Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper.
Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper.
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DOI：
10.1186/s12870-015-0476-7
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发表时间：
2015-03-28
影响因子：
5.3
通讯作者：
Masuta C
中科院分区：
文献类型：
--
作者：
Ogawa K;Murota K;Shimura H;Furuya M;Togawa Y;Matsumura T;Masuta C
辣椒素类物质，包括辣椒素及其类似物，是辣椒（辣椒属）果实辛辣的原因。尽管辣椒素是人类熟悉和日常使用的，但参与辣椒素生物合成途径的基因尚未得到很好的表征。推测的氨基转移酶（pAMT）和刺激基因1（Pun 1）蛋白被认为分别催化途径中的倒数第二步和最后一步，使得Pun 1蛋白成为推测的辣椒素合酶。然而，没有直接证据表明Pun 1具有辣椒素合成酶活性。为了验证Pun 1蛋白实际上在辣椒素生产中起作用，我们产生了针对大肠杆菌合成的Pun 1蛋白的抗Pun 1抗体，并使用它们来拮抗内源性Pun 1活性。为了证实抗Pun 1抗体的特异性，我们使用病毒诱导的基因沉默靶向Pun 1 mRNA。在Pun 1下调的胎盘组织中，使用抗Pun 1抗体在蛋白质印迹上鉴定的Pun 1蛋白的累积水平降低，同时，在相同组织中辣椒素累积减少。在从头辣椒素合成的体外无细胞测定中，使用从胎盘组织分离的原生质体，辣椒素合成通过添加抗Pun 1抗体来抑制。我们接下来分析了pAMT和Pun 1在各种辣椒品种中的表达谱，发现辣椒素的高水平积累总是伴随着pAMT和Pun 1的高表达水平，这表明这两个基因对辣椒素合成都很重要。然而，香草胺（辣椒素的前体）和辣椒素之间的辛辣和nonpentric品种的积累水平的比较表明，香草胺水平在辛辣的品种是非常低的，可能是由于其快速转换为辣椒素Pun 1合成后不久，在nonpentric品种，香草胺积累到相当高的水平，由于缺乏Pun 1。使用新开发的基于原生质体的从头辣椒素合成和抗Pun 1抗体的测定，我们成功地证明了Pun 1基因及其基因产物参与辣椒素合成。香草胺积累相对于辣椒素的分析表明，Pun 1是其积累水平的主要决定因素。本文的在线版本（doi：10.1186/s12870-015-0476-7）包含补充材料，可供授权用户使用。
Capsaicinoids, including capsaicin and its analogs, are responsible for the pungency of pepper (Capsicum species) fruits. Even though capsaicin is familiar and used daily by humans, the genes involved in the capsaicin biosynthesis pathway have not been well characterized. The putative aminotransferase (pAMT) and Pungent gene 1 (Pun1) proteins are believed to catalyze the second to last and the last steps in the pathway, respectively, making the Pun1 protein the putative capsaicin synthase. However, there is no direct evidence that Pun1 has capsaicin synthase activity. To verify that the Pun1 protein actually plays a role in capsaicin production, we generated anti-Pun1 antibodies against an Escherichia coli-synthesized Pun1 protein and used them to antagonize endogenous Pun1 activity. To confirm the anti-Pun1 antibodies’ specificity, we targeted Pun1 mRNA using virus-induced gene silencing. In the Pun1-down-regulated placental tissues, the accumulated levels of the Pun1 protein, which was identified on a western blot using the anti-Pun1 antibodies, were reduced, and simultaneously, capsaicin accumulations were reduced in the same tissues. In the de novo capsaicin synthesis in vitro cell-free assay, which uses protoplasts isolated from placental tissues, capsaicin synthesis was inhibited by the addition of anti-Pun1 antibodies. We next analyzed the expression profiles of pAMT and Pun1 in various pepper cultivars and found that high levels of capsaicin accumulation always accompanied high expression levels of both pAMT and Pun1, indicating that both genes are important for capsaicin synthesis. However, comparisons of the accumulated levels of vanillylamine (a precursor of capsaicin) and capsaicin between pungent and nonpungent cultivars revealed that vanillylamine levels in the pungent cultivars were very low, probably owing to its rapid conversion to capsaicin by Pun1 soon after synthesis, and that in nonpungent cultivars, vanillylamine accumulated to quite high levels owing to the lack of Pun1. Using a newly developed protoplast-based assay for de novo capsaicin synthesis and the anti-Pun1 antibodies, we successfully demonstrated that the Pun1 gene and its gene product are involved in capsaicin synthesis. The analysis of the vanillylamine accumulation relative to that of capsaicin indicated that Pun1 was the primary determinant of their accumulation levels. The online version of this article (doi:10.1186/s12870-015-0476-7) contains supplementary material, which is available to authorized users.
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影响因子：
4.3
作者：
del Rosario Abraham-Juarez, Ma;del Carmen Rocha-Granados, Ma;Ochoa-Alejo, Neftali
通讯作者：
Ochoa-Alejo, Neftali
影响因子：
15
作者：
LEETE, E;LOUDEN, MCL
通讯作者：
LOUDEN, MCL
栽培辣椒和野生辣椒的全基因组测序为辣椒驯化和专业化提供了见解
DOI：
10.1073/pnas.1400975111
发表时间：
2014-04-08
影响因子：
11.1
作者：
Qin, Cheng;Yu, Changshui;Zhang, Zhiming
通讯作者：
Zhang, Zhiming
影响因子：
7.2
作者：
Stewart, C;Kang, BC;Jahn, MM
通讯作者：
Jahn, MM
DOI：
10.1080/00021369.1979.10863843
发表时间：
1979-01-01
期刊：
AGRICULTURAL AND BIOLOGICAL CHEMISTRY
影响因子：
--
作者：
IWAI, K;SUZUKI, T;FUJIWAKE, H
通讯作者：
FUJIWAKE, H