Functional Analysis of the <i>Brassica napus</i> L. Phytoene Synthase (PSY) Gene Family

<div><p>Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including <i>Brassica napus</i> 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 <i>Arabidopsis thaliana</i>, 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 <i>B. napus</i> 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 <i>B. napus</i> 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 <i>B. napus</i> 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 <i>B. napus</i> 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.</p></div

<i>B. napus</i> PSY gene expression during seed development.

<p>A. <i>BnaX.PSY</i> gene expression was determined by RT-PCR using homologue-specific primers. B. <i>B</i>. <i>napus 18S</i> gene expression (loading control). C. Sampling stages of <i>B. napus</i> seed development (from left to right: 20, 35, 40 and 60 days post anthesis) and leaf tissue. L: 100 bp ladder; WC: water control; gDNA: <i>B</i>. <i>napus</i> gDNA control; C+: PSY homologue-specific plasmid controls (positive); C-: PSY homoelogue plasmid controls (negative). RT-PCR (40 cycles) was performed in two biological replicates, only one is shown for simplicity.</p

Phylogenetic relationship of <i>B. napus</i> and selected monocot and dicot PSY enzymes.

<p>The evolutionary history was inferred using the neighbor-joining method using MEGA4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114878#pone.0114878-Tamura1" target="_blank">[38]</a>. The percentage of replicate trees in which the associated taxa clustered together in the 500-bootstrap replication test is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units (number of amino acid substitutions per site) as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114878#pone.0114878-Zuckerkandl1" target="_blank">[39]</a>. Arabidopsis (AtPSY, AAA32836), cassava (MePSY1, ACY42666; MePSY2; ACY42670), maize (ZmPSY1, P49085; ZmPSY2, AAQ91837; ZmPSY3, ABC75827), pepper (CaPSY1, ACE78189.1), rice (OsPSY1, AAS18307; OsPSY2, AAK07735; OsPSY3, ABC75828), sorghum (SbPSY1, AAW28996; SbPSY2, XP002442578; SbPSY3, AAW28997) and tomato (SlPSY1, P08196.2; SlPSY2, ABV68559.1; SlPSY3, XP_004228928.1). The cassava MePSY3 protein sequence was obtained from Phytozome (<a href="http://www.phytozome.net/cassava.php" target="_blank">http://www.phytozome.net/cassava.php</a>) as described in Arango <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114878#pone.0114878-Arango1" target="_blank">[12]</a>.</p

Functional complementation of <i>B. napus</i> PSY genes.

<p><i>E. coli</i> cells were transformed with: (A) pDS1B, a pBAD33 vector carrying <i>E. uredovora</i> carotenogenic genes <i>crtE</i>, <i>crtB</i>, <i>crtI</i>, <i>crtY</i> and <i>CrtX</i>; (B) pDS1B-Δ<i>crtB</i> which has a deletion of the <i>Eu crtB</i> gene + pETBlue1 (empty vector); (C) pDS1B-Δ<i>crtB</i> + pETBnaC.PSY.a; (D) pDS1B- Δ<i>crtB</i> + pETBnaA.PSY.b; (E) pDS1B- Δ<i>crtB</i> + pETBnaA.PSY.c; (F) pDS1B- Δ<i>crtB</i> + pETBnaA.PSY.d; (G) pDS1B- Δ<i>crtB</i> + pETBnaC.PSY.e; and (H) pDS1B- Δ<i>crtB</i> + pETBnaC.PSY.f and HPLC chromatograms obtained at 450 nm are shown for each transformation. The spectral fine spectrum for beta carotene (peak 1) is shown as an example in the control panel (A). The amount (mg) of β-carotene produced by each complementation assay expressed as per gram of dry weight (DW) is shown in (I). Bars represent standard deviation calculated from three replications. For each vector combination, different letters indicate significant differences at p<0.05 determined by Tukey's HSD test.</p