Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles
Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles
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DOI：
10.1111/j.1365-313x.2005.02625.x
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发表时间：
2006-01-01
期刊：
影响因子：
7.2
通讯作者：
Nakayama, T
中科院分区：
文献类型：
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作者：
Ono, E;Hatayama, M;Nakayama, T
金鱼草素合成酶是一种多酚氧化酶(PPO)，它特异性地催化查尔酮氧化形成极光，是植物黄酮类化合物，也是金鱼草花黄色的原因。所有已知的PPO都定位于叶绿体中，而类黄酮的生物合成被认为发生在细胞质[或内质网(ER)的细胞质表面]。然而，金黄色葡萄糖素合成酶的主要结构特征及其一些分子特性反对该酶定位于质体和细胞质。本研究对黄鱼花瓣细胞中该酶的亚细胞定位进行了研究。蔗糖密度梯度分析和差速离心法分析表明，该酶(39 kDa成熟型)不存在于叶绿体或内质网中。利用绿色荧光蛋白(GFP)嵌合体与PPO前体的假定前肽融合的瞬时分析表明，该酶定位于液泡腔内。我们还发现，空泡靶向PPO所需的信息编码在53个残基的N-末端序列(NTPP)中，而不是在前体的C-末端序列中。通过NTPP-GFP嵌合体与拟南芥GTPase Sar1的显性负突变体或与单体红色荧光蛋白(MRFP)融合的高尔基体(拟南芥的H+转移无机焦磷酸酶)的共表达，证实了NTPP介导的内质网向液泡的转运。我们在前体的NTPP中确定了一个序列特异性的空泡分选决定因素。我们已经证明了类黄酮骨架在液泡中的生物合成。这种代谢区划的发现可能为克服细胞质中前体查尔酮的生化不稳定性提供一种策略，从而导致极光在花中的有效积累。
Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.