A Preliminary Study On The Effects Of SlCOLD1 On Cold Tolerance In Tomato

The yearly lowest temperature in 2/3 of the land area of the earth is below 0℃,and half of these areas have a lowest temperature below-20℃.During the plant`s whole life cycle,temperature stresses can affect plant`s growth and development.Among temperature stresses,cold stress can reduce plant growth,decrease yield and productivity,and extremely cold stress can cause plant death.By using quantitative trait locus（QTL）analysis,researchers in Chong Kang’s lab found a gene that can be used to improve cold tolerance in rice.They performed a QTL analysis for chilling-tolerance divergence in recombinant inbred lines（RILs）obtained from a cross between Nipponbare（Japonica）and 93-11（Indica）cultivars,and found a key gene,COLD1.They found that overexpression of COLD1^jap enhanced cold tolerance,while loss-of-function of COLD1^jap reduced cold tolerance in rice.On the other hand,overexpression of COLD1^ind in japonica rice reduced cold resistance,while overexpression of COLD1^ind in the cold1 mutants failed to improve cold tolerance.Based on sequencing of COLD1 in one hundred cultivated and wild rice,they found that the 187th amino acid of COLD1 in the rice from the lower temperature region is lysine,but in rice from higher temperature region was not.Thus,they concluded that the 187th lysine is necessary foe COLD1 to regulate cold resistance in rice.Due to its small genome and relatively easoier to generate trangenic plants,Tomato is an ideal plant for plant genetics,plant development,physiology and plant pathology studies.Tomato is originated in tropical and subtropical regions and is one of the crops that are highly sensitive to low temperature.Through sequencing analysis,we found that there is a COLD1 homologous protein in the tomato,but the 187th amino acids was not lysine.We named it SlCOLD1.Phylogenetic analysis results indicate that SlCOLD1 is more closely related to GTG1and GTG2 in Arabidopsis thaliana.RT-PCR results showed that the expression of SlCOLD1 is induced by low temperature,but not ABA.In order to study whether SlCOLD1 is involved in the regulation of cold resistance in tomato,we made SlCOLD1and SlCOLD1-S187K constructs through gene cloning and mutagenesis for protein localization and plant transformation.We found that SlCOLD1 was located in cell membrane,and 187th amino acid subsitution from serine to lysine in SlCOLD1 did not affect its localization in protoplasts.Through plant transforming,we obtained transgenic tomatoes that over expressing SlCOLD1 and SlCOLD1-S187K,respectively.We also generated slcold1 homozygous mutant by using CRISPR/Cas9-mediated gene editing.We found that there was no significant difference between slcold1 and wild-type in response to cold treatment.Considering that only two amino acids changed in SlCOLD1 in the slcold1 mutant,we cold not rule out the possibility that loss-of-function of SlCOLD1 may affect cold resistance in tomato.In addition to tomato plants,we also generated transgenic Arabidopsis thaliana plants overexpressing SlCOLD1-S187K.We found that the transgenic plants are morphological similar to the wild type plants,and no significant difference were observed between the transgenic and wild-type plants under cold stress conditions.We therefore concluded that overexpression of SlCOLD1-S187K may not affect cold resistance in Arabidopsis thaliana.