Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses

Abstract

Background: Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3) that have been widely characterized in rice, maize and sorghum.

Results: In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress.

Conclusions: We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses.

Figure 1

Carotenoid biosynthetic pathway (adapted from Luet al.2008). The isopentenyl diphosphate (IPP) is synthesized via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. IPP and DMAPP form the central intermediate geranylgeranyl diphosphate (GGDP) for carotenoid biosynthetic pathway. Phytoene synthase (PSY) acts at the condensation of two molecules of GGDP to form the phytoene, the first step of the carotenoid pathway. Names of compounds are highlighted in black and of enzymes in red. Abbreviations: G3P, glyceraldheyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; ABA, abscisic acid; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate; IPI, isopentenyl diphosphate isomerase; GGPS, geranylgeranyl diphosphate synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotene isomerase; LCYB, β-cyclase; LCYE, ϵ-cyclase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase.

Figure 2

PSY3 gene characterization in bread wheat. (a) The physical position of wheat PSY3 on chromosome group 5 and associated deletion bin are shown. Vertical bars on wheat chromosomes represent chromosome bins. The orthologous chromosomes in rice (Chr9), sorghum (Chr2) and Brachypodium (Chr4) are illustrated by conserved genes linked with black lines. (b) Annotated BAC clones are illustrated by conserved genes (black boxes) linked with black lines as well as Copia (in green), Gypsy (in blue) and class II (in red) transposable elements. (c) Comparison of PSY3 gene structures in Grasses (maize, rice, sorghum, Brachypodium and wheat) is shown based on coloured boxes highlighting conserved exons. Intron and exon sizes are shown as well as the total gene (in brackets) and CDS sizes. Maize and rice PSY3 share the same structure with six exons of conserved sizes and five introns. Their third intron shows a difference in size but in both rice and maize its 5′ starts with the same GC motif (highlighted in red), instead of GT. Sorghum and Brachypodium PSY3 show a structure of five exons and four introns. Wheat PSY3 have lost two introns probably due to the illegitimate recombination between the inversed repeated motives, TGG|CCA and CGG|CCG, identified at the deletion breakpoints (highlighted in red). White boxes represent MITEs identified on rice PSY3 sequence. Blue arrows show the position of primers designed on Brachypodium PSY3 sequence and used for BAC library screening. Red arrows correspond to specific primers, designed on intron sequence and used to assign BAC clones to A, B and D sub-genomes in wheat

Figure 3

Mechanism driving intron-exon shuffling ofPSY3 genes in Grasses. (a)PSY3 gene evolution among Grasses. The modern structure of the PSY3 gene identified in rice, wheat, Brachypodium, maize and sorghum is shown at the bottom with coloured boxes representing conserved exons. The ancestral as well as pooideae and panicoideae PSY3 is structured in five introns and lineage-specific intron losses and consequently exon fusions in wheat, Brachypodium and sorghum are illustrated according to the text description. (b) Representation of intron loss mechanism identified for the wheat PSY3 with inverted and repeated motives that may have driven intron loss through replication slippage via the formation and a DNA loop. (c) Motifs identified at the deletion breakpoints and involved in intron loss due to illegitimate recombination at the splicing site are shown in red.

Figure 4

PSY gene evolutionary scenario in Angiosperms. The modern monocots (exemplified by the rice genome structured in 12 chromosomes) and eudicots (exemplified by the grape genome structured in 19 chromosomes) are illustrated at the bottom using a color code that reflect the ancestral genome structure of respectively 5 and 7 protochromosomes according to Abrouk et al.[28]. The angiosperm ancestor may contain two PSY gene copies, i.e. PSY1 and PSY2. The two copies are located on the protochromosomes A10 and A19 of the eudicot ancestor and both retained in single copies during the WGD shared by the eudicots. In modern eudicots, the PSY gene family is structured in two copies, i.e. PSY1 and PSY2. In monocots PSY1 has been duplicated during a first WGD (referenced as 1) so that the monocot ancestral genome contains three PSY gene copies, i.e. PSY1, PSY2 and PSY3. The three PSY genes are located on ancestral chromosomes A4, A11 and A8 and all retained in single copies during the WGD shared by the monocots (referenced as 2). In modern monocots, the PSY gene family is structured in three copies, i.e. PSY1, PSY2 and PSY3.

Figure 5

Effect of ABA treatment on wheat PSY expression. (a) Experimental design. (b) Expression level of PSYs in non-stressed leaves and roots. Transcript levels were normalized using the wheat spastin and RNase L inhibitor-like cDNA. (c) (d) (e) Figures show PSY response (y-axis) to ABA treatments at the three different tested concentrations (50 μM in c panel, 100 μM in d panel and 150 μM in e panel) in both leaves and roots (see legend) from 0 to 8 hours (x-axis). cDNA and expressed relative to the level detected in not stressed tissues. Each stage has been compared two by two with a confidence level of 95%. The statistical significance in gene expression is shown by an astrisck (*). (f) Efficiency of ABA on PSY3 induction in wheat roots.