Study On The Identification And Molecular Characterization Of LcIDL1 And Transcription Factor LcKNOX23 Involved In Fruit Abscission In Litchi

Litchi(Litchichinensis Sonn.),a famous tropic and subtropic fruit tree originated from China,is of great economic and nutritional value.However,the litchi tree is easily subject to massive physiological fruit drop,thus,addressing this problem is key to promote litchi industry development.To date,the information about the mechanism underlying the fruit abscission,especially for the molecular pathway,is limited.Previously our group has screened 3066 genes involved infruit abscission in litchi,including INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE(IDL)genes and KNOTTED-LIKE HOMEOBOX(KNOX)transcription factors,which have been demonstrated to play critical roles in organ abscission in Arabidopsis and tomato.Here,based on RNA-seq data regarding to the fruit abscission of 'Feizixiao' litchi we used a series of experimental technologies,including bioinformatics,transgenic plants and real-time PCR,to mine the key LcIDLs and LcKNOXs involved in control of the fruit abscission in litchi,and to reveal their functional characterization.The main results are as follows:1.Three LcIDLs genes(LcIDL1,LcIDL2 and LcIDL3)were cloned,among these genes LcIDL1 showed the closest homology and functioned similarly to Arabidopsis At IDA and was localized in cell membrane and cytoplasm.In-depth analyzes indicated that LcIDL1 likely played a key role in control of the organ abscission via regulating the p H value and cell wall remodelling genes in abscission zone cells.The main evidences included:(1)Real-time PCR analysis showed that the expression level of LcIDL1 accumulated gradually following flower abscission,and it was obviously induced by fruit abscission-promoting treatments(ETH and GPD).(2)Overexpression of LcIDL1,regardless of whether the LcIDL1 was driven by Ca MV35 S promoter or Arabidopsis At IDA promoter,induced the floral organ abscission in wild type and ida-2 mutant plants.(3)Previous work revealed that the cytosolic p H increase in AZ cells occurs concomitantly with the execution of organ abscission.We further observed the p H-value in the abscission zone of the floral organs using a p H-sensitive indicator,BCECF.The results showed that transgenic plants overexpressing LcIDL1 displayed stronger and earlier BCECF green fluorescence when compared with wild type.(4)In contrast to wild type,four cell wall remodelling genes(TCH4,GAD4,EXO and EXL1)were significantly increased in abscission zone cells of LcIDL1 overexpression line.2.Seven LcKNOXs genes(LcKNOX23,LcKNOX82,LcKNOX03,LcKNOX16,LcKNOX53,LcKNOX35 and LcKNOX28)were screened and cloned,among these genes LcKNOX23 showed the closest homology to Arabidopsis KNAT1/BP and tomato KD1,which have been found to play key roles in organ abscission.LcKNOX23 was localized in nucleus and possessed transcriptional repression activity.Further study indicated that LcKNOX23 likely function as a repressor in fruit abscission via regulating the regulating the p H value and cell wall remodelling genes in abscission zone cells.The main evidences included:(1)Real-time PCR analysis showed that the expression level of LcKNOX23 decreased gradually following flower abscission,and it was obviously inhibited by fruit abscission-promoting treatments(ETH and GPD).(2)Overexpression of LcKNOX23 in Arabidopsis,regardless of whether the LcKNOX23 was driven by Ca MV35 S promoter or Arabidopsis BP promoter,inhibited the floral organ abscission in wild type and bp-9 mutant plants.Furthermore,transgenic plants overexpressing LcKNOX23 displayed much weaker BCECF green fluorescence when compared with wild type.(3)Overexpression of LcKNOX23 in tomato significantly delayed the flower abscission and displayed weaker BCECF green fluorescence at the same time point after the flower removed than that in wild type.(4)Finally,RNA-seq analyzes showed that 213 genes were significantly altered following the whole abscission process after the flowers removed.In-depth analyzes revealed that 34 genes were regarded as the down stream genes regulated by LcKNOX23,which can be clustered into 12 different functional categories,such as plant hormone responsive genes,transcriptional factors and cell wall hydrolases.Interestingly,all the genes(8 genes)related to cell wall hydrolase were down-regulated.