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

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-ß-carotene, 9-Z-ß-carotene, E-ß-carotene, lutein, violaxanthin, and ß-cryptoxanthin) were highest in the leaves. The highest content was observed for lutein, followed by E-ß-carotene, and 9-Z-ß-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.

Transcriptome Analysis and Metabolic Profiling of Green and Red Mizuna (Brassica rapa L. var. japonica).

Mizuna (Brassica rapa L. var. japonica), a member of the family Brassicaceae, is rich in various health-beneficial phytochemicals, such as glucosinolates, phenolics, and anthocyanins. However, few studies have been conducted on genes associated with metabolic traits in mizuna. Thus, this study provides a better insight into the metabolic differences between green and red mizuna via the integration of transcriptome and metabolome analyses. A mizuna RNAseq analysis dataset showed 257 differentially expressed unigenes (DEGs) with a false discovery rate (FDR) of <0.05. These DEGs included the biosynthesis genes of secondary metabolites, such as anthocyanins, glucosinolates, and phenolics. Particularly, the expression of aliphatic glucosinolate biosynthetic genes was higher in the green cultivar. In contrast, the expression of most genes related to indolic glucosinolates, phenylpropanoids, and flavonoids was higher in the red cultivar. Furthermore, the metabolic analysis showed that 14 glucosinolates, 12 anthocyanins, five phenolics, and two organic acids were detected in both cultivars. The anthocyanin levels were higher in red than in green mizuna, while the glucosinolate levels were higher in green than in red mizuna. Consistent with the results of phytochemical analyses, the transcriptome data revealed that the expression levels of the phenylpropanoid and flavonoid biosynthesis genes were significantly higher in red mizuna, while those of the glucosinolate biosynthetic genes were significantly upregulated in green mizuna. A total of 43 metabolites, such as amino acids, carbohydrates, tricarboxylic acid (TCA) cycle intermediates, organic acids, and amines, was identified and quantified in both cultivars using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS). Among the identified metabolites, sucrose was positively correlated with anthocyanins, as previously reported.

Identification and analysis of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation in watercress (Nasturtium officinale R. Br.).

Watercress (Nasturtium officinale R. Br.) is a cruciferous plant consumed by people worldwide. This vegetable contains numerous health-benefiting compounds; however, gene information and metabolomic profiling of individual parts for this plant species are scarce. In this study, we investigated the expression patterns of phenylpropanoid biosynthetic genes and the content of phenylpropanoids in different parts of watercress. We identified 11 genes encoding enzymes related to the phenylpropanoid biosynthetic pathway and analyzed the expression patterns of these genes in the leaves, stems, roots, flowers, and seeds of watercress. The result showed that most of the genes were expressed at the highest levels in the flowers. HPLC analysis performed in samples from these same parts revealed the presence of seven phenylpropanoid-derived compounds. The content of total phenylpropanoids was the highest in flowers, followed by that in the leaves, whereas the lowest level was generally detected in the stems. Rutin was the most abundant phenylpropanoid in all plant segments, while quercetin was detected only in the flowers and roots. This study provides useful information for further molecular and functional research involving N. officinale and closely related species.

Comparative analysis of glucosinolate production in hairy roots of green and red kale (Brassica oleracea var. acephala).

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that function in plant defense and provide benefits to human health. In this study, using Agrobacterium rhizogenes R1000, green and red kale hairy roots were established. The expression levels of GSLs biosynthesis genes and their accumulation in both kale hairy roots were analyzed by quantitative real-time PCR and HPLC. The results showed that the expression of most indolic GSLs biosynthesis genes was higher in the hairy roots of green kale than in that of red kale. In contrast, the expression of BoCYP83A1 and BoSUR1 encoding key enzymes aromatic GSL biosynthesis was significantly higher in red kale hairy root. The HPLC analysis identified six GSLs. The levels of 4-methoxyglucobrassicin, glucobrassicin, and 4-hydroxyglucobrassicin were 6.21, 5.98, and 2 times higher, respectively, in green kale than in red kale, whereas the levels of neoglucobrassicin and gluconasturtiin were 16.2 and 3.48 times higher, respectively, in red kale than in green kale. Our study provides insights into the underlying mechanisms of GSLs biosynthesis in kale hairy roots and can be potentially used as "biological factories" for producing bioactive substances such as GSLs.

Enhancement of Glucosinolate Production in Watercress ( Nasturtium officinale) Hairy Roots by Overexpressing Cabbage Transcription Factors.

Glucosinolates are secondary metabolites that play important roles in plant defense and human health, as their production in plants is enhanced by overexpressing transcription factors. Here, four cabbage transcription factors (IQD1-1, IQD1-2, MYB29-1, and MYB29-2) affecting genes in both aliphatic and indolic glucosinolates biosynthetic pathways and increasing glucosinolates accumulation were overexpressed in watercress. Five IQD1-1, six IQD1-2, five MYB29-1, six MYB29-2, and one GUS hairy root lines were created. The expression of all genes involved in glucosinolates biosynthesis was higher in transgenic lines than in the GUS hairy root line, in agreement with total glucosinolates contents, determined by high-performance liquid chromatography. In transgenic IQD1-1 (1), IQD1-2 (4), MYB29-1 (2), and MYB29-2 (1) hairy root lines, total glucosinolates were 3.39-, 3.04-, 2.58-, and 4.69-fold higher than those in the GUS hairy root lines, respectively. These results suggest a central regulatory function for IQD1-1, IQD1-2, MYB29-1, and MYB29-2 transcription factors in glucosinolates biosynthesis in watercress hairy roots.

Comparative analysis of glucosinolates and metabolite profiling of green and red mustard (brassica juncea) hairy roots.

Here, accumulation of glucosinolates and expression of glucosinolates biosynthesis genes in green and red mustard hairy roots were identified and quantified by HPLC and qRT-PCR analyses. The total glucosinolates content of green mustard hairy root (10.09 µg/g dry weight) was 3.88 times higher than that of red mustard hairy root. Indolic glucosinolates (glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) in green mustard were found at 30.92, 6.95, and 5.29 times higher than in red mustard hairy root, respectively. Conversely, levels of glucotropaeolin (aromatic glucosinolate) was significantly higher in red mustard than in green mustard. Accumulation of glucoraphasatin, an aliphatic glucosinolate, was only observed only in red mustard hairy roots. Quantitative real-time PCR analysis showed that the expression level of genes related to aliphatic and aromatic glucosinolate biosynthesis were higher in red mustard, exception BjCYP83B. The expression of BjCYP79B2, which encodes a key enzyme involved in the indolic glucosinolate biosynthetic pathway, was higher in green mustard than in red mustard. Additionally, to further distinguish between green mustard and red mustard hairy roots, hydrophilic and lipophilic compounds were identified by gas chromatography-mass spectrometry and subjected to principal component analysis. The results indicated that core primary metabolites and glucosinolate levels were higher in the hairy roots of green mustard than in those of red mustard.

De novo transcriptome analysis and glucosinolate profiling in watercress (Nasturtium officinale R. Br.).

BACKGROUND: Watercress (Nasturtium officinale R. Br.) is an aquatic herb species that is a rich source of secondary metabolites such as glucosinolates. Among these glucosinolates, watercress contains high amounts of gluconasturtiin (2-phenethyl glucosinolate) and its hydrolysis product, 2-phennethyl isothiocyanate, which plays a role in suppressing tumor growth. However, the use of N. officinale as a source of herbal medicines is currently limited due to insufficient genomic and physiological information. RESULTS: To acquire precise information on glucosinolate biosynthesis in N. officinale, we performed a comprehensive analysis of the transcriptome and metabolome of different organs of N. officinale. Transcriptome analysis of N. officinale seedlings yielded 69,570,892 raw reads. These reads were assembled into 69,635 transcripts, 64,876 of which were annotated to transcripts in public databases. On the basis of the functional annotation of N. officinale, we identified 33 candidate genes encoding enzymes related to glucosinolate biosynthetic pathways and analyzed the expression of these genes in the leaves, stems, roots, flowers, and seeds of N. officinale. The expression of NoMYB28 and NoMYB29, the main regulators of aliphatic glucosinolate biosynthesis, was highest in the stems, whereas the key regulators of indolic glucosinolate biosynthesis, such as NoDof1.1, NoMYB34, NoMYB51, and NoMYB122, were strongly expressed in the roots. Most glucosinolate biosynthetic genes were highly expressed in the flowers. HPLC analysis enabled us to detect eight glucosinolates in the different organs of N. officinale. Among these glucosinolates, the level of gluconasturtiin was considerably higher than any other glucosinolate in individual organs, and the amount of total glucosinolates was highest in the flower. CONCLUSIONS: This study has enhanced our understanding of functional genomics of N. officinale, including the glucosinolate biosynthetic pathways of this plant. Ultimately, our data will be helpful for further research on watercress bio-engineering and better strategies for exploiting its anti-carcinogenic properties.