Tian Li, Magallanes-Lundback Maria, Musetti Valeria, DellaPenna Dean
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Plant Cell. 2003 Jun;15(6):1320-32. doi: 10.1105/tpc.011403.
Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of beta- and epsilon -ring carotenoid hydroxylases. Two genes that encode beta-ring hydroxylases (beta-hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward beta-rings but only weakly active toward epsilon -rings. A third distinct activity required for epsilon -ring hydroxylation has been defined by mutation of the LUTEIN1 (LUT1) locus, but LUT1 has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to beta-hydroxylases 1 and 2 (b1 and b2, respectively) were isolated and all possible hydroxylase mutant combinations were generated. beta-Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known beta-hydroxylase enzymes, still contains significant levels of beta-carotene-derived xanthophylls, suggesting that additional beta-ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of beta-carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated beta-rings. This finding suggests that it is the presence of minimal amounts of hydroxylated beta-rings, rather than minimal amounts of specific beta-carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.
叶黄素和玉米黄质是二羟基叶黄素,它们是由相应的类胡萝卜素前体通过β-环和ε-环类胡萝卜素羟化酶的作用产生的。在拟南芥基因组中已鉴定出两个编码β-环羟化酶的基因(β-羟化酶1和2),它们对β-环具有高活性,但对ε-环只有微弱活性。ε-环羟化所需的第三种独特活性已通过叶黄素1(LUT1)位点的突变得到定义,但LUT1尚未被克隆。为了研究拟南芥三种类胡萝卜素羟化酶活性在体内的个体功能和重叠功能,分离了与β-羟化酶1和2相对应的T-DNA敲除突变体(分别为b1和b2),并产生了所有可能的羟化酶突变体组合。β-羟化酶单突变体没有表现出明显的生长缺陷,并且相对于野生型对类胡萝卜素组成的影响有限,这表明所编码的蛋白质在体内具有显著程度的功能冗余。令人惊讶的是,缺乏两种已知β-羟化酶的b1 b2双突变体仍然含有大量源自β-胡萝卜素的叶黄素,这表明体内存在额外的β-环羟化活性。双羟化酶和三羟化酶突变体的表型表明,这种活性至少有一部分存在于LUT1基因产物中。尽管源自β-胡萝卜素的叶黄素严重减少(在lut1 b1 b2三突变体中高达90%),但双羟化酶和三羟化酶突变体仍然含有至少50%野生型量的羟化β-环。这一发现表明,对于体内捕光复合体II的组装和功能而言,至关重要的是存在微量的羟化β-环,而不是微量的特定源自β-胡萝卜素的叶黄素。野生型种子中的类胡萝卜素谱以及单羟化酶和多羟化酶突变的影响与光合组织中的不同,这表明每个基因产物的活性在这两种组织中有所不同。总体而言,羟化酶突变体为类胡萝卜素羟化酶在体内意想不到的重叠活性提供了深入了解。