Ultramicrostructural and compositional analyses of fruit cuticular wax in different avocado varieties

Abstract：【Objective】The fruit surface is covered with the cuticular wax layer composed of very longchain fatty acids (VLCFA) and their derivatives and alicyclic compounds. The fruit cuticular wax can reduce postharvest water loss, and prevent the invasion of microorganisms and pathogens, playing an important role in the growth, development and post-harvest storage of fruit. Avocado (Persea americana Mill.) is one of the typical climacteric fruit, and postharvest water loss and rot of fruit often occur, causing huge economic losses. However, there have been few reports on the study of avocado fruit cuticular wax. This study aimed to clarify the microstructure and chemical composition of the fruit cuticular wax in different avocado varieties, and provide basic data for further research on the cuticular wax functions. 【Methods】In this work, 5 avocado varieties (Peterson, RKYYL6- 2, Pollock, Walter Hole and RKYYL8-13) were used for the analysis of the fruit cuticular wax. Firstly, we observed the ultrastructure of the avocado fruit cuticular wax using the scanning electron microscope, and then separated and extracted the epicuticular wax and intracuticular wax of the avocado fruit. The extraction methods of the epicuticular wax and intracuticular wax were as follows: firstly the gum arabic was applied evenlyon the surface of the avocado fruits to separate the epicuticular wax; then, the fruits were immersed to extract the intracuticular wax of the fruits. The chemical composition and content of epicuticular wax and intracuticular wax of avocado fruit were further analyzed by GC-MS and GC-FID.【Results】The observation of scanning electron microscope showed that the cuticular wax crystals on the avocado fruit surface were mainly plates and granules. At ×30 000 and ×100 000 magnification, the plates wax crystals were clearly found on the fruit surface on Peterson and RKYYL6-2. The crystals of the cuticular wax of Pollock were relatively small, but the plates structure could also be observed at ×100 000 magnification. The crystals of the cuticular wax of Walter Hole was relatively large, and the continuous cuticular wax crystals could be observed at ×5000 magnification, and the crystal structure of cuticular wax of Walter Hole was also plates at ×30 000 and ×100 000 magnification. The crystal structure of the cuticular wax of RKYYL8-13 was different from those of the above-mentioned varieties. At ×30 000 and × 100 000 magnification, the crystals of the cuticular wax of RKYYL8-13 appeared granular and closely stacked. Subsequently, we analyzed the content and chemical composition of the epicuticular wax and intracuticular wax of the 5 avocado varieties. The chemical composition of the avocado epicuticular wax consisted of six types of compounds, namely alkanes, aldehydes, fatty acids, alcohols, steroids and monoacylglycerols; besides the above compounds, the chemical composition of the avocado intracuticular wax also included tocopherols. In terms of the carbon chain distribution of each component of the epicuticular wax, the carbon chain numbers of the alkanes included C23, C25, C27, C29 and C31, and the aldehydes were C30 and C32, the fatty acids were C28, C30 and C32, and the alcohols were C26, C28, C30, C32 and C34, the steroids included stigmasterol and β-Sitosterol, and monoacylglycerols included 1-Monopalmitin and 1-Monostearin. Besides the above wax components of the different carbon chain lengths, the intracuticular wax also contained C26 fatty acid, C34 fatty acid, δ-tocopherol, γ-tocopherol and α-tocopherol. In terms of the cuticular wax content, the epicuticular wax content of the 5 avocado varieties ranged from 0.17 to 0.87 μg · cm- 2 , and the intracuticular wax content ranged from 2.00 to 8.22 μg· cm-2 ; the content of epicuticular wax and intracuticular wax of the RKYYL6-2 was the lowest among the 5 varieties, while Walter Hole was the highest. In most varieties, the alkane had the highest content among the components of the epicuticular wax and intracuticular wax. The intracuticular wax embedded in the cutin and the epicuticular wax covered the outermost layer and together formed the cuticular wax. In terms of the total cuticular wax (the sum of epicuticular wax and intracuticular wax), the variety with the lowest total cuticular wax content was RKYYL6-2, only 2.17 μg·cm^-2 ; Walter Hole had the highest total wax content, up to 9.09 μg·cm^-2 ; and Peterson, Pollock and RKYYL8- 13 had similar total cuticular wax content of 4.13, 4.29 and 4.73 μg·cm^-2 , respectively. In terms of the proportion of the fruit epicuticular wax and intracuticular wax in the total cuticular wax, among the 5 avocado varieties, the content of the epicuticular wax of avocado only accounted for 8.02%-12.81% of the total wax content, while the content of the intracuticular wax accounted for 87.19%-91.99%. Therefore, the proportion of the intracuticular wax in the total cuticular wax was much higher than that of the epicuticular wax.【Conclusion】The crystal structure of avocado wax was usually plates or granular, and different avocado varieties apparently differed in the content and chemical composition of the epicuticular wax and intracuticular wax. The research results would be helpful for further clarification of the biological functions of the cuticular wax of avocado fruits.