Cuticular waxes are complex mixtures of very-long-chain fatty acids (VLCFAs) and their derivatives, forming a natural barrier on aerial surfaces of terrestrial plants against biotic and abiotic stresses. In VLCFA biosynthesis, β-ketoacyl-CoA synthase (KCS) is the key enzyme, catalyzing the first reaction in fatty acid elongation and determining substrate specificity. We isolated a rice (Oryza sativa) wax crystal-sparse leaf 4 (WSL4) gene using a map-based cloning strategy. WSL4 is predicted to encode a KCS, a homolog of Arabidopsis (Arabidopsis thaliana) CER6. Complementation of the mutant wsl4-1 with WSL4 genomic DNA rescued the cuticular wax-deficient phenotype, confirming the function of WSL4. The load of wax components longer than 30 carbons (C30) and C28 were reduced markedly in wsl4-1 and wsl4-2 mutants, respectively. Overexpression of WSL4 increased the cuticular wax load in rice leaves. We further isolated a cofactor of WSL4, OsCER2, a homolog of Arabidopsis CER2, by coimmunoprecipitation and confirmed their physical interaction by split-ubiquitin yeast two-hybrid experiments. Expression of WSL4 alone in elo3 yeast cells resulted in increased C24 but did not produce VLCFAs of greater length, whereas expressing OsCER2 alone showed no effect. Coexpression of WSL4 and OsCER2 in elo3 yeast cells yielded fatty acids up to C30. OsCER2 with a mutated HxxxD motif (H172E, D176A, and D176H) interrupted its interaction with WSL4 and failed to elongate VLCFAs past C24 when expressed with WSL4 in elo3 yeast cells. These results demonstrated that WSL4 was involved in VLCFA elongation beyond C22 and that elongation beyond C24 required the participation of OsCER2.
角質(zhì)層蠟質(zhì)是極長(zhǎng)鏈脂肪酸（VLCFA）以及它們衍生物的混合體，在陸生植物地上部的表面形成天然的屏障，抵御生物和非生物脅迫。在VLCFA合成中，β-酮脂酰輔酶A合成酶（KCS）是一個(gè)關(guān)鍵酶，催化脂肪酸延長(zhǎng)的第一步反應(yīng)，決定底物特異性。我們利用圖位克隆的策略分離了一個(gè)水稻WSL4基因。WSL4預(yù)測(cè)編碼一個(gè)KCS，它是擬南芥CER6的同源蛋白。利用WSL4基因組DNA互補(bǔ)能夠恢復(fù)wsl4-1的角質(zhì)層蠟質(zhì)缺陷的表型，證實(shí)WSL4的功能。wsl4-1和wsl4-2突變體中，超過(guò)30個(gè)碳（C30）和28個(gè)碳（C28）的蠟質(zhì)組分含量分別顯著降低。過(guò)表達(dá)WSL4會(huì)增加水稻葉片中角質(zhì)層蠟的含量。我們通過(guò)免疫共沉淀進(jìn)一步分離了WSL4的輔因子OsCER2，它是擬南芥CER2的同源蛋白，并通過(guò)分離泛素酵母雙雜實(shí)驗(yàn)證實(shí)了它們的物理互作。在elo3酵母細(xì)胞中單獨(dú)表達(dá)WSL4會(huì)導(dǎo)致C24增加，但不會(huì)產(chǎn)生更長(zhǎng)的VLCFAs，但是單獨(dú)表達(dá)OsCER2沒(méi)有作用。在elo3酵母細(xì)胞中共表達(dá)WSL4和OsCER2產(chǎn)生最長(zhǎng)為C30的脂肪酸。OsCER2的HxxxD基序（H172E、D176A和D176H）突變會(huì)破壞它與WSL4的互作，并且當(dāng)與WSL4在elo3酵母細(xì)胞中時(shí)，不能延長(zhǎng)VLCFAs超過(guò)C24。這些結(jié)果表明，WSL4參與VLCFA的延長(zhǎng)超過(guò)C22，VLCFA延長(zhǎng)超過(guò)C24時(shí)還需要OsCER2的參與。