The Effect And Mechanism Of Arabidopsis Xths And Pmeis In Freezing Tolerance

The plant cell wall is mainly a complex structure composed of cellulose,hemicellulose,pectin and structural proteins.The cell wall plays an important role in the plant response to abiotic stresses..Plant cell wall metabolism and related enzyme activities will change when response to cold-stress.The temperature change can dynamically adjust the pectin content and degree of methyl esterification of pectin,while the degree of pectin methyl esterification is co-regulation by pectin methyl esterase(PME)and pectin methyl esterase inhibitory protein(PMEI).Xyloglucan endo-?-transglucosylases/hydrolases(XTH)catalyzes the cleavage and re-ligation of xyloglucan molecules to modify the cellulose-xyloglucan complex structure of plant cell walls.It is found that the expression of XTH and PMEI genes will change when plant response to cold-stress.However,the mechanism of XTH and PMEI influence on plant cold resistance is not yet clear.In this paper,molecular genetics is used to study the effect and mechanism of XTH and PMEI on plant freezing tolerance in Arabidopsis thaliana.The results obtained in this study are as follows:1?35S:XTH5/7 and 35S:PMEI7/11 expression vectors were constructed.Then transferred to wild type Arabidopsis Col-0 obtained XTH5,XTH7,PMEI7,PMEI11 gene overexpression transgenic plants.The experimental results of plant survival under low temperature treatment showed that over-expressed XTH transgenic lines had a 25% increase in plant survival at-5°C(1h)compared with wild-type Col-0,and PMEI transgenic lines over-expressed at-5 °C(1h).The survival rate of plant under the condition decreased about 25% compared with the wild type Col-0.The results were statistically significant.It showed that over-expression of XTH gene could enhance the frost resistance of plants,while over-expression of PMEI gene reduced the frost resistance of Arabidopsis thaliana.2?It was analyzed whether the effect of anti-freezing of transgenic plants was related to the changes of cell wall cellulose,hemicellulose and pectin content.The results showed that overexpressing the XTH gene increased the cell wall hemicellulose content by about 28%(significantly different from the wild-type Col-0),and the cellulose and pectin content remained unchanged.This process may increase the rigidity of the cell wall.Thus,the rigidity and strength of the cell wall are enhanced to make the plant more resistant to stress.Overexpression of PMEIgene resulted in a slight decrease of pectin content in the cell wall,and there was a positive correlation between the increase of pectin content and plant frost resistance,which may be part of the reason for the reduction of frost resistance of oxPMEI genotype plants.However,the over-expression of PMEI gene in Arabidopsis caused the decrease in frost resistance.The over-expression of PMEI gene increased the degree of methyl esterification of cell wall pectin.The low methyl esterified pectin can release free carboxyl groups and Ca2+ cross-linked to produce hard coagulation.The glue enhances the rigidity of the cell wall,which makes the plant more resistant to stress.3?The effect of overexpression of XTH and PMEI genes on Arabidopsis root length was analyzed.The results showed that the root length of the oxXTHs genotype plants increased by about 12% compared to the wild-type Col-0 at 22°C for 1 week.At 8°C and 3 weeks of growth,the root length of oxXTHs genotype plants increased by 13% compared with wild-type Col-0,with significant differences.It shows that overexpression of XTH promotes Arabidopsis root growth at both low and normal temperatures.At 22 ? for 1 week,there was no significant difference between the root length of the oxPMEIs genotype and wild-type Col-0.At 8°C and 3weeks of growth,the root length of oxPMEIs genotypes increased by about 20% compared with wild-type Co I-0,with significant differences.Shows that overexpression of PMEI promotes Arabidopsis root growth at low temperature.4?Analyzed whether the effect of anti-freezing of transgenic plants was produced by the content of soluble substances.The results showed that the proline content in the four plant lines overexpressing the XTH gene was 45,65,67,and 84 ug/g,respectively,compared with the wild type Col-0(proline content 30 ug/g);The proline contents of 40 plants overexpressing PMEI gene were 40,36,37,and 19 ug/g,respectively,and there was no significant change in wild-type Col-0(proline content 30 ug/g);There was no significant difference between the total soluble sugar content in the transgenic lines and the wild-type strains;The oxXTH genotype of sucrose and fructose was slightly higher than that of oxPMEI genotype.The above results indicate that the difference in frost resistance between transgenic lines may be related to the change in osmotic pressure caused by changes in the content of soluble substances in cells.5 ? It was analyzed whether the effects of overexpression of XTH gene and PMEI gene antifreeze were related to changes in antioxidant enzyme activity.Antioxidant enzyme activity analysis showed that the activities of SOD,POD and CAT enzymes in the transgenic lines were basically the same as those in the wild-type Col-0 enzyme,with no significant difference.The effect of anti-freezing of transgenes was not directly related to the activity of intracellular antioxidant enzymes.6?The qPCR and antioxidant enzyme activity analysis results showed that the difference in frost resistance between the transgenic lines was not directly related to the activity of intracellular antioxidant enzymes,and was inconsistent with the CBF antifreeze signaling pathway.This paper analyzes the effect and mechanism of anti-freezing of two genes(XTH and PMEI)in Arabidopsis thaliana.It was found that when the expression levels of XTH and PMEI genes related to cell wall metabolism increase,they may affect plant freezing resistance by regulating cell wall composition and structure.