Gain‐of‐function mutations in beet DODA2 identify key residues for betalain pigment evolution

First author: Alexander Bean; Affiliations: The University of Texas at Austin (得克萨斯大学奥斯汀分校): Austin, USA

Corresponding author: Alan Lloyd

The key enzymatic step in betalain biosynthesis (甜菜红色素生物合成) involves conversion of l‐3,4‐dihydroxyphenylalanine (l‐DOPA; 3,4-二羟苯丙氨酸，俗名：L-多巴) to betalamic acid (甜菜醛氨酸). One class of enzymes capable of this is 3,4‐dihydroxyphenylalanine 4,5‐dioxygenase (DODA; 3,4-二羟苯丙氨酸4,5-双加氧酶). In betalain‐producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain‐ onfunctional DODA into a betalain‐functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets (甜菜), Arabidopsis and yeast, and gene silencing was performed by virus‐induced gene silencing. Site‐directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris(甜菜) andMirabilis jalapa(紫茉莉) both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site‐directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales (石竹目) species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain‐ onfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants.

甜菜红色素生物合成最关键的酶促反应在于将L-3,4-二羟苯丙氨酸转化为甜菜醛氨酸，该步反应由一类3,4-二羟苯丙氨酸4,5-双加氧酶DODA基因催化。在产甜菜红色素的物种中，DODA基因具有多个旁系同源基因。本文的研究揭示了哪些旁系同源基因作用于甜菜红色素的生物合成，以及哪些关键的氨基酸残基变化导致这些旁系同源基因演化成具有酶活的DODA。作者在甜菜、拟南芥和酵母中测试了两组DODA旁系同源基因的表达，并通过病毒诱导型沉默实施基因沉默。通过对位点的直接突变鉴定了一些对于甜菜红色素生物合成必要的氨基酸残基。甜菜和紫茉莉都有能够在甜菜红色素生物合成中发挥功能的DODA1基因，并且同时具有另外一个不具备功能的DODA2基因。通过对于之前已知的一个非功能性的DODA进行位点突变使得甜菜红色素能够正常进行生物合成，说明了DODA基因能够在甜菜红色素生物合成过程中发挥作用的关键氨基酸残基。DODA旁系同源基因的功能分化，一个能够参与甜菜红色素的生物合成，而另一个不能，这种特性在大多的石竹目物种中都存在。至少需要改变七个氨基酸残基才能够使得非功能性的DODA旁系同源基因获得甜菜红色素酶活性，揭示了植物中参与甜菜红色素通路的演化。

通讯：Alan Lloyd (https://cns.utexas.edu/directory/item/16-molecular-biosciences/193-lloyd-alan-m?Itemid=349)

研究方向：植物发育机制和色素合成通路。

doi: https://doi.org/10.1111/nph.15159

Journal: New Phytologist

First Published date: 13 May, 2018

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