Dissection of Tomato Lycopene Biosynthesis through Virus-Induced Gene Silencing
Dissection of Tomato Lycopene Biosynthesis through Virus-Induced Gene Silencing
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DOI：
10.1104/pp.113.224733
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发表时间：
2013-10-01
期刊：
影响因子：
7.4
通讯作者：
Giuliano, Giovanni
中科院分区：
文献类型：
--
作者：
Fantini, Elio;Falcone, Giulia;Giuliano, Giovanni
番茄（Solanum lycopersicum）果实中番茄红素的生物合成是由八氢番茄红素合成酶（PSY）、两种去饱和酶（八氢番茄红素去饱和酶[PDS]和ζ-胡萝卜素去饱和酶[ZDS]）和两种顺反异构酶（ζ-胡萝卜素异构酶[ZISO]和番茄红素原异构酶[CrtISO]）催化的多顺式途径进行的。这些酶的作用机制已在大肠杆菌中进行了研究，但缺乏对其体内功能的系统研究。我们研究了9个候选基因（PSY 1，PSY 2，PSY 3，PDS，ZDS，ZISO，CrtISO，CrtISO-Like 1和CrtISO-Like 2）的功能，使用病毒诱导的基因沉默（VIGS）结合高分辨率液相色谱法，二极管阵列检测器和质谱法，这使得45种不同的类胡萝卜素异构体，包括线性叶黄素的鉴定和定量。这些数据证实了VIGS信号对沉默果实的限制，以及PSY 1和CrtISO沉默果实的表型与黄色果肉和黄色果肉突变体的表型的相似性。光照能够恢复ZISO沉默的果实中番茄红素的生物合成。在不同的代谢步骤中沉默的果实的异构体组成表明存在三个功能单元，包括PSY 1，PDS/ZISO和ZDS/CrtISO，并分别负责15-cis-八氢番茄红素，9，9 '-di-cis-zeta-胡萝卜素和全反式-番茄红素的合成。去饱和酶（PDS或ZDS）的沉默导致在相同功能单元（分别为ZISO或CrtISO）中异构酶的诱导。全反式-zeta-胡萝卜素在非沉默的水果中检测到，在ZDS沉默的水果中大大增加，在CrtISO-Like 1-/CrtISO-Like 2-沉默的水果中消失，这表明存在代谢侧分支，包括该化合物并由后者酶启动。
Lycopene biosynthesis in tomato (Solanum lycopersicum) fruits has been proposed to proceed through a poly-cis pathway catalyzed by phytoene synthase (PSY), two desaturases (phytoene desaturase [PDS] and zeta-carotene desaturase [ZDS]), and two cis-trans isomerases (zeta-carotene isomerase [ZISO] and prolycopene isomerase [CrtISO]). The mechanism of action of these enzymes has been studied in Escherichia coli, but a systematic study of their in vivo function is lacking. We studied the function of nine candidate genes (PSY1, PSY2, PSY3, PDS, ZDS, ZISO, CrtISO, CrtISO-Like1, and CrtISO-Like2) using virus-induced gene silencing (VIGS) coupled to high-resolution liquid chromatography coupled with diode array detector and mass spectrometry, which allowed the identification and quantitation of 45 different carotenoid isomers, including linear xanthophylls. The data confirm the confinement of the VIGS signal to the silenced fruits and the similarity of the phenotypes of PSY1- and CrtISO-silenced fruits with those of the yellow flesh and tangerine mutants. Light was able to restore lycopene biosynthesis in ZISO-silenced fruits. Isomeric composition of fruits silenced at different metabolic steps suggested the existence of three functional units, comprising PSY1, PDS/ZISO, and ZDS/CrtISO, and responsible for the synthesis of 15-cis-phytoene, 9,9'-di-cis-zeta-carotene, and all-trans-lycopene, respectively. Silencing of a desaturase (PDS or ZDS) resulted in the induction of the isomerase in the same functional unit (ZISO or CrtISO, respectively). All-trans-zeta-carotene was detectable in nonsilenced fruits, greatly increased in ZDS-silenced ones, and disappeared in CrtISO-Like1-/CrtISO-Like2-silenced ones, suggesting the existence of a metabolic side branch, comprising this compound and initiated by the latter enzymes.