Studies On Cuticular Wax Variation And The Expression Of Key Wax Genes During The Fruit Development And Storage Of Kumquat

The Huapi kumquat,Rong’an kumquat and Suichuan kumquat were used as experimental materials.Scanning electron microscopy and gas chromatography-mass spectrometry were used to study the changes of wax structure and composition during the fruit development and storage of kumquat.The Real-Time quantitative PCR（RT-qPCR）technique was used to analyze the expression of wax-related regulatory genes during fruit development and storage.The structure and composition characteristics of kumquat wax and the wax changes during development and storage as well as the difference in waxiness between different kumquat varieties were discussed at the biochemical and molecular levels.The main findings are as follows:1.The results of the analysis of the appearance quality,peel wax and expression of wax related regulatory genes of the kumquat fruits during development were as follows:（1）During the whole development period,the vertical and horizontal diameter,single fruit weight,and peel surface area of the kumquat were all the largest,and there was no significant difference in the fruit shape index from 90 days after flowering to maturity.At60 days after flowering,there was no significant difference in the L value between the Huapi and Suichuan kumquat,but the L value of both are significantly smaller than Rong’an kumquat;from 90 to 150 days after flowering,there was no significant difference in the L values of Rong’an and Suichuan kumquat,but they were significantly larger than those of the Huapi kumquat.At 180 days after flowering,there was no significant difference in the L value among the three.（2）The epidermal wax layer of the three kumquat varieties was composed of an amorphous wax structure and lamellar wax crystals scattered thereon.The Huapi kumquat genitals formed crystals earlier,at a faster rate,and with larger lamellar crystals at maturity than the other two varieties.During the development process,the external wax of the three kumquat varieties accounted for more than 68%of total wax.The main chemical components of the outer and inner wax include fatty acids,alcohols,alkanes,sterols and triterpenes compounds,but aldehydes were also included in the outer wax,and the distribution of C₃₄ saturated fatty acids was present,but not detected in the inner wax.The change trend of wax content of three varieties during fruit development was different and there were some differences in the proportion and content of the outer and inner wax of the three varieties,but the content of triterpenoids gradually increased with the development of the fruit,and the content was highest at maturity（inner and outer wax accounts for more than 30%）.（3）Analysis of expression levels of related genes regulating the wax formation of kumquat peel:during the development process,the ACC1,LACS4,KCS6,CER3,CER1-LIKE1,CER4-LIKE1,CER4-LIKE2,FAR2-LIKE1,ABCG11-LIKE1,ABCG11-LIKE2,ABCG11-LIKE3,LUP2,LUP4-LIKE1,MYB16,MYB94 and MYB96genes were basically consistent with the changes in the total amount or components of kumquat wax,and the relative expression level in Huapi kumquat was higher than the other two varieties,which was also consistent with the difference in total wax content or composition of the three kumquats.2.The results of the changes in the wax and related genes expression of Huapi kumquat and Rong’an kumquat during cold storage were as follows:（1）The decay rate of Huapi kumquat was lower than that of Rong’an kumquat for most of the storage period,especially in the later period of storage.The weight loss rate of Rong’an kumquat was significantly higher than Huapi kumquat after 30 days of storage.（2）Scanning electron microscopy observations of the two kumquat varieties were similar during storage:when they were stored at low temperatures for 30 days,the wax crystals on both surfaces appeared to melt and the wax crystals covered less.After that,cracks appeared on the surface of the wax layer.After 90 days of storage,new wax formation was found to fill the cracks,and new lamellar crystals gradually appeared.However,after 150 days of storage,the wax crystals of the Huapi kumquat were larger than those of Rong’an kumquat.（3）Both showed a significant decrease at 30 days after storage.During the whole storage period,the wax content of Huapi kumquat was significantly higher than that of Rong’an kumquat,and in the later storage period,the total wax amount of Huapi kumquat exceeded that before storage.However,there was no significant change in Rong’an kumquat before and after storage.In addition,the content of alkanes,alcohols,aldehydes and fatty acids in the Huapi kumquat were significantly higher than that of the Rong’an kumquat during most of the storage period.And the ratio of aliphatic compounds/triterpenes,（alkanes+aldehydes）/triterpenes,and aldehydes/triterpenes in the former were significantly higher than the latter.（4）When stored for 30 days,the ACC1,CER1-LIKE1,CER4-LIKE1,CER4-LIKE2,FAR2-LIKE1,ABCG11-LIKE3,MYB16,MYB94,MYB96 gene expression of both decreased.The ACC1,CER4-LIKE1,FAR2-LIKE1,ABCG11-LIKE2,ABCG11-LIKE3,MYB16,MYB96 genes related to wax synthesis,transport and transcription of Rong’an kumquat were down-regulated relative to Huapi kumquat during storage.3.The effect of heat treatment on the change of wax and wax-related genes expression in Suichuan kumquat were as follows:（1）During the whole storage process,the decay rate of heat treatment fruit was lower than that of the control,and the time of rotten fruit appeared delayed.And the weight loss rate was lower than that of the control after 15 days of storage,and after 25 days,there was a significant difference in weight loss rate between the two.（2）Heat treatment before storage would not cause changes in the content of various wax components and the total wax content identified in the wax of Suichuan kumquat,but the wax crystals of the heat treatment fruit were dissolved and flattened on the surface of the wax layer after being soaked in hot water.The wax layer on the surface of the control fruit had cracks at 30 days after storage,but no crack appeared on the fruit surface after heat treatment.In addition,after storage,the content of triterpenoids in the wax of the control Suichuan kumquat was significantly higher than that of heat treatment.The total aliphatic compounds and the ratio of the aliphatic components to the triterpenes except fatty acids in the former were significantly lower than the latter.（3）By analyzing the expression of genes related to wax formation in the peels of control and heat treatment Suichuan kumquat,we found that:The changes in the expression of ABCG11-LIKE1,ABCG11-LIKE2,MYB16,MYB96,ACC1,LACS4,KCS6,CER3,CER1-LIKE1,CER4-LIKE1,CER4-LIKE2,LUP2,LUP4-LIKE1 gene were consistent with the changes in their total wax content and various wax components,respectively.In addition,the expression levels of LUP2 and LUP4-LIKE1 genes in heat treatment fruits were lower than the control at 30 days after storage,significantly lower than the control at 90 days after storage,and the expression of LUP4-LIKE1 gene was significantly lower that of the control at 60 days after storage.