Genome-wide identification and characterization of the GDP-L-galactose phosphorylase gene family in bread wheat

Abstract

Background: Ascorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting enzyme of the L-galactose pathway-the dominant ascorbate biosynthetic pathway in plants-and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5'-untranslated region (5'UTR). The GGP genes have yet to be identified in bread wheat (Triticum aestivum L.), one of the most important food grain sources for humans.

Results: Bread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes (TaGGP2-A/B/D and TaGGP1-A/B/D, respectively) and a highly conserved uORF was present in the long 5'UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta.

Conclusions: We have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium/Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta. The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.

Keywords: Ascorbic acid; Gene expression; Phylogeny; Synteny; Transient expression; Upstream open reading frame; Vitamin C.

Fig. 1

Gene structure of the TaGGP genes from bread wheat cv. Chinese Spring. The uORF (orange box), coding sequence (CDS, black box), introns (grey box), and UTR (lines) of the TaGGP genes are depicted and the length (bp) of each section indicated

Fig. 2

Amino acid sequence alignment of GGP proteins from a range of graminaceous species. Green, olive green, yellow and white background colour represents 100%, 80 to 100%, 60 to 80%, and less than 60% conservation of amino acids between species, respectively. The HIT motif (HφHφQ, where φ is a hydrophobic amino acid) of the HIT protein superfamily and the KKRP NLS are outlined in red. The 13 residues across the protein that distinguish GGP1 proteins from GGP2 proteins within the graminaceous species are indicated by an asterisk. The prefixes for the graminaceous species are as follows: Aet is Aegilops tauschii; Bd is Brachypodium distachyon; Hv is Hordeum vulgare; Os is Oryza sativa; Sb is Sorghum bicolor; Ta is Triticum aestivum; and Zm is Zea mays

Fig. 3

Amino acid sequence alignment of GGP uORFs from a range of graminaceous and non-graminaceous species. Green, olive green, yellow, and white background colour represents 100%, 80 to 100%, 60 to 80%, and less than 60% conservation of amino acids between species, respectively. The 45th residue of the consensus sequence distinguishing the graminaceous (A; alanine) from the non-graminaceous (E; glutamic acid) species is indicated with an asterisk. The truncated 11 residues from the TaGGP2, HvGGP2, BdGGP2, and AetGGP2 uORF peptide sequences relative to the TaGGP1, HvGGP1, BdGGP1, and AetGGP1 uORF peptide sequences are outlined in red. The prefixes for the graminaceous species are the same as those presented in Fig. 1. The prefixes for the non-graminaceous species are as follows: Ad is Actinidia deliciosa; At is Arabidopsis thaliana; Cs is Cucumis sativus; Fv is Fragaria vesca; Gm is Glycine max; Ls is Lactuca sativa; Md is Malus x domestica; Mt is Medicago truncatula; Nb is Nicotiana benthamiana; Pt is Populus trichocarpa; Sl is Solanum lycospersicum; and Vv is Vitis vinifera

Fig. 4

An unrooted phylogenetic tree of GGP proteins from a range of graminaceous and non-graminaceous species. Black nodes (●) represent weak bootstrap values (< 75%). The scale bar corresponds to evolutionary distance in substitutions per site and the numbers correspond to bootstrap percentage. The prefixes for the species are the same as those presented in Figs. 1 and 2

Fig. 5

Microsynteny analysis of the TaGGP genes from bread wheat cv. Chinese Spring. (a) TaGGP1 and (b) TaGGP2 homoeologous SRs on chromosomal groups 5 and 4, respectively. Syntenic genes are represented by arrow colour. The orientation of the genes is indicated by the direction of the arrows. The physical length (Mbp) of each of the TaGGP SRs is provided for each chromosome. The physical position (Mbp) of the TaGGP genes and the centromeres (black oval) are also presented. The physical distance (Mbp) between the proximal gene of the SR and the respective centromere (black dotted line) is 58.2, 22.2 and 4.4 for the TaGGP1-A, TaGGP1-B, and TaGGP1-D SRs, respectively, and 127.5, 203.8 and 104.6 for the TaGGP2-A, TaGGP2-B, and TaGGP2-D SRs, respectively. The list of genes used in this analysis and their respective position and gene function are provided in Additional file 2: Table S1

Fig. 6

Quantitative reverse transcription-PCR analysis of the TaGGP genes from bread wheat cv. Chinese Spring. Relative expression of the (a) TaGGP1 and (b) TaGGP2 homoeologs is provided in: (1) embryonic root; (2) mesocotyl; (3) seedling root; (4) crown; (5) seedling leaf; (6) bracts; (7) anthers; (8) pistil; (9) caryopsis 3–5 DAP; and (10) embryo 22 DAP. The geometric mean of TaActin, TaCyclophilin, and TaELF were used as normalisation factors. Error bars indicate SEM of three technical replicates derived from a bulk of three independent biological samples

Fig. 7

Transient transformation of the TaGGP coding sequences in N. benthamiana. a Schematic representation of the T-DNAs used for constitutive overexpression of the TaGGP genes in N. benthamiana. RB, right border; 2 x 35S, dual CaMV 35S promoter; TaGGP, coding sequence of TaGGP1-A (1,293 bp), TaGGP1-B (1,293 bp), TaGGP1-D (1,293 bp), TaGGP2-A (1,296 bp), and TaGGP2-B (1,296 bp); nos T, nopaline synthase terminator; 2 x 35S enhanced, dual CaMV 35S promoter enhanced; hptII, hygromycin phosphotransferase II; pA, CaMV poly(A) signal; LB, left border. b Ascorbate concentrations of N. benthamiana leaves co-infiltrated with A. tumefaciens (GV3101 MP90) containing the constructs of interest and a P19 construct to prevent post-transcriptional gene silencing. The control was infiltrated with A. tumefaciens containing the P19 construct alone. The AcGGP gene from kiwifruit driven by a single 35S promoter in the pGreen vector system was used as a positive control. Bars represent mean ± SEM of three infiltrated young leaves. Means that do not share a letter are significantly different (one-way ANOVA followed by Tukey post-hoc test with 95% confidence level)