| Maturation of the fruit is a complex process with strict regulation. As typicalclimacteric fruit, tomato is an important part of human diet. In addition, tomato hadbecome a model to study the mechanism of fruit maturation and ethylene regulationbecause it had relatively simple genomes, a large number of mutants and efficienttransgenic technology. Previous studies have shown that, ethylene, as an important planthormone, played a key role during tomato fruit development and ripening. There weretwo patterns of ethylene biosynthesis in tomato; one pattern is system I which is thebasal low rate of ethylene production present in vegetative tissues and nonclimactericfruits. The other one is system II which is the high rate of ethylene production observedduring ripening in climacteric fruits and in certain senescent flowers. The pathway ofethylene biosynthesis has been well established in higher plants and the ACC synthase(ACS) and ACC oxidase (ACO) were two key enzymes in this process. So far, therewere five members of ACO gene family reported (LeACO1-5). ACS gene family andLeACO1to LeACO4expression during flower maturation leaves senescence and fruitripening had been extensively characterized. However, little information had been doneon the regulation of LeACO5by ethylene and the role of LeACO5in ethylenebiosynthesis. We investigated the roles of ACO gene family played in ethylenebiosynthesis in tomato fruit for providing the basis theoretical to regulate tomato fruitripening and extend fruit shelf life. The main contents and results of the experiment areas follows1. Cloning of tomato ACO gene familyAccording to sequence information in NCBI and TIGR database, we cloned thecDNA of LeACO1-5and a novel member of ACO gene family named LeACO6byspecific primers.2. Sequence alignment of LeACO gene familyLeACO6mRNA is1293bp in length and contained an open reading frame of960bp.The pairwise comparison (%)of deduced amino acid sequences of LeACO6compared with LeACO1-5were79.6%,76.6%,78.1%,85.6%and45.3%respective.Moreover, phylogenetic cladogram analysis showed that LeACO6is most closelyrelated to LeACO4and the similarity of LeACO5is lowest than other members ofLeACO gene family. 3. Analysis of expression pattern of LeACO1-6in wild-type tomato fruit bysemi-quantitative PCRThe expression patterns of LeACO1-6in wild-type tomato fruit at five periodsof immature green (IMG)，mature green (MG)，breaker(B)，breaker+4day (B+4)，breadker+7(B+7) were investigated. The results confirmed that LeACO1is functioningin system I and system II ethylene biosynthesis. LeACO3gene mainly participated intransition stage ethylene biosynthesis which between in the system I and system II.Moreover, the results also revealed that LeACO5and LeACO6were both functioning insystem I ethylene biosynthesis and LeACO6also played a role in transition stage toinitiate the system II ethylene biosynthesis with other genes which had high leveltranscripts accumulation in transition stage.4Analysis of expression patterns of LeACO1-6in response to exogenous ethyleneand wounding by semi-quantitative PCRThe expression patterns of LeACO1-6in MG fruit in response to exogenousethylene were studied. The results showed that expression of LeACO1,3,6wereinduced in MG tomato fruit after8h treating by exogenous ethylene while theexpression of LeACO4was suppressed. It suggested that the regulation of LeACO1,3,6in transition stage was In front of that of LeACO4. Furthermore, after leaves weretreated2h by wounding reposed, the expression of LeACO1-6were studied and theresults indicated that LeACO1,3,4were related to the response to wounding in leaves.5. Analysis of expression patterns of LeACO5,6in tomato fruit by quantitativereal-time RT-PCR.The expression patterns of LeACO5,6in wild-type, rin and Nr fruit during fruitdevelopment were investigated. The expression pattern of LeACO5,6gene in thewild-type fruit obtained by qRT-PCR confirmed the result obtained by sq-PCR. BothLeACO5and LeACO6genes have transcripts accumulation in rin mutant fruit during35dpa to55dpa which different from expression pattern in wild-type fruit. LeACO5gene showed only a slight delay to maximal expression in Nr fruit while the high levelexpression of LeACO6was part depends on fruit ethylene sensitivity. These resultsrevealed that LeACO5is a ripening-related gene response for system I ethylenebiosynthesis in tomato and LeACO6, functioning in system I ethylene synthesis andtransition stage, is regulated by both ripening-related factor included RIN and ethylenein tomato fruit.6. The roles of LeACO1-6in tomato fruit ethylene biosynthesis were characterized based on the reports and conclusions of this article... |
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