Characterization of Aldehyde Oxidase (AO) Genes Involved in the Accumulation of Carotenoid Pigments in Wheat Grain

Abstract

Aldehyde Oxidase (AO) enzyme (EC 1.2.3.1) catalyzes the final steps of carotenoid catabolism and it is a key enzyme in the abscisic acid (ABA) biosynthesis. AO isoforms are located in the cytosolic compartment of tissues in many plants, where induce the oxidation of aldehydes into carboxylic acid, and in addition, catalyze the hydroxylation of some heterocycles. The goal of the present study was to characterize the AO genes involved in the accumulation of carotenoid pigments in wheat grain, an important quantitative trait controlled by multiple genes. The cDNAs corresponding to the four AO isoforms from Arabidopsis thaliana and five AO isoforms from Brachypodium distachyon were used as query in 454 sequence assemblies data for Triticum aestivum cv. Chinese Spring (https://urgi.versailles.inra.fr/blast/blast.php) to obtain the partial or whole orthologous wheat AO sequences. Three wheat isoforms, designated AO1, AO2, and AO3 were located on the chromosome groups 2, 5, and 7, respectively, and mapped on two consensus wheat maps by SNP markers located within the AO gene sequences. To validate the possible relationships between AO3 genes and carotenoid accumulation in wheat, the expression levels of AO-A3 and AO-B3 gene were determined during the kernel maturation stage of two durum wheat cultivars, Ciccio and Svevo, characterized by a low and high carotenoid content, respectively. Different AO-A3 gene expression values were observed between the two cultivars indicating that the AO-A3 allele present in Ciccio was more active in carotenoid degradation. A gene marker was developed and can be used for marker-assisted selection in wheat breeding programs.

Keywords: SNP; aldehyde oxidase; carotenoid genes; wheat; yellow pigments.

Figure 1

Simplified representation of the abscisic acid pathway. The conversion of β-carotene to abscisic acid is catalyzed by the β-carotene hydroxylases (BCH1/2), zeaxanthin epoxidase (ZEP), violaxanthin de-epoxidase (VDE), neoxanthin synthase (NXS), 9-cis-epoxycarotenoid dioxygenase (NCDE) and short-chain alcohol dehydrogenase (ABA2). The last step involves the aldehyde oxidese (AO), responsible of the synthesis of abscisic acid (black arrow).

Figure 2

Schematic representation of the durum wheat linkage map and AO markers. Each linkage map derives from the durum consensus map (Maccaferri et al., 2015) and has been represented by a SNP marker every about 20 cM. SSR markers have been also inserted every about 20 cM to compare the consensus SNP map with published SSR-based maps.

Figure 3

Comparison of AO3 gene structures in rice, Brachypodium, and wheat is shown based on colored boxes highlighting conserved exons. Intron and exon sizes are shown as well as the whole gene (in brackets the total length). Rice and Brachypodium AO share the same structure with ten exons of conserved sizes and nine introns. Brachypodium and wheat AO3 show an high similarity in sequence and structure for eight exons. Black dashed line indicates the absence of intron sequence since only a cDNA sequence has been found for AO-B3.

Figure 4

Comparison of the expression levels of AO-A3 and AO-B3 genes in kernel tissue of cv. Svevo and cv. Ciccio by qRT-PCR. The y ax shows the normalized fold expression. The error bars indicate the ± SE of the mean.

Figure 5

Expression analysis from PLEXdb database of all wheat AO genes in a wide range of tissues and developmental stages in wheat (1–13). The asterisk signals indicate, respectively the values higher or lower than the mean values ± 2 SD.

Figure 6

Optimization of DHPLC analysis for SNP detection between cvs. Ciccio and Svevo at locus IWB59875. The chromatograms correspond to the elution profiles of homoduplex molecules (cv Ciccio in black, cv Svevo in dark blue), and of homoduplexes plus heteroduplexes derived from 1:1 mixed DNA (pink line).