Involvement Of Ethylene Signal Transduction Components In Kiwifruit Ripening And Postharvest Abiotic Stress Response

Kiwifruit(Actinidia) was used to study ethylene signaling related genes expression during kiwifruit development, ripening and softening. Treatment of fruit with ethylene (100μl/L),1-MCP (0.5μl/L), low-temperature (0℃), high-temperature (35℃), high CO2 (5%) and dry air (～10%RH) was used to follow responses of these genes in relation to fruit ripening and postharvest stress. Transcription factor and promoter interaction and functional transgenic works were also included in the experiment. The main results are as follows:1. Five ethylene receptor genes, two CTR1 like genes, four AdEILs and fourteen AdERFs were isolated based on kiwifruit EST database. Among the 25 genes, only AdERF2 was not detected in fruit tissue, while the others were widely expressed in various kiwifruit plant tissues. Temporal expression patterns of the genes indicated that most of them were strongly expressed at fruit early stages of development. This is consistent with our knowledge of ethylene-dependent activity in early stages of organ development.2. Expression studies of the five ethylene receptor genes showed that AdERS1a, AdETR2 and AdETR3 were increased during fruit ripening, oppositely, AdETR1 was down-regulated by ethylene. According to ethylene receptor negative regulation model, AdERS1a, AdETR2 and AdETR3 might act as ethylene binder, while AdETR1 might act as sensor with implications for responsiveness of the ethylene signalling pathway. The expression patterns of AdETR1 also could partially explain the mechanism of kiwifruit high ethylene sensitivity. The relative insensitivity to ethylene of AdERS1b and its pattern associated with softening invites the speculation that it might have a separate involvement in promoting some aspect of later fruit softening.3. Contrast to AdETRs, AdEILs were constitutively expressed during fruit ripening. Gene interaction experiments indicated that AdEIL2/3 act as transcription activators by inducing AdACO1 and AdXET5 promoter activity. Further study on AdEIL2/3 function found that ethylene production was triggered in AdEIL2/3 over-expressed Arabidopsis plants. In order to study the function of different motifs in gene interaction, four deletions were designed in promoter of AdXET5. AdEIL2 might recognize the first and third motif in AdXET5 promoter, while the first and second motifs were important for AdEIL3. These results indicated AdEIL2/3 could transcriptionally regulate ripening related genes, but the target motifs might be different.4. Within AdERFs gene family, AdERF2 was excluded in fruit regime experiment. The other thirteen AdERFs genes were differentially expressed during fruit ripening, which could be divided into four patterns:(Ⅰ) AdERF10 and 14 were increased during fruit ripening, (Ⅱ) AdERF4 and 6 expression was relatively high at the post-climacteric stage, (Ⅲ) Expression of AdERF5 and 9 was constant and not affected by exogenous ethylene, (Ⅳ) the other seven AdERFs genes followed a decreasing pattern during ripening. Combined results from ethylene treatment,1-MCP treatment and ACO knock-out fruit, decreases of AdERF1/7/8/11/12 transcripts were likely to be ripening specific, and be independent of ethylene. Promoter of AdEXP1 contains a GCC box, however, no significant interaction was found between AdERFs and AdEXPl promoter. Interestingly, AdERF9 was confirmed as a transcriptional repressor by repressing AdXET5 promoter, although no GCC box was found in this promoter. Based on AdXET5 promoter deletion results, the unknown target for AdERF9 located at-265 to-1 region of AdXET5 promoter.5. Low-temperature (0℃) significantly delay kiwifruit ripening and softening, while low temperature generally resulted in increased gene expression, it is not yet clear how much it is associated with ripening changes including softening, whilst that response is more likely to be stress response. It is worth emphasizing the novel response of EIN3-like genes to low temperature, which provides some insight into the relationship between ethylene signalling and low temperature. AdEIL3 and AdERF1/6/9/13 were over-expressed in Arabidopsis respectively, and all of the transgenic lines manifest higher low-temperature tolerance. More postharvest abiotic stresses were applied to kiwifruit, and AdETRs and AdERFs were choosed to study the genes stress response. AdETR3 and AdERF1/3/4/11/12/14 had similar response to different abiotic stress.In conclusion, different ethylene signalling components of kiwifruit are not only involved in regulation on kiwifruit ripening, but also involved in response to fruit postharvest abiotic stress.