Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases and correlation with grain protein content

Abstract

Background: Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutamine synthetase (GS), GOGAT maintains the flow of nitrogen from NH4 (+) into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis.

Results: The aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively) indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes.

Conclusion: The three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved - consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16-17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence conservation within enzyme functional domains remains high. Higher expression levels of NADH-GOGAT are associated with higher grain protein content in two durum wheats.

Figure 1. Exon/intron organization of wheat NADH-GOGAT genes.

A. A genome NADH-GOGAT, B. B genome NADH-GOGAT and C. D genome NADH-GOGAT. Exons are indicated by numbered boxes and introns by intervening lines. Start and stop codon positions of the full-length coding regions are marked.

Figure 2. GOGAT sequence comparisons.

NADH-GOGAT DNA sequences from start to stop codons, including both exons and introns are compared to show relative conservation of sequences. A. Sequences of THE NADH-GOGAT genes from the A- and B-genomes. Match criterion of 80% match over a 20 residue window. B. DNA sequences from start to stop codons were compared between indicated plants using a criterion on 80% match over a 20 base window. Dot blot diagonals are arrayed horizontally for comparison. In both frames the color-coded heat map represents a composite of the degree and length of sequence similarity.

Figure 3. Comparison of three hexaploid NADH-GOGAT amino acid sequences.

The three cv Chinese Spring NADH-GOGAT polypeptide sequences are aligned. Genomes are indicated on the right by letter of the genome (A, B, D). Differences in amino acid residues are indicated by yellow shading.

Figure 4. Dicot vs Moncot NADH-GOGAT amino acid sequences.

The derived amino acid sequence of the wheat B-genome NADH-GOGAT protein is compared to sequences derived from DNA sequences of other plants (poplar, grape, Arabidopsis (Arab.), maize, rice, and Brachypodium (Brachy.). Amino acid residues conserved in all aligned proteins are shaded in yellow. Amino acids unique to monocots are shaded blue. Vertical red lines indicate exon boundaries. Exon numbers, such as ‘X5’ for exon 5, are to the right of the exon boundary lines. Known or presumptive functional domains of the NADH-GOGAT enzyme are below the alignment – with approximate domain boundaries indicated by vertical green lines.

Figure 5. Physical mapping of the NADH-GOGAT gene on chromosome 3A.

A gene specific marker was amplified in cvs Ciccio (C), Svevo (S), Chinese Spring (CS) and a set of nullitetrasomic and bin deletion lines. The 1 kb fragment was absent in the nulli-3A-tetra-3D line, in the ditelo-3AS, and in the bin line 3AL-4-0.42, as indicated by arrows.

Figure 6. NADH-GOGAT primers.

NADH-GOGAT-3A and NADH-GOGAT-3B specific primer combinations used in qRT-PCR analysis. The boxed base indicates the change in the NADH-GOGAT-3B forward primer to increase specificity. Primers are listed in Table 2.

Figure 7. qRT-PCR of NADH-GOGAT.

qRT-PCR conducted for NADH-GOGAT-3A and NADH-GOGAT-3B genes with specific probes. The figure shows: A. The expression levels of the two genes in the roots of the two durum wheat cvs Ciccio and Svevo (C and S, respectively); B. Comparison of the two homoeologous gene expressions in cv Ciccio during the stage of first leaf, flowering and grain filling; D. Comparison of the two homoeologous gene expressions in cv Svevo during the stage of first leaf, flowering and grain filling;. The error bars indicate the ± SE of the mean.