Mechanisms Of Brassinosteroids And Strigolactones In The Regulation Of Tomato Cold Tolerance

Cold stress seriously affects the growth and development,yield and quality of thermophilic crops like Solanum lycopersicum,and has very bad effects on the nutritional value and economic benefits of greenhouse vegetables in China.Therefore,it is of great significance to explore ways to alleviate cold-induced damage in vegetable crops to improve the yield and quality of vegetables.Brassinosteroids(BRs)and strigolactones(SLs)are important plant hormones,which are widely involved in plant life activities and stress responses.The involvement of BRs and SLs in cold responses has been reported,but the specific regulatory mechanism remains unclear.In this study,tomato,a thermophilic vegetable crop,was used to study the specific functions of BRs and SLs in the response to cold by means of genetics,molecular biology and plant physiology.The mechanism of BR-signaling transcription factor BZR1 regulating autophagy and its role in cold stress were elucidated.The roles and relationships of key components in cold resistance induced by SLs,such as C-repeat binding factor 1(CBF1),antioxidant enzymes and abscisic acid(ABA)were discussed.The main findings are as follows:1.BRs enhance tomato resistance to cold stress by inducing autophagy to promote the degradation of ubiquitinated proteins.Compared with the wild type(WT),dwf mutants were more sensitive to cold stress,showing increased values of relative electrolyte leakage(REL),decreased values of maximum quantum yield of PSII(Fv/Fm)and D1 protein abundance.In contrast,overexpression plants(DWFOE)and WT plants treated with exogenous BL(Brassinolide)reduced the sensitivity to cold stress,showing decreased REL values and increased Fv/Fm values and D1 protein abundance.These results confirmed that BRs could enhance tomato cold resistance.Meanwhile,dwf mutants had a higher accumulation of insoluble proteins and ubiquitinated proteins,while DWFOE or WT plants treated with exogenous BL had a lower accumulation of insoluble proteins and ubiquitinated proteins under cold conditions.Autophagy was then detected by a variety of methods,and we found that cold could induce autophagy formation,and the accumulation of autophagosomes was positively correlated with the BR levels.In addition,the accumulation of selective autophagy receptor NBR1 protein was increased in response to cold,and this effect was promoted by BRs,suggesting that BRs are involved in regulating the degradation of NBR1-mediated ubiquitinated proteins.In conclusion,both endogenous BRs and exogenous BRs can induce the formation of autophagy and reduce the accumulation of ubiquitinated proteins,thus alleviating the damage caused by cold stress in tomato.2.BZR1 mediates BR-regulated tomato cold resistance by transcriptional regulation of autophagy-related genes(ATGs)and NBR1.Compared with WT plants,the relative electrolyte leakage of bzr1 mutants was significantly increased,while the Fv/Fm value was significantly decreased,suggesting that the cold resistance was significantly reduced in bzr1 mutants.On the contrary,the relative electrolyte leakage of BZR1OE plants was significantly decreased,and the Fv/Fm value was significantly increased,which means that the cold resistance was significantly induced as compared to the WT.It is worth mentioning that exogenous BL treatment alleviated cold damage to WT and BZR1OE plants,however it did not improve the cold tolerance of bzr1 mutants.Then we found that compared with WT plants,bzr1mutants had lower accumulation of autophagosomes,lower abundance of Atg8-PE and NBR1 protein and lower transcription levels of ATG and NBR1 genes,and had no response to exogenous BL under cold conditions.In contrast,BZR1OE plants exhibited stronger autophagy signaling under cold conditions and the autophagy signaling was induced by exogenous BL.Meanwhile,according to the expression of ATG and NBR1 genes,ATG2,ATG6,NBR1a and NBR1b,which were not only regulated by BL but also closely related to BZR1 level in response to cold,were screened out.Cold and BL could increase the accumulation of BZR1 protein,meanwhile Y1H and Ch IP experiments demonstrated that enhanced BZR1 could bind to the promoters of ATG2,ATG6,NBR1a and NBR1b,thus inducing the formation of autophagy.These results suggest that BZR1 promotes the accumulation of autophagosomes through transcriptional activation of ATG2,ATG6,NBR1a and NBR1b,and mediates the regulation of BRs on tomato cold resistance.3.The role of ATGs and NBR1 on BR-induced cold tolerance in tomato.ATG2,ATG6,NBR1a and NBR1b genes were silenced by virus-induced gene silencing(VIGS).Compared with the control plants,the silenced plants were more sensitive to cold stress,with increased relative electrolyte leakage,decreased Fv/Fm value and increased accumulation of ubiquitinated proteins.Moreover,exogenous BL could not alleviate the damage caused by cold in silenced plants.Cold and BL enhanced the accumulation of autophagosomes in control plants,whereas cold-and BL-induced autophagosome formation was inhibited in silenced plants.In addition,cold and BL could significantly induce the accumulation of proteins involved in photoprotection(Psb S,VDE,D1)in the control plants,but these proteins were not significantly induced in the silenced plants.These results suggest that ATG2-,ATG6-,NBR1a-and NBR1b-mediated autophagosome formation and photoprotection are involved in the regulation of BRs on cold tolerance in tomato.4.SLs positively regulate tomato cold resistance in an ABA-dependent manner through CBF and antioxidant responses.Compared with WT plants,SL deficient mutants ccd7 exhibited reduced resistance in response to cold.The ccd7 plants showed more severe water loss and wilting,had higher relative electrolyte leakage,higher malondialdehyde(MDA)content as well as lower Fv/Fm and?_PSIIvalues.Importantly,exogenous SL analogues(GR24^5DS)mitigated these injuries.Meanwhile,compared with WT plants,the ccd7 mutants accumulated more ROS(H₂O₂and O₂^-)under cold conditions,and the activities of antioxidant enzymes and the transcription levels of CBF1 and antioxidant genes were lower.Interestingly,GR24^5DStreatment reduced ROS accumulation in both WT and ccd7 plants under cold stress,and enhanced the activities of antioxidant enzymes and the expression of CBF1 and antioxidant genes.In addition,ccd7 plants had less ABA accumulation and lower expression of genes involved in ABA biosynthesis and signaling under cold conditions.Exogenous GR24^5DStreatment enhanced ABA content in both WT and ccd7 plants,as well as the transcriptional accumulation of ABA biosynthesis and responsive genes.These results suggest that SLs mitigate cold injury through CBF,antioxidant metabolism and ABA pathways.We found that the ABA deficient mutant not(notabilis)had reduced resistance in response to cold,and exogenous GR24^5DScould not restore the phenotype of cold injury in not plants.Meanwhile,cold-and GR24^5DS-induced activities of antioxidant enzymes and expression of CBF1 and antioxidant genes were significantly inhibited in not plants.In conclusion,SLs rely on ABA to enhance tomato resistance to cold stress through CBF and antioxidant responses.