Transcriptome Profiling Of Genes Involved In Ethylene-induced Fruit Abscission, Cloning And Characterization Of Ethylene-regulated Genes In Citrus Sinensis

Abscission is an important determinant of citrus yield. Ethylene is a major abscission hormone that regulates many biological processes involved in abscission such as cell division, cell expansion and cell wall decomposition. The biosynthesis of ethylene has been elucidated. Previous researches indicated that two ethylene production systems, systemⅠandⅡ, exist in both citrus fruit and leaves. During the first phase (3～5d in mature leaves and 6～7d in young leaves, after detachment)systemⅠis operating. During the second phase, which started 5d and 7d, respectively, in young leaves and mature leaves after harvest, systemⅡis initiated and ethylene evolution becomes autocatalytic and climacteric-like, which is typical in systemⅡ. Mature citrus fruits, which are generally considered as non-climacteric, only exhibit systemⅠ-like behavior, and respond to exogenous ethylene by undergoing ripening-related pigment changes and accelerated respiration. Young citrus fruitlets exhibit a climacteric-like rise in ethylene production after detachment, indicating an autocatalytic systemⅡ-like ethylene biosynthesis.To explore the mechanism of ethylene action in the abscission of mature fruit, changes in transcriptome in the abscission zone of mature fruit during ethylene-induced abscission were studied. Several highly ethylene-regulated genes, CsPRP4,CsERF1,CsUnknow and CsCysP, were full-length cloned. Bio-informatics researches, together with quantitative RT-PCR studies on their expression changes, were performed on these genes to probe their possible roles in ethylene-induced abscission. Construction of overexpression and RNAi vectors was conducted for these genes, and transformation was done via agrobacterium-mediated transformation using the constructs and sweet orange explants. Regenerated antibiotic buds were obtained and some of them were successfully grafted.1. Profiling genes involved in ethylene-induced abscission in mature citrus fruitEthylene responsive genes in mature Valencia orange fruit abscission zones were analyzed by using Affymetrix GeneChip citrus genome array. Fruit were treated with 20μL/L of ethylene for 0,4 and 24h and RNA was extracted from abscission zones and subjected to genechip analysis. GCOS software was used to analyze the original genechip hybridization data. RMA (robust multiarray analysis) was used to filtrate the unreliable dada. Of the 30395 total probe sets that exceed the probe pair threshold on the microarray,20639 detected changed expression of their corresponding genes.569 and 326 genes were identified to be ethylene-responsive as shown by more than 4-fold changes in their expression levels after 4 and 24 h of ethylene treatment at FDR (false-discovery rate)≤0.02 level. Of these genes,243 and 176 were up-regulated, whereas 326 and 190 were down-regulated, respectively, after 4 and 24h of ethylene treatment. Results from relative quantitative RT-PCR analysis performed on 16 selected genes were highly agreeable with those observed from microarray analysis. Classification based on their functions showed that these ethylene-responsive genes are related to a variety of different processes including responses to stress, responses to abiotic or biotic stimulus, protein metabolism, transport, signal transduction and transcription. The most significant changes in expression were found for genes encoding proline-rich protein 4, ethylene response factor 1 and cysteine proteinase, and their possible involvement in abscission was further studied.2. Cloning and Expression Analysis of CsPRP4,CsERF1,CsUnknow and CsCysP genesPrimers were designed from sequences deposited in HarvEst Citrus (Version 1.25) database, and the full-length cDNAs of CsPRP4,CsERF,CsUnknow and CsCysP genes were cloned by using methods of RT-PCR and SMART-RACE.Sequence analysis revealed that the full-length cDNAs of CsPRP4, CsERF, CsUnknow and CsCysP are 1251bp,1453bp,1545bp and1452bp respectively, and their corresponding ORFs are 585bp,810bp,1101bp and 1050bp in length which would encode 194,269,365 and 347 amino acids with calculated molecular masses of 20.79KD,29.25KD,41.6KD and 38.4KD, respectively. Protein physiochemistry analysis suggested that CsPRP4 and CsERF1 would be stable while CsUnknow and CsCysP would be unstable.Bioinformatics and expression analysis showed that CsPRP4 was probably the most highly induced cell-wall gene and expressed differentially between leaves and fruit abscission zones when induced by exogenous ethylene. CsERF1 is a member of the AP2 supperfamily possessing transcription activator activity. ERF1, with GCC-box binding activity and as a component of the ethylene signal transduction pathway, plays an important role in modulating the expression of genes downstream of it by binding the GCC boxes located in the promoters of many ethylene responsive genes, and may be one of the key factors that eventually cause abscission. The function of CsUnknow encoded protein is unknown, but its expression was highly induced by ethylene. The sequence of CsUnknow suggested that it probably located in endomembrane system and possessed GCC-box binding motif. CsCysP, a transmembrane protein located in vacuolar membrane, is one of the important hydrolytic proteases with endopeptidase activity. The expression of CsCysP was strongly repressed by exogenous ethylene treatment, suggesting that it negatively regulates downstream gene(s) and that the accumulation of proteins targeted by CsCysP may be required in abscission. 3. Construction of CsPRP4,CsERF1 and CsCPA RNAi vectors and transformationBy using RT-PCR, the targeted cDNA sequences of PRP4,ERF1 and CysP were amplified from citrus and T/A cloned. Binary vector pFGC594 and target sequences were sequentially digested twice with endonucleases and then the target sequences were inserted in sense and antisense directions, respectively, into the two sides of the chalcone synthase gene intron that was carried in the vector to form inverted repeats. Various methods such as PCR by using bacteria containing the constructs, digestion of the isolated plasmids with endonucleases, and sequencing verified that all the constructs were correct. The sense constructs used in the construction of RNAi expression vectors were used as over-expression constructs. Antibiotic resistant buds were regenerated after transformation of citrus explants via agrobacterium-mediated transformation, and some of the buds have been successfully grafted onto rootstock seedlings, which should have paved a way towards the elucidation of the genes' functions.4. Conclusion remarks In this work, a large-scale study on the changes in transcriptome profiles of ethylene-induced genes during fruit abscission was carried out and a large quantity of data about the genes involved in abscission and their expression changes were generated. Comprehensive analysis of the data provided information of the biological pathways involved in abscission, and deepened our understanding of the molecular mechanism on gene regulation during ethylene-induced abscission.The full-length cDNAs of 4 ethylene-regulated genes CsPRP4,CsERF1 CsUnknow and CsCysP during ethylene-induced abscission were obtained, and were analyzed with bioinformatics methods. In combination with the expression analysis, the roles of the genes in abscission were assumed.The overexpression and RNAi expression vectors were constructed for CsPRP4,CsERF1,CsUnknow and CsCysP. Regenerated antibiotic buds were obtained through agrobacterium-mediated transformation, and some of the buds were survived after grafting, which placed a base for further characterization of the genes'functions.