Identification of a regiospecific S-oxygenase for the production of marasmin in traditional medicinal plant Tulbaghia violacea.

Marasmin [S-(methylthiomethyl)-L-cysteine-4-oxide] is a pharmaceutically valuable sulfur-containing compound produced by the traditional medicinal plant, Tulbaghia violacea. Here, we report the identification of an S-oxygenase, TvMAS1, that produces marasmin from its corresponding sulfide, S-(methylthiomethyl)-L-cysteine. The amino acid sequence of TvMAS1 showed high sequence similarity to known flavin-containing S-oxygenating monooxygenases in plants. Recombinant TvMAS1 catalyzed regiospecific S-oxygenation at S4 of S-(methylthiomethyl)-L-cysteine to yield marasmin, with an apparent K m value of 0.55 mM. TvMAS1 mRNA accumulated with S-(methylthiomethyl)-L-cysteine and marasmin in various organs of T. violacea. Our findings suggest that TvMAS1 catalyzes the S-oxygenation reaction during the last step of marasmin biosynthesis in T. violacea.

The ability of callus tissues induced from three Allium plants to accumulate health-beneficial natural products, S-alk(en)ylcysteine sulfoxides.

S-Alk(en)ylcysteine sulfoxides (CSOs), such as methiin, alliin, and isoalliin, are health-beneficial natural products biosynthesized in the genus Allium. Here, we report the induction of multiple callus tissue lines from three Allium vegetables, onion (A. cepa), Welsh onion (A. fistulosum), and Chinese chive (A. tuberosum), and their ability to accumulate CSOs. Callus tissues were initiated and maintained in the presence of picloram and 2-isopentenyladenine as auxin and cytokinin, respectively. For all plant species tested, the callus tissues almost exclusively accumulated methiin as CSO, while the intact plants contained a substantial amount of isoalliin together with methiin. These results suggest that the cellular developmental conditions and the regulatory mechanisms required for the biosynthesis of methiin are different from those of alliin and isoalliin. The methiin content in the callus tissues of onion and Welsh onion was much higher compared to that in the intact plants, and its cellular concentration could be estimated as 1.9-21.7 mM. The activity of alliinase that degrades CSOs in the callus tissues was much lower than that of the intact plants for onion and Welsh onion, but at similar levels as in the intact plants for Chinese chive. Our findings that the callus tissues of onion and Welsh onion showed high methiin content and low alliinase activity highlighted their potential as a plant-based system for methiin production.

S-Alk(en)ylcysteine sulfoxides in the genus Allium: proposed biosynthesis, chemical conversion, and bioactivities.

S-Alk(en)ylcysteine sulfoxides are sulfur-containing natural products characteristic of the genus Allium. Both the flavor and medicinal properties of Allium plants are attributed to a wide variety of sulfur-containing compounds that are generated from S-alk(en)ylcysteine sulfoxides. Previous radiotracer experiments proposed that S-alk(en)ylcysteine sulfoxides are biosynthesized from glutathione. The recent identification of γ-glutamyl transpeptidases and a flavin-containing S-oxygenase involved in the biosynthesis of S-allylcysteine sulfoxide (alliin) in garlic (Allium sativum) provided insights into the reaction order of deglutamylation and S-oxygenation together with the localization of the biosynthesis, although the rest of the enzymes in the pathway still await discovery. In intact plants, S-alk(en)ylcysteine sulfoxides are stored in the cytosol of storage mesophyll cells. During tissue damage, the vacuolar enzyme alliinase contacts and hydrolyzes S-alk(en)ylcysteine sulfoxides to produce the corresponding sulfenic acids, which are further converted into various sulfur-containing bioactive compounds mainly via spontaneous reactions. The formed sulfur-containing compounds exhibit bioactivities related to pathogen defense, the prevention and alleviation of cancer and cardiovascular diseases, and neuroprotection. This review summarizes the current understanding of the occurrence, biosynthesis, and alliinase-triggered chemical conversion of S-alk(en)ylcysteine sulfoxides in Allium plants as well as the impact of S-alk(en)ylcysteine sulfoxides and their derivatives on medicinal, food, and agricultural sciences.

Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

Transcriptomic landscape of Pueraria lobata demonstrates potential for phytochemical study.

Pueraria lobata (Willd.) Ohwi has a long and broad application in the treatment of disease. However, in the US and EU, it is treated as a notorious weed. The information to be gained from decoding the deep transcriptome profile would facilitate further research on P. lobata. In this study, more than 93 million fastq format reads were generated by Illumina's next-generation sequencing approach using five types of P. lobata tissue, followed by CLC de novo assembly methods, ultimately yielding about 83,041 contigs in total. Then BLASTx similarity searches against the NCBI NR database and UniProtKB database were conducted. Once the duplicates among BLASTx hits were eliminated, ID mapping against the UniProt database was conducted online to retrieve Gene Ontology information. In search of the putative genes relevant to essential biosynthesis pathways, all 1,348 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. Enzymes related to the isoflavonoid and flavonoid biosynthesis pathways were focused for detailed investigation and subsequently, quantitative real-time reverse transcription polymerase chain reaction was conducted for biological validation. Metabolites of interest, puerarin and daidzin were studied by HPLC. The findings in this report may serve as a footstone for further research into this promising medicinal plant.

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots.

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.