Ahrazem Oussama, Diretto Gianfranco, Argandoña Javier, Rubio-Moraga Ángela, Julve José Manuel, Orzáez Diego, Granell Antonio, Gómez-Gómez Lourdes
Instituto Botánico, Departamento de Ciencia y Tecnología Agroforestal y Genética, Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain.
Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Campus Tecnológico de la Fábrica de Armas, Avda, Carlos III s/n, E-45071 Toledo, Spain.
J Exp Bot. 2017 Jul 20;68(16):4663-4677. doi: 10.1093/jxb/erx277.
Crocetin, one of the few colored apocarotenoids known in nature, is present in flowers and fruits and has long been used medicinally and as a colorant. Saffron is the main source of crocetin, although a few other plants produce lower amounts of this apocarotenoid. Notably, Buddleja davidii accumulates crocetin in its flowers. Recently, a carotenoid dioxygenase cleavage enzyme, CCD2, has been characterized as responsible for crocetin production in Crocus species. We searched for CCD2 homologues in B. davidii and identified several CCD enzymes from the CCD1 and CCD4 subfamilies. Unexpectedly, two out of the three CCD4 enzymes, namely BdCCD4.1 and BdCCD4.3, showed 7,8;7',8' activity in vitro and in vivo over zeaxanthin. In silico analyses of these enzymes and CCD2 allowed the determination of key residues for this activity. Both BdCCD4 genes are highly expressed during flower development and transcripts levels parallel the accumulation of crocins in the petals. Phylogenetic analysis showed that BdCCD4.2 grouped with almost all the characterized CCD4 enzymes, while BdCCD4.1 and BdCCD4.3 form a new sub-cluster together with CCD4 enzymes from certain Lamiales species. The present study indicates that convergent evolution led to the acquisition of 7,8;7',8' apocarotenoid cleavage activity in two separate CCD enzyme families.
藏红花素是自然界中已知的少数几种有色类胡萝卜素之一，存在于花朵和果实中，长期以来一直被用于医药和作为着色剂。藏红花是藏红花素的主要来源，尽管其他一些植物也能产生少量这种类胡萝卜素。值得注意的是，大叶醉鱼草在其花朵中积累藏红花素。最近，一种类胡萝卜素双加氧酶裂解酶CCD2已被鉴定为负责番红花属植物中藏红花素的产生。我们在大叶醉鱼草中搜索了CCD2同源物，并从CCD1和CCD4亚家族中鉴定出了几种CCD酶。出乎意料的是，三种CCD4酶中的两种，即BdCCD4.1和BdCCD4.3，在体外和体内对玉米黄质均表现出7,8；7',8'活性。对这些酶和CCD2的计算机分析确定了这种活性的关键残基。两个BdCCD4基因在花发育过程中均高度表达，转录本水平与花瓣中藏红花素的积累平行。系统发育分析表明，BdCCD4.2与几乎所有已鉴定的CCD4酶归为一类，而BdCCD4.1和BdCCD4.3与某些唇形目物种的CCD4酶一起形成了一个新的亚簇。本研究表明，趋同进化导致在两个不同的CCD酶家族中获得了7,8；7',8'类胡萝卜素裂解活性。
