Preliminary Study Of Specific Low Temperature-Induced Terpene Aroma Release And Transcriptional Regulation In ’Hongyang’ Kiwifruit

Kiwifruit（Actinidia spp.）is popular with consumers for its excellent flavor and nutritional qualities.As an important component of the fruit flavor,aroma quality is an important indicator of the eating quality of kiwifruit and has a significant impact on consumer acceptance.Kiwifruit aroma quality is derived from a subtle mixture of volatile organic compounds,and in the mature fruit of A.chinensis,terpene volatiles such as 1,8-cineole give the fruit its distinctive’minty’aroma.As a kind of respiratory climacteric fruit,kiwifruit is susceptible to exogenous ethylene,which in turn activates the synthesis of large amounts of endogenous ethylene,resulting in rapid softening of the flesh and loss of edibility within a short period of time.In recent years,however,researchers have found that kiwifruit fruit also exhibit softening at specific low temperature storage,and the changes in aroma quality of kiwifruit fruit under this characteristic are still unclear.The monoterpene synthase Ac TPS1b is responsible for the synthesis of a series of monoterpene volatiles,mainly 1,8-cineole in A.chinensis,and the transcriptional regulation mechanism is still unexplored.Therefore,in this study,kiwifruit fruits of the red-fleshed variety’Hongyang’were subjected to different storage temperatures and ethylene treatments,and volatiles were determined and analyzed using SPME-GC-MS and rapid gas chromatography with electron nose to investigate the effect of specific low-temperature softening pathways on fruit aroma quality;in addition,transcription factor prediction,c DNA library screening and yeast-one-hybrid,gel migration assay and dual luciferase assay were used to investigate the transcriptional regulation mechanism of Ac TPS1b gene.The main results of the study are as follows:1.Specific low temperatures induce the release of volatiles such as 1,8-cineole from’Hongyang’kiwifruit.1)Aroma formation was specifically induced at 10°C compared to that at 1°C and 20°C:softening of’Hongyang’kiwifruit was significantly accelerated at 10°C compared to that at 1°C and 20°C,accompanied by an increase in soluble solids and a decrease in titratable acids.SPME-GC-MS showed that the release of the terpene volatiles 1,8-cineole and linalool,the aldehyde volatiles hexanal and（E）-2-hexenal was significantly higher at the first week of storage at 10°C than that at other temperatures,while the major ester volatiles were released slowly at10°C.2)Specific induction of aroma formation at 10°C compared to that under ethylene treatment:rapid GC e-nose analysis showed that the release of 1,8-cineole,ethyl hexanoate,hexanal,（E）-3-hexen-1-ol and（Z）-3-hexenol from’Hongyang’kiwifruit was significantly higher than that from ethylene-treated fruit under storage conditions at 10°C during the edible period.3)Aroma synthesis gene expression was affected by specific temperature:q RT-PCR results showed that the expression abundance of Ac TPS1b and LOX1 was significantly induced from the second week of storage at 10°C;AAT expression was significantly higher at 10℃than that at 1°C and20°C when the fruit was stored until the 2^nd to 3^th week.2.Transcription factors Ac WOX13 and AcRAP2.4 regulate Ac TPS1b in response to specific low temperatures.1)Interacting transcription factor screening:Through transcription factor prediction and c DNA library screening combined with yeast-one-hybrid experiments,we found that transcription factors Ac WOX13 and AcRAP2.4 can interact with the Ac TPS1b promoter.Further EMSA experiments showed that both transcription factors could bind to the Ac TPS1b promoter in vitro.2)Subcellular localization and gene expression analysis:subcellular localization showed that Ac WOX13 and AcRAP2.4 were localized in the nucleus.Gene expression analysis showed that Ac WOX13 and AcRAP2.4 were both induced to be expressed at 10℃.Analysis of cis-acting elements showed that both promoter regions had low temperature response elements.3)Transcription factors induced promoter activity in response to specific low temperatures:dual luciferase assays were conducted as follows:co-injection of transcription factors and Ac TPS1b promoters into tobacco,and then temperature treatment was performed,the results revealed that activation of the Ac TPS1b promoter by both transcription factors was induced at 5°C or 10°C.These results suggested that specific low temperatures can promote the release of terpene volatiles in A.chinensis,and two transcription factors were identified that activate the expression of Ac TPS1b in response to specific low temperatures.This study provides a basis for further investigation of the regulatory mechanisms underlying changes in fruit flavor and quality in kiwifruit in response to specific low temperatures.