding water. For annual ryegrass, seeds of the transgenic lines and WT were sowed in pots. After 5 weeks, drought stress was imposed to the plants by withholding water.During the drought treatment period the WT plants wilted earlier and displayed more severe stunted growth than the transgenic ryegrass plants. The transgenic ryegrass plants had much better developed root system and significantly higher biomass than the WT plants under drought treatment. After a long time drought treatment, all the plants in experiment severely wilted with roll folded leaves. However, the transgenic ryegrass lines recovered more rapidly and showed a higher survival ratio than the WT plants after re-watered. These morphological responses to drought stress suggested the enhanced drought tolerance in transgenic ryegrass plants.During the early 7-day drought treatment period, various physiological characteristics were measured to investigate the mechanism of the enhanced drought tolerance in transgenic ryegrass plants. Before stress there was no significant difference between transgenic ryegrasess and WT. On the 7th day of drought treatment, the GB content in the transgenic ryegrass plants was significantly higher than that in WT. And the GB content of the transgenic perennial ryegrass L3 (17.34μmol g-1 FW) was 2.19 folds higher than that of WT (7.91μmol g'FW). The GB content of the transgenic annual ryegrass L3 (6.12μmol g-1 FW) was 1.42 folds higher than that of WT (4.3μmol g-1 FW). It was found that after drought stress the GB accumulation levels were positively correlated with the expression level of the ApGSMT2 and ApDMT2 genes in the transgenic ryegrass plants. The leaf relative water content (RWC) of all plants decreased gradually, but the magnitude of decrease in leaf RWC in the transgenic ryegrass lines was significantly smaller than that in the WT plants. Correspondingly, the ion leakage and the MDA levels in the transgenic ryegrass plants were significantly lower than that in WT, suggesting less membrane damage in the transgenic ryegrass lines. The results implied that increased accumulation level of GB might help to maintain the stability of cell membranes. In addition, compared with the WT plants, the net photosynthesis (Pn) were higher in transgenic ryegrass plants, which might be partly ascribed to higher stomatal conductance (gs) under moderate drought stress. Increased GB content in transgenic ryegrass plant might protect the PS II core complex more effectively. Therefore, the Fv/Fm values of the transgenic ryegrass plants were measured and it was found they were higher than that of WT under drought stress. The transgenic ryegrass plants accumulated more soluble sugars than WT, which might be resulted from the better photosynthetic performance.There was a strong coordination among different amino acid biosynthesis pathways in higher plants and changes on a single amino acid could ripple the amino acid pool. Considering the transgenic ryegrass plants could consume glycine to biosynthesize GB, it is necessary to analyze the changes of free amino acid levels before and after drought stress in transgenic plants. It was found that there was no difference in the contents of each free amino acid between transgenic ryegrass plants and WT before drought stress treatment. On the 7th day of drought treatment the Gly, Ser and Thr contents were significantly lower in transgenic annual ryegrass plants than in WT, and only the Gly content in transgenic perennial plants was lower than in WT. The results suggested that considerable amounts of Gly were used to synthesis GB, and the reaction rate from Gly to Ser were elevated. Plants accumulated free amino acid, especially Pro, as osmotic protectants under drought stress. However, there was no significant difference in the total free amino acids contents and Pro contents between the transgenic ryegrass plants and WT after drought stress treatment. The results indicated that the enhanced drought tolerance in transgenic ryegrass plants was not related to the total free amino acids contents and Pro contents.To investigate the effect of GB accumulation on the gene expression in transgenic perennial ryegrass plants during drought stress treatment, several stress-related genes from perennial ryegrass were chosen according to some reports. The expression trends of some genes were not different between transgenic plants and WT. The genes were two homologues of DREB/CBF transcription factor genes, D1 protein gene and choline phosphate cytidylytransferase gene which catalyzes the choline biosynthesis offering substrate for GB accumulation. Aquaporins are water channel proteins and their protective role during drought stress is well known. Only two of the four selected aquaporin genes, LpTIR2:1 and LpTIR1:2, showed different expression trends in transgenic plants and might be induced by increased GB content under drought stress treatment.In this section of the studies, the glycine-methylation biosynthetic pathway of GB was introduced into ryegrass by transferring the ApGSMT2 and ApDMT2 genes for the first time. It was confirmed that heterologous expression of ApGSMT2 and ApDMT2 genes could increase GB accumulation level and enhance the drought tolerance in transgenic ryegrass plants. The physiological and molecular analysis revealed that GB might not only stabilize the integrity of the cell membrane and PSⅡcore complex, but also induce the expression of some stress-related genes to contribute to the enhancement of the drought tolerance.In conclusion, this study has cloned two useful genes, ApGSMT2 and ApDMT2, which catalyze the glycine-methylation biosynthetic pathway of GB, broadening the gene resources for breeding GB-accumulating transgenic plants. And some valuable ryegrass materials for ryegrass drought tolerance breeding have been created in this study. Moreover, the physiological and gene expression analysis under drought stress provides some important information for better understanding the molecular mechanisms of the transgenic ryegrass resisting drought stress.