Abstract: 【Objective】This study is aiming at characterizing the mature fruits wax from citrus cultivated species(Citrus reticulata Blanco cv. Ponkan, C. limon cv. Eureka and C. grandis Osbeck cv. Shatian Yu) which are widespread in China, establishing the database of citrus fruits wax components, and providing important information for waxing for the specifical cultivar of citrus fruits. 【Method】Wax crystal structure of three citrus cultivars were observed by scanning electron microscopy, and cuticle thickness observed by transmission electron microscope. Subsequently, the fruits waxes of these cultivars were analyzed by gas chromatography-mass spectrometry (GC-MS). Then, the expression levels of wax associated genes were detected in different cultivars using qPCR. 【Result】 Scanning electron microscopy showed that larger and more wax platelets were deposited on the surface of tight-skin citrus Shatian Pummelo than on the loose-skin citrus Ponkan fruits, while wax platelets on lime fell in between. The total waxes of the three citrus cultivars were mostly composed of aldehydes, alkanes, fatty acids, and primary alcohols, while their proportions and carbon number distribution varied among different cultivars. The epicuticular wax concentrations in fruits of Citrus reticulata Blanco, C. limon cv Eureka and C. grandis Osbeck were 1.1, 2.2 and 9.3 μg·cm^-2, respectively. The proportions of aldehydes, fatty acids, alkanes and primary alcohols in the epicuticular wax in Citrus reticulata Blanco were 50%, 16%, 28% and 6%, respectively. Their proportions in C. limon cv Eureka were 42%, 30%, 18% and 11%, respectively, while their proportions in C. grandis Osbeck were 50%, 31%, 12% and 7%, respectively. Hexacosanal (C26) and octacosanoic acid (C28) were the most abundant compounds in the epicuticular wax in Ponkan, while in Lemon, their carbon number distributed evenly. Higher carbon number aliphatic compounds were found in Shatian Pummelo, for example, dotriacontanoic acid, hentriacontane and tritriacontane were dominated. The total wax concentrations in fruits of Ponkan and Lemon were 4.3 and 4.6 μg·cm^-2, respectively. The proportions of aldehydes, fatty acids, alkanes and primary alcohols in total wax in Ponkan were 57%, 14%, 23% and 4%, respectively. Their proportions in Lemon were 53%, 15%, 19% and 8%, respectively. The chain length distribution of total wax was similar with that of epicuticular wax in both of the cultivars. In addition, terpenoids showed great differences among these cultivars. The most predominant triterpenoid compounds were friedelin, lupeol, α-amyrin and β-amyrin in C. limon cv Eureka. However, there was no friedelin in total wax in Ponkan. Farnesol and squalene were exclusive in tense-skin citrus. qPCR analysis indicated that expression profile of member of beta-ketoacyl-CoA synthase family KCS6 showed a similar trend with wax amounts of the five cultivars, while member of the BAHD superfamily of acyl-transferases CER2 was expressed highest in Shatian Pummelo, which was consistent with high abundance of constituents with carbon number higher than 30. In this study, the wax of citrus fruits was mostly composed of aldehydes, followed by fatty acids or alkanes. Primary alcohols were the least predominant compounds. By comparing the distribution of wax components, it was found that, within the very-long-chain aliphatic wax components, primary alcohols and very-long-chain fatty acides tend to accumulate to higher percentages in the epicuticular layer, while alkanes occurred at lower percentages in it. With terpenoids, triterpenoids and sterols were only present in the total wax, however, squalene and farnesol were detected in both epicuticular and intracuticular wax.【Conclusion】Wax components were regulated by inheritance and peel anatomical structure synergistically. Therefore, loose-skin citrus and tight-skin citrus showed different characteristics of wax profile, which suggested that different synthetic waxes should be adopted in those two types of citrus fruits, respectively.

Key words: citrus fruits, wax, scanning electron microscope, GC-MS, expression analyses