Regulation Of Tomato Fruit Softening By LOB1 And Other Transcription Factors

Fruit softening is an irreversible feature accompany with fruit ripening which increases damage during transportation and shelf life.Extensive studies on fruit softening have been done on tomato as a fleshy fruit model and most of the studies focused on cell wall degrading enzymes and encoding genes.However,the fruit softening specific transcription factor was unknown.This study investigated the role of SILOB1 and SIMYB1/2 in transcriptional regulation of fruit texture and cell wall compound,also proved LOB1 is an important regulator of fruit softening.1.S1LOB1 is an important transcription factor that control fruit softening.Based on available RNA-seq database,we selected a fruit specific expressed gene,named SlLOB1,also shows gel-expressed ahead pericarp expression pattern,which is positively correlated with fruit softening.Furthermore,knockdown LOB1 results in remarkable slower softening rate during ripening;meanwhile,35S:LOB1 accelerate fruit softening.In contract with previously characterized ripening mutants(rin and Cnr)which exhibit minimal softening,LOB1 RNAi and OE fruits show normal ripening features,RNAi fruits even with additional enhanced lycopene accumulation.2.Exploration of targeting genes for SILOB1.We identified numerous cell wall DEGs(down-regulated)in LOB1 RNAi through transcriptomic-analysis,including cell wall degrading enzyme,such as pectate lyase(PL1-27),mannase(MAN),endoglucanase(CEL2),xylosidase(XY),expansin(EXP1)and other aspect of cell wall genes,like cell wall structural protein(AGP2),cell wall modifying enzyme(TBL),cell wall signaling candidates(LRR),also other cell wall related transcription factors.Ten genes were selected and validated the expression pattern by time-course Q-RT PCR in RNAi fruits,which is consistent with RNA-seq result and we also analyzed in OE plant.All the expression data indicate that LOB1 is a transcriptional activator,by controlling cell wall gene expression.3.Regulatory effects of SILOB1 on cell wall related genes.Combing the luciferase assay and gene expression in RNAi and OE plant,we thought that EXP1 is a direct target of LOB1 protein,whose promoter activity was triggered by LOB1 up to 67 times compared to control.The transcript of EXP1 was significantly inhibited in RNAi fruit and western blot showed that EXP1 protein was undetectable in RNAi fruit,meanwhile,EXP1 was induced in both OE fruit and young leave.We also characterized the cis-element by deletion of EXP1 promoter,which is TC-rich repeats.4.Identified two tomato MYB genes,SIMYB1/2,which are orthologs of the Arabidopsis MYB83/46.The expression levels of SIMYB1/2 and SICESA4/5/6 were analyzed in different tissues of tomato plants.Dual luciferase assay indicated the transcriptional function of SIMYB1/2 in regulating three CESA gene promoters.This work also showed the role of SIMYB1 and SIMYB2 in cellulose accumulation in transient and stable heterologous expressing in tobacco.