Abstract
Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.
Highlights
The first committed step of the carotenoid biosynthetic pathway, the formation of phytoene from two molecules of geranylgeranyl pyrophosphate (GGPP), is catalyzed by the enzyme phytoene synthase (PSY)
PSY is encoded by a single copy gene (AtPSY, At5g17230) in Arabidopsis thaliana, controlling the flux and responses of the pathway is limited to regulating this unique enzyme [5]
We have recently reported that PSY genes have been retained in a triplicated state in both A- and C- Brassica diploid genomes and that B. napus bears the largest plant PSY gene family described to date [14]
Summary
The first committed step of the carotenoid biosynthetic pathway, the formation of phytoene from two molecules of geranylgeranyl pyrophosphate (GGPP), is catalyzed by the enzyme phytoene synthase (PSY). Since GGPP serves as a precursor for the synthesis of tocopherols, chlorophylls, plastoquinones and gibberellins, PSY gene expression is highly regulated and represents an important checkpoint for controlling the flux (of carbon) into the carotenoid biosynthetic pathway [1,2,3,4,5,6]. Contain small PSY gene families composed of two or three members [7,8,9,10,11,12] In these plant species, subfunctionalization of PSY gene expression has been described mainly between photosynthetic and non photosynthetic tissues [7, 8, 13, 14]. It has been shown that specific PSY paralogues can mediate the stress-induced production of abscisic acid (ABA) in roots [11, 12, 15]
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