| Plant cell wall plays important roles in cold acclimation and freezing tolerance of plants. Plant cells increase their wall thickness and strength to reduce cell volume shrinking or prevent cell from deformation. These changes are fulfilled by increases in cell wall contents, such as cellulose, pectin, lignin, and glycoprotein contents. In plant cold acclimation, Pectin methylesterases (PMEs) (EC 3.1.1.11) activity was increased to de-esterificate pectins, allowed extracellular Ca2+ ions to cross-link the free carboxyl groups to form a semi rigid pectate gel, increased the stiffness of cell walls and resulted in an increase in cold and freezing tolerance of plant. Although PME transcription and activities have been reported to be up-regulated by cold stress.little is known about underlying mechanisms. To investigate whether brassinosteroids participate in regulating expression and activity of PME under chilling stress. We tested the activity and expression pattern of PME in Col-0 and BR-related mutants under chilling stress, the physiological effects of Arabidopsis under chilling acclimation on PME activity impaired, and the relation between chilling stress and BR signaling pathway by enzyme activity assay, semi-quantitative RT-PCR and western blot. All the results as following:1,Exogenous eBL treatments had a positive effect on enhancing PME activity. PME activity induced by exposing 1μM eBL continued to increase and reached a peak after 12 hours of eBL treatment..2,Chilling-induced increase in PME activity depends on BR signaling pathway. In our studies, PME activity increased under chilling stress in seedlings of Arabidopsis and bzrl-1D, a constitutive BR signaling activation mutant. Chilling-stress stimulation of PME activity not only was not found in bril-116 mutant, a null allele of BR receptor kinase BR11, but also resulted in decrease of PME activity.3,Chilling-induced expression of AtPME41 depends on BR signaling pathway. Semi-quantitative RT-PCR analysis confirmed that AtPME41 was up-regulated by BR. Our results of RT-PCR analysis also showed that the expression of AtPME41 was up-regulated by chilling stress, but this effect was impaired in bril-116. It was worth noticing that the peak of AtPME41 transcripts induced by chilling treatment (6 hours) was later than by eBL treatment (3 hours). 4,Our studies showed the physiological means of AtPME41 expression and brassinosteroids during cold acclimation. Chilling stress resulted in significant increases in ion leakage in pme41 and bril-116, compare with wild type. Low temperature treatment also led to obvious increases in extent of lipid peroxidation in wide type and bril-116. But the changes of MDA content in col-0 and pme41 showed the same trend before and after chilling treatment. These results suggested that decrease of BR singaling significantly decreased chilling telorance in Arabidopsis. Meanwhile, decrease of PME activity lead to increase in ion leakage, but not caused accumulation of MDA content, implying the protection of PME to cell member.5,Chilling stress induced BR signaling output. By analyzing the phosphorylation states of BR bio-marker BZR1, our results showed that chilling stress induced BR signaling output through BR receptor BRI1 pathway6,Our studies showed the relationship between NO and BR. NO acted downstream of, or at the same level as BR signaling pathway by analyzing the phosphorylation states of BR bio-marker BZR1.In conclusion, our results indicated that chilling stress regulation of PMEs in Arabidopsis was BR dependent, probably by regulating the expression of AtPME41. The decrease of PME activity increased ion leakage in the seedling of Arabidopsis under chilling stress. This result suggested the protection of PME activity to cell member. Chilling stress induced BR signaling output. Meanwhile decrease of the BR signaling significantly decreased chilling tolerance of Arabidopsis. These result suggested the important physiological means of brassinosteroids during cold acclimation.
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