Docimo Teresa, Francese Gianluca, Ruggiero Alessandra, Batelli Giorgia, De Palma Monica, Bassolino Laura, Toppino Laura, Rotino Giuseppe L, Mennella Giuseppe, Tucci Marina
Consiglio Nazionale delle Ricerche, Istituto di Bioscienze e Biorisorse UOS Portici, Italy.
Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Centro di Ricerca per l'Orticoltura Pontecagnano, Italy.
Front Plant Sci. 2016 Jan 28;6:1233. doi: 10.3389/fpls.2015.01233. eCollection 2015.
Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena) fruits. Chlorogenic acid (CGA) accounts for 70-90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena. Higher contents of CGA, Delphinidin 3-rutinoside, and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR, and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group six MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties. In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation. Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9) resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of the C-terminal region of SmMyb1 does not limit its capability to regulate CGA accumulation, but impairs anthocyanin biosynthesis. To our knowledge, this is the first study reporting a functional elucidation of the role of the C-term conserved domain in MYB activator proteins.
苯丙烷类化合物是茄子（Solanum melongena）果实中的主要次生代谢产物。绿原酸（CGA）占果肉组织中总酚类物质的70 - 90%，而花青素主要存在于果实表皮中。为了有助于理解这些对健康有益的代谢产物在茄子中的特殊积累情况，我们报道了茄子中代谢物丰度、CGA和花青素生物合成的调控以及候选CGA生物合成基因的特征。与其他组织相比，茄子果实中CGA、矢车菊素3 - 芸香糖苷和芦丁的含量更高，这与PAL、HQT、DFR和ANS等结构基因转录本丰度的升高相关，表明活跃的原位生物合成有助于果实组织中花青素和CGA的积累。从一个西方茄子传统品种中分离出了两个CGA生物合成基因PAL和HQT的假定直系同源基因，以及一个与第六组MYB具有同一性的MYB1转录因子变体，并证明其与亚洲品种已发表的序列不同。对分离出的SmPAL1、SmHQT1、SmANS和SmMyb1启动子的电子分析揭示了生物合成基因存在几个Myb调控元件以及该转录因子的独特元件，表明其除了参与苯丙烷类生物合成调控外还参与其他生理作用。SmMyb1和C端截短形式的SmMyb1（SmMyb1Δ9）在本氏烟草叶片中的瞬时过表达仅导致SmMyb1农杆菌浸润叶片积累花青素。酵母双杂交试验证实了SmMyb1和SmMyb1Δ9与一种与花青素相关的马铃薯bHLH1转录因子之间的相互作用。有趣的是，在SmMyb1和SmMyb1Δ9农杆菌浸润的叶片中均检测到CGA含量翻倍，因此表明SmMyb1的N端区域足以激活其合成。这些数据表明，SmMyb1 C端区域的缺失并不限制其调节CGA积累的能力，但会损害花青素的生物合成。据我们所知，这是第一项报道对MYB激活蛋白中C端保守结构域作用进行功能阐释的研究。
