Department of Animal Science, University of British Columbia, Vancouver, BC, Canada V6T1Z4.
Phytochemistry. 2010 Feb;71(2-3):168-78. doi: 10.1016/j.phytochem.2009.10.011. Epub 2009 Nov 24.
Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of alpha- and beta-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo beta-carotene hydroxylations of beta,beta-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the epsilon-ring of alpha-carotene or the beta-ring of beta,epsilon-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the beta- and epsilon-rings of alpha-carotene and beta-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) beta-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 beta-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of alpha-carotene derivatives, accompanied by a reduction of alpha-carotene, were observed. These data suggest possible interactions between the beta-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.
类胡萝卜素是一组广泛分布的色素，它们来源于通用的异戊二烯生物合成途径，在植物的初级和次级代谢中具有多种功能。α-和β-胡萝卜素骨架的修饰部分取决于环羟化。两个铁氧还蛋白依赖的非血红素二铁单加氧酶（AtB1 和 AtB2）主要催化体内β-胡萝卜素的β，β-类胡萝卜素的羟化，两个含血红素的细胞色素 P450（CYP）单加氧酶（CYP97A3 和 CYP97C1）优先羟化α-胡萝卜素的ε-环或β，ε-类胡萝卜素的β-环，已通过分析功能丧失突变体表型在拟南芥中得到鉴定。我们通过过表达 AtB1、CYP97A3、CYP97C1 和羟化酶候选 CYP97B3，进一步研究了这两类羟化酶在α-胡萝卜素和β-胡萝卜素的β-和ε-环修饰中的功能作用。由于β-酮基胡萝卜素酮酶 bkt1（CrtO）需要类胡萝卜素羟化才能生成酮类胡萝卜素，因此所有羟化酶构建体也被引入表达 Haematococcus pluvalis bkt1 β-胡萝卜素酮酶的拟南芥系中。分析过表达单个羟化酶的系中的叶类胡萝卜素图谱表明 CYP97B3 在类胡萝卜素生物合成中起作用，证实并扩展了基于敲除突变体的羟化酶活性的先前发现，并表明多功能酶在类胡萝卜素生物合成中的作用。羟化酶过表达与 bkt1 结合并没有导致酮类胡萝卜素的积累，而是观察到了意想不到的α-胡萝卜素衍生物模式，同时α-胡萝卜素的减少。这些数据表明，β-胡萝卜素酮酶 bkt1 和羟化酶之间可能存在相互作用，影响碳通量分配到不同的类胡萝卜素分支途径。
