Roles And Mechanisms Of Arabidopsis Auxinresponsive SAUR6,SAUR14 And SAUR16 In Freezing Tolerance

Enhanced stress tolerance is commonly accompanied with growth inhibition in plants.Low temperature,as an important abiotic stress,is adverse to plant normal growth and development processes.However,the relationship between plant growth and freezing tolerance under cold stress and the underlying mechanisms are still unknown.In previous laboratory studies three early auxin-responsive SAUR(SMALL AUXIN UP RNA)family members in Arabidopsis thaliana,SAUR6,SAUR14 and SAUR16,were repressed by BZR1,a BR signaling transcription factor.Since auxin is a most important hormone that affecting plant growth and development,and BR and BZR1 play positively roles in freezing tolerance,it is probable that SAURs might take part in trade-off between growth and freezing tolerance at low temperature.In this work,the effects of the SAURs on freezing tolerance and the possible mechanisms were studied in Arabidopsis thaliana with wild type Col-0 and transgenic plants overexpressing At SAUR6,At SAUR14 or At SAUR16 at normal temperature(driven by driven by 35 S promoter)or induced by cold(driven by RD29 A promoter),by means of phenotype observation,physiological analysis and molecular biological technology.The results were shown below.1.Responses of SAURs to low temperature were analyzed by q PCR.The results showed that m RNA levels of SAUR6,SAUR14 and SAUR16 were significantly reduced in wide type Col-0 after cold treatment,indicating that cold stress represses the expression of SAUR6,SAUR14 and SAUR16.2.SAURs transgenic plants driven by two types of promoters were used in this work: 35S::SAUR can express in normal temperature,while RD29A::SAUR express only in cold conditions which are ideal materials for dissecting the roles of SAURs overexpression at low temperature.As compared with Col-0,the number of leaves and fresh weight decreased in 35S::SAUR16,and no difference in 35S::SAUR6 and35S::SAUR14 when grown at 22? for 4 weeks.Plant growth is poor at low temperature(4?).There was no difference in the number of leaves and fresh weight between all RD29A::SAURs and Col-0 that grown at 4? for 12 weeks.The results indicated the roles of SAUR6,SAUR14 and SAUR16 in growth are different even though they belong to the same gene family.3.Next,the effects of SAURs on freezing tolerance were investigated via survival rate and semi-lethal temperature(LT50).For survival rate analysis,35S::SAURs plants were cultivated at 22? for 3 weeks and RD29A::SAURs cultivated addition to 4? for4 days,respectively,then treated-5? or-11? for 1 hour.The survival rates after recovered 48 hours at 22? could reflect holistic freezing resistance in plant.For LT50 analysis,electrolyte leakage of leaves treated with a series of temperatures were measured.According to electrolyte leakage,the semi-lethal temperature could be calculated,reflecting physiologically freezing resistance of plants.Compared with Col-0 cultivated in the same conditions,the survival rates were significantly lower in SAURs overexpressing plants grown at normal temperature and low temperature,indicating that SAURs overexpression lead to lower freezing tolerance.However,higher semilethal temperature(LT50)which means lower freezing tolerance,were found only in transgenic plants with SAUR16 overexpression compared to Col-0 at 22?.No significant differences of LT50 were observed in all other transgenic plants as compared to Col-0 at 22? or 4?.Given a higher credibility of survival rate in revealing holistic freezing resistance in plant compared to LT50,it was deducible that SAUR6,SAUR14 and SAUR16 negatively regulate freezing tolerance in Arabidopsis.4.Osmolytes are important for plant freezing tolerance.To test whether SAURs reduce freezing tolerance by down-regulating osmolytes,the contents of soluble sugars and proline in transgenic plants and their changes after cold treatment were analyzed.Accumulations of total soluble sugar,sucrose,fructose and proline and the hydrolysis of starch in Col-0 were found after cold treatment.As for soluble sugars,compared to Col-0 grown at 22?,SAUR6 overexpression(driven by 35 S promoter)had no influence on sugars and starch,while SAUR14 and SAUR16 overexpression led to in increases in starch and sucrose,respectively.Compared to Col-0 under 4?: coldinduced SAUR6 overexpression(driven by RD29 A promoter)led to a decrease in total soluble sugar;no significant variations in sugars and starch contents were found in SAUR14 overexpressing plants;more starch was found in cold-induced SAUR16 overexpressing plants.For proline,compared to Col-0 in same cultivation conditions,more accumulation of proline occurred after constitutively overexpression and coldinduced overexpression of SAUR6;no proline changes were observed when SAUR14 overexpressed under normal temperature or low temperature;proline content increased in 35S::SAUR16 but decreased RD29A::SAUR16.These results illustrated that the osmolytes could be influenced by SAURs overexpression,but there was no consistently decreased trend of osmolytes contents among SAURs transgenic plants.Therefore,osmolytes might not play an important role in freezing tolerance related to SAURs.5.Cold stress caused secondary oxidative stress,and higher anti-oxidative capacity favors freezing tolerance while lower capacity may have an adverse effect.The results showed that cold treatment led increased peroxidase(POD)activity in Col-0,while the activities of catalase(CAT),ascorbate peroxidase(APX),glutathione reductase(GR)remained unchanged.Compared to Col-0 grown at 22?,SAUR6 or SAUR14 overexpressing plants showed no change in POD and CAT activities,while POD activity was increased in SAUR16 overexpression plants.Compared to Col-0treated with 4?,cold-induced SAUR6 overexpression caused lower POD activity and similar CAT activity;cold-induced SAUR14 overexpression caused higher POD activity and lower catalase(CAT)activity;lower CAT activity appeared in cold-induced SAUR16 overexpressing plants.Compared to Col-0 with same cultivation,the ascorbate peroxidase(APX)and glutathione reductase(GR)activities were unaltered by SAUR6,SAUR14 and SAUR16 overexpression at both normal and low temperature.Since these results reveal no consistent trend of antioxidative enzymes activities in SAURs overexpressing plants,it inferred that there is no direct correlation between antioxidant enzymes and freezing tolerance related to the SAUR genes.6.Cell wall is a major barrier to limit spread of ice crystals.As main components of plant cell wall,pectin,cellulose and hemicellulose contribute not only to cell wall plasticity but also to freezing tolerance.The results showed that pectin increased and hemicellulose decreased in Col-0 after cold treatment.Compared to Col-0 grown at22?,no significant changes in pectin and hemicellulose contents were found in transgenic plants overexpressing SAUR6,SAUR14 or SAUR16.Compared to Col-0 at4?,no significant variances in cell wall components were found cold-induced SAUR6 overexpressing plants,while increased pectin and decreased hemicellulose contents were found in SAUR14 and SAUR16 transgenic plants.Except for SAUR6,which overexpression in normal temperature resulted in an increase in cellulose,other SAURs had no effect on cellulose content as compared to Col-0 in the same condition.These results revealed that overexpression of SAURs might alter cell wall components,but their responses to cold were similar to those in Col-0,suggesting that cell wall components may not contribute to freezing tolerance related to SAURs.7.The expression of CBF(C-repeat Binding Factor)under cold conditions are vital for plant freezing tolerance.Expression analysis of CBFs and CORs(Coldregulated genes)is helpful for clarification the molecular mechanism of SAURs-related freezing tolerance.The results showed that CBFs and CORs were significantly induced by cold.Under low temperature,compared to Col-0,no difference of CBF1,CBF2 and CBF3 expression emerged when overexpressed SAURs.Compared to Col-0,at 4?,the expression of COR15 A and COR47 but not COR78 enhanced after SAUR6 or SAUR16 overexpression,but the expression of CORs didn't change when low temperature induced SAUR14 overexpression.The results indicated that the expression of CORs would be impacted partly by SAURs,but SAURs-related freezing tolerance might not relate to CBF-dependent pathway.In general,this work found cold stress represses the expression of SAURs and SAURs overexpression resulted in impaired freezing tolerance,indicating important roles of SAURs in plant cold response and inhibition of SAURs under cold stress is probably beneficial to enhance freezing tolerance.All results related to mechanism of freezing tolerance indicated that SAURs overexpression did not give rise to consistent reduction of osmolytes contents,antioxidant enzymes activities,cell wall components contents and the expressions of CBFs and CORs.The results suggested that even though belonging to the same gene family,SAUR6,SAUR14 and SAUR16 may have different roles in plants.Furthermore,SAURs may affect plant freezing tolerance via mechanisms differ from the “traditional” freezing tolerant mechanisms.In conclusion,above results clearly indicated that SAUR6,SAUR14 and SAUR16 play negative roles in freezing tolerance of Arabidopsis,but further study is need to reveal the related mechanisms.