Identification, Characterization, and Expression Analysis of Carotenoid Biosynthesis Genes and Carotenoid Accumulation in Watercress (Nasturtium officinale R. Br.)

Abstract

Watercress (Nasturtium officinale R. Br.) is an important aquatic herb species belonging to the Brassicaceae family. It has various medicinal properties and has been utilized for the treatment of cancer and other diseases; however, currently available genomic information regarding this species is limited. Here, we performed the first comprehensive analysis of the carotenoid biosynthesis pathway (CBP) genes of N. officinale, which were identified from next-generation sequencing data. We identified and characterized 11 putative carotenoid pathway genes; among these, nine full and two partial open reading frames were determined. These genes were closely related to CBP genes of the other higher plants in the phylogenetic tree. Three-dimensional structure analysis and multiple alignments revealed several distinct conserved motifs, including aspartate or glutamate residues, carotene-binding motifs, and dinucleotide-binding motifs. Quantitative reverse transcription-polymerase chain reaction results showed that the CBP was expressed in a tissue-specific manner: expression levels of NoPSY, NoPDS, NoZDS-p, NoCrtISO, NoLCYE, NoCHXE-p, and NoCCD were highest in the flower, whereas NoLCYB, NoCHXB, NoZEP, and NoNCED were highest in the leaves. Stems, roots, and seeds did not show a significant change in the expression compared to the leaves and flowers. High-performance liquid chromatography analysis of the same organs showed the presence of seven distinct carotenoid compounds. The total carotenoid content was highest in the leaves followed by flowers, seeds, stems, and roots. Among the seven individual carotenoids, the levels of six carotenoids (i.e., 13-Z-β-carotene, 9-Z-β-carotene, E-β-carotene, lutein, violaxanthin, and β-cryptoxanthin) were highest in the leaves. The highest content was observed for lutein, followed by E-β-carotene, and 9-Z-β-carotene; these carotenoids were much higher in the leaves compared to the other organs. The results will be useful references for further molecular genetics and functional studies involving this species and other closely related species.

Figure 1

Schematic view of the CBP in Nasturtium officinale. Enzymes are shown in blue, and the pink asterisk represents the gene identified and characterized in this study. Solid black arrows denote biosynthesis, and dotted black arrows denote degradation of carotenoids. IPI-Isopentenyl pyrophosphate isomerase. The pathway scheme is adapted and modified from the study by Sathasivam et al.

Figure 2

Phylogenetic analysis based on the concatenated amino acid sequences of 11 CBP genes. The NJ phylogenetic tree was drawn with the Poisson-correction distance. The number at each node denotes the percentage in the bootstrap analysis (1000 replicates), whereas the numbers below the branch points represent bootstrap values. The outgroup is Eutrema salsugineum.

Figure 3

Predicted 3D structure of upstream CBP genes of Nasturtium officinale. (A) NoPSY, (B) NoPDS, (C) NoZDS-p (partial ORF), and (D) NoCrtISO structures were generated using Chimera 1.14 software. The amino (NH₂) and carboxyl (COOH) terminals are presented in blue and dark red, respectively. In these 3D structures, α-helices and β-strands are shown in light sea green and hot pink, respectively. For the sequence alignment of each gene, see Φιγυρε Σ3.

Figure 4

Predicted 3D structure of downstream CBP genes of Nasturtium officinale. (A) NoLCYB, (B) NoLCYE,(C) NoCHXB-p, (D) NoZEP, (E) NoCCD, and (F) NoNCED structures were generated using Chimera 1.14 software. The amino (NH₂) and carboxyl (COOH) terminals are shown in blue and dark red, respectively. In these 3D structures, α-helices and β-strands are shown in light sea green and hot pink, respectively. For sequence alignment of each gene, see Φιγυρε Σ3.

Figure 5

Comparison of tomato and watercress PSY nucleotide sequences. The tomato PSY sequence was retrieved from the previous manuscript published by Gady et al. (A) Multiple alignments of the SlPSY and NoPSY protein sequence were performed with the BioEdit program. The yellow highlighted represents the change in the amino acid sequence. Predicted 3D structures, (B) SlPSY, and (C) NoPSY. The amino (NH₂) and carboxyl (COOH) termini are presented in blue and dark red, respectively. In these structures, α-helices and β-strands are shown in light sea green and hot pink, respectively. The changes in the amino acid sequence in the structural region are indicated.

Figure 6

Relative gene expression profiles of 11 CBP genes of Nasturtium officinale. (A) Transcriptional levels of CBP genes were analyzed in different tissues such as leaf, stem, root, flower, and seed using qRT-PCR analysis. The relative gene expression was calculated using ubiquitin-conjugating enzyme 9 (UBC9). Results are given as the means of triplicates ± SD. Letters a–e denote significant differences (p < 0.05). (B) Heat map showing the expression profiles of CBP genes in five different tissues namely leaf, stem, root, flower, and seed. The heat map was generated using fold change values obtained from qRT-PCR. The tree view of hierarchical clustering was used to show the organ-specific expression of CBP genes. A gradient color bar at the top is used to illustrate whether the CBP genes are upregulated (red) or downregulated (green).

Figure 7

Carotenoid content in the different tissues of Nasturtium officinale. For HPLC analysis, samples were harvested from 2-month-old plants. Results are given as the means of triplicates ± SD. Letters a–e denote significant differences (p < 0.05).

Figure 8

Overview of carotenoid pathway gene expression and carotenoid accumulation changes in different plant organs of N. officinale. Each colored box (left to right) under each gene and compound represents F-Flower; L-Leaf; R-Root; S-Seed; St-Stem. The scale bar indicates the transformed average value of gene expression level and metabolites, and the colored square boxes (gene expression level (green to red) and carotenoid content (dark blue to dark red)) represent the relative gene expression level and metabolite abundance in different plant organs.