Spatio-temporal expression of candidate genes for nectar spur development in Tropaeolum (Tropaeolaceae: Brassicales).

BACKGROUND AND AIMS: Tropaeolaceae (Brassicales) comprise ~100 species native to South and Central America. Tropaeolaceae flowers have a nectar spur, formed by a late expansion and evagination of the fused proximal region of the perianth (i.e. the floral tube). This spur is formed in the domain of the tube oriented towards the inflorescence axis, which corresponds to the adaxial floral region. However, little is known about the molecular mechanisms responsible for the evolution of spurs in Tropaeolaceae. METHODS: In this study, we examined the spatio-temporal expression of genes putatively responsible for differential patterns of cell division between the adaxial and abaxial floral regions in Tropaeolaceae. These genes include previously identified TCP and KNOX transcription factors and the cell division marker HISTONE H4 (HIS4). KEY RESULTS: We found a TCP4 homologue concomitantly expressed with spur initiation and elaboration. Tropaeolaceae possess two TCP4-like (TCP4L) copies, as a result of a Tropaeolaceae-specific duplication. The two copies (TCP4L1 and TCP4L2) in Tropaeolum longifolium show overlapping expression in the epidermis of reproductive apices (inflorescence meristems) and young floral buds, but only TlTCP4L2 shows differential expression in the floral tube at early stages of spur formation, restricted to the adaxial region. This adaxial expression of TlTCP4L2 overlaps with the expression of TlHIS4. Later in development, only TlTCP4L2 is expressed in the nectariferous tissue of the spur. CONCLUSIONS: Based on these results, we hypothesize that Tropaeolaceae TCP4L genes had a plesiomorphic role in epidermal development and that, after gene duplication, TCP4L2 acquired a new function in spur initiation and elaboration. To better understand spur evolution in Tropaeolaceae, it is critical to expand developmental genetic studies to their sister group, the Akaniaceae, which possess simultaneously an independent duplication of TCP4L genes and a spurless floral tube.

Tropaeolum majus L. and low dose gamma radiation suppress liver carcinoma development via EGFR-HER2 signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers around the world and remain asymptomatic in early stage. An alcoholic extract prepared from leaves of Tropaeolum majus L. (Tropaeolaceae) was assessed for its potential activity against diethylnitrosamine-induced liver carcinoma in vivo. Oral administration of the extract significantly decreased the inflammatory marker translation NF-kB and supressed HCC progression in combination with 0.5 Gy gamma radiation via EGF-HER-2 pathway. Histopathological and immunohistopathological features also showed the recovery of a hepatic architecture. Immunohistochemical study showed the T. majus and LDR enhancement effect on proapoptotic markers (caspase-3 and Bax) and inhibition of anti-apoptotic factor (BCl2). HPLC-DAD-MSn analysis of the extract revealed the annotation of twelve compounds. T. majus could mediate a defensive influence against diethylnitrosamine-induced hepatocarcinogenesis and serve as a respectable option in amelioration of the hepatocellular carcinoma development in combination with low dose of gamma radiation.

Tropaeolum majus R2R3 MYB Transcription Factor TmPAP2 Functions as a Positive Regulator of Anthocyanin Biosynthesis.

Anthocyanins are an important group of water-soluble and non-toxic natural pigments with antioxidant and anti-inflammatory properties that can be found in flowers, vegetables, and fruits. Anthocyanin biosynthesis is regulated by several different types of transcription factors, including the WD40-repeat protein Transparent Testa Glabra 1 (TTG1), the bHLH transcription factor Transparent Testa 8 (TT8), Glabra3 (GL3), Enhancer of GL3 (EGL3), and the R2R3 MYB transcription factor Production of Anthocyanin Pigment 1 (PAP1), PAP2, MYB113, and MYB114, which are able to form MYB-bHLH-WD40 (MBW) complexes to regulate the expression of late biosynthesis genes (LBGs) in the anthocyanin biosynthesis pathway. Nasturtium (Tropaeolum majus) is an edible flower plant that offers many health benefits, as it contains numerous medicinally important ingredients, including anthocyanins. By a comparative examination of the possible anthocyanin biosynthesis regulator genes in nasturtium varieties with different anthocyanin contents, we found that TmPAP2, an R2R3 MYB transcription factor gene, is highly expressed in "Empress of India", a nasturtium variety with high anthocyanin content, while the expression of TmPAP2 in Arabidopsis led to the overproduction of anthocyanins. Protoplast transfection shows that TmPAP2 functions as a transcription activator; consistent with this finding, some of the biosynthesis genes in the general phenylpropanoid pathway and anthocyanin biosynthesis pathway were highly expressed in "Empress of India" and the 35S:TmPAP2 transgenic Arabidopsis plants. However, protoplast transfection indicates that TmPAP2 may not be able to form an MBW complex with TmGL3 and TmTTG1. These results suggest that TmPAP2 may function alone as a key regulator of anthocyanin biosynthesis in nasturtiums.

Formation and stability of isothiocyanate protein conjugates at different pH values and bread types enriched with nasturtium (Tropaeolum majus L.).

Brassicaceae vegetables are rich in glucosinolates (GLS), which degrade into various breakdown products, including isothiocyanates (ITC), during food processing. ITC are associated with health-promoting properties; therefore, producing food products enriched with a high content of these compounds is of interest for improving and maintaining human health. The present study aimed at evaluating a potential increase in ITC formation in Brassicaceae-enriched bread while minimizing ITC-protein conjugates. The influence of pH on GLS degradation was evaluated in different bread types and pH-adjusted model breads. In all samples, ITC, nitriles, and ITC-amino acid conjugates were analyzed by GC-MS and LC-ESI-MS/MS, respectively. The highest ITC-Lys levels with 33.9 µmol BITC-Lys/g bread could be found in a (more alkaline soda) bread with a pH of 7.2, while "free" BITC content was the lowest. However, this finding could not be directly correlated to pH value, because in model breads no significant relation between the pH and the BITC content could be identified. Especially the baking process impacted the ITC content as it was reduced from dough to the bread by 98%. Therefore, a heated food product is not suitable for an enrichment with GLS-rich vegetables to achieve a high content on BITC. Nevertheless, in the bread matrix itself the degradation products and BITC-Lys conjugates were stable during storage.

Proteomic analysis of mashua (Tropaeolum tuberosum) tubers subjected to postharvest treatments.

Mashua (Tropaeolum tuberosum) is an important food in certain areas of the Andean region, where it is popularly believed to possess medicinal properties. Several studies have previously shown the potential of this tuber as a source of bioactive compounds. Traditionally, the tuber is exposed to the sun before consumption, in order to reduce its bitterness. The present work aims to study, at the proteome level, the differential abundance of proteins in tubers subjected to different postharvest treatments: sun-exposure (SUN), shade (SHA), refrigeration (COLD) and shade combined with sun-exposure (SHA-SUN) compared to recently harvested tubers (INIT). Results showed that sun exposure for prolonged times (9 days) resulted in increased abundance of proteins classified as heat shock proteins, intracellular traffic, disease/defense and protein degradation. Our results reflect that the sun treatment activates defense systems and osmoprotection adjustment against water loss and reactive oxygen species.

Glucosinolate composition of Colombian accessions of mashua (Tropaeolum tuberosum Ruíz & Pavón), structural elucidation of the predominant glucosinolate and assessment of its antifungal activity.

BACKGROUND: The content of individual and total glucosinolates in 65 mashua tuber accessions (Tropaeolum tuberosum) from the germplasm bank at Universidad Nacional de Colombia was determined by reverse phase high-performance liquid chromatography on enzymatically desulfated extracts. The predominant glucosinolate was identified and the possible structure of the glucosinolate present in lower proportion was postulated from evidence obtained by high-performance liquid chromatography/mass spectrometry, 1 H and 13 C nuclear magnetic resonance and bi-dimensional experiments. The biological action of the hydrolysis products generated from the glucosinolates in the accessions that showed a higher content of these compounds was assessed in the presence of Fusarium oxysporum f. sp. dianthi, Rhizoctonia solani and Phytophthora infestans. RESULTS: The total content of glucosinolates ranged between >3.00 × 10-1 and 25.8 µmol g-1 dry matter. p-Methoxybenzyl glucosinolate was identified as the predominant glucosinolate in Colombian mashua accessions; besides, the possible presence of p-hydroxybenzyl glucosinolate was postulated. In vitro assays established an important fungal growth inhibition of the potato pathogen P. infestans. CONCLUSION: The biological action from p-methoxybenzyl glucosinolate and p-hydroxybenzyl glucosinolate found in Colombian mashua accessions depends on their concentration, with the Tt30 accession, characterized for showing the highest content of glucosinolates, being the most promising to control the assessed pathogens. © 2016 Society of Chemical Industry.

Nutritional composition and antioxidant capacity in edible flowers: characterisation of phenolic compounds by HPLC-DAD-ESI/MSn.

Edible flowers are commonly used in human nutrition and their consumption has increased in recent years. The aim of this study was to ascertain the nutritional composition and the content and profile of phenolic compounds of three edible flowers, monks cress (Tropaeolum majus), marigold (Tagetes erecta) and paracress (Spilanthes oleracea), and to determine the relationship between the presence of phenolic compounds and the antioxidant capacity. Proximate composition, total dietary fibre (TDF) and minerals were analysed according to official methods: total phenolic compounds (TPC) were determined with Folin-Ciocalteu's reagent, whereas antioxidant capacity was evaluated using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In addition, phenolic compounds were characterised by HPLC-DAD-MSn. In relation to the nutritional value, the edible flowers had a composition similar to that of other plant foods, with a high water and TDF content, low protein content and very low proportion of total fat-showing significant differences among samples. The levels of TPC compounds and the antioxidant capacity were significantly higher in T. erecta, followed by S. oleracea and T. majus. Thirty-nine different phenolic compounds were tentatively identified, with flavonols being the major compounds detected in all samples, followed by anthocyanins and hydroxycynnamic acid derivatives. In T. erecta small proportions of gallotannin and ellagic acid were also identified.

RNA-Seq analysis of developing nasturtium seeds (Tropaeolum majus): identification and characterization of an additional galactosyltransferase involved in xyloglucan biosynthesis.

A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis. cDNA sequences were generated from developing nasturtium (Tropaeolum majus) seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was identified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog (AT5G62220) revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 (mur3.1) and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.

Turning the 'mustard oil bomb' into a 'cyanide bomb': aromatic glucosinolate metabolism in a specialist insect herbivore.

Plants have evolved a variety of mechanisms for dealing with insect herbivory among which chemical defense through secondary metabolites plays a prominent role. Physiological, behavioural and sensorical adaptations to these chemicals provide herbivores with selective advantages allowing them to diversify within the newly occupied ecological niche. In turn, this may influence the evolution of plant metabolism giving rise to e.g. new chemical defenses. The association of Pierid butterflies and plants of the Brassicales has been cited as an illustrative example of this adaptive process known as 'coevolutionary armsrace'. All plants of the Brassicales are defended by the glucosinolate-myrosinase system to which larvae of cabbage white butterflies and related species are biochemically adapted through a gut nitrile-specifier protein. Here, we provide evidence by metabolite profiling and enzyme assays that metabolism of benzylglucosinolate in Pieris rapae results in release of equimolar amounts of cyanide, a potent inhibitor of cellular respiration. We further demonstrate that P. rapae larvae develop on transgenic Arabidopsis plants with ectopic production of the cyanogenic glucoside dhurrin without ill effects. Metabolite analyses and fumigation experiments indicate that cyanide is detoxified by ß-cyanoalanine synthase and rhodanese in the larvae. Based on these results as well as on the facts that benzylglucosinolate was one of the predominant glucosinolates in ancient Brassicales and that ancient Brassicales lack nitrilases involved in alternative pathways, we propose that the ability of Pierid species to safely handle cyanide contributed to the primary host shift from Fabales to Brassicales that occured about 75 million years ago and was followed by Pierid species diversification.

The glucosinolate-myrosinase system in nasturtium (Tropaeolum majus L.): variability of biochemical parameters and screening for clones feasible for pharmaceutical utilization.

Leaves of Tropaeolum majus L. contain high amounts of the glucosinolate glucotropaeolin. They are used in traditional medicine to treat infections of the urinary tract. When Tropaeolum leaves are consumed, glucotropaeolin is hydrolyzed to yield mustard oils, which are absorbed in the intestine and excreted in the urine, exhibiting their antimicrobial activity. For a corresponding phytopharmacon, a sufficiently high glucotropaeolin concentration is required and any degradation of glucosinolates while drying must be minimized, i.e. the post mortal cleavage by myrosinases, which are activated by ascorbic acid. In extensive screenings, the dominant parameters determining the glucotropaeolin content in the dried leaves were quantified. It turned out that the glucotropaeolin concentration in the dried leaves represented the most suitable screening parameter. The screening of several hundred Tropaeolum plants resulted in the selection of eight high-yield varieties, from which in vitro plants had been generated and propagated as a source for large field trials.

Analysis of accumulation, extractability, and metabolization of five different phenylarsenic compounds in plants by ion chromatography with mass spectrometric detection and by atomic emission spectroscopy.

Phenylated arsenic compounds occur as highly toxic contaminants in former military areas where they were formed as degradation products of chemical warfare agents. Some phenylarsenic compounds such as roxarsone and aminophenylarsonic acids were applied as food additive and veterinary drugs in stock-breeding and therefore pose an environmental risk in agricultural used sites. Very few data exist in the literature concerning uptake and effects of phenylarsenic compounds in plants growing on contaminated soils. In this study, the accumulation, extractability, and metabolization of five different phenylarsenic compounds, phenylarsonic acid, p- and o-aminophenylarsonic acid, phenylarsine oxide, and 3-nitro-4-hydroxyphenylarsonic acid called roxarsone, by the terrestrial plant Tropaeolum majus were investigated. Ion chromatography coupled to inductively coupled plasma mass spectrometry was used to differentiate these arsenic compounds, and inductively coupled plasma atomic emission spectroscopy was used for total arsenic quantification. All compounds considered were taken up by the roots and transferred to stalks, leaves, and flowers. The strongest accumulation was observed for unsubstituted phenylarsonic acid followed by its trivalent analogue phenylarsine oxide that was mostly oxidized in soil whereas the amino- or nitro- and hydroxy-substituted phenylarsonic acids were accumulated to a smaller degree. The highest extraction yield of 90% for ground leaf material was achieved by 0.1M phosphate buffer, pH 7.7, in a two-step extraction with a total extraction time of 24h. The extraction of higher amounts of arsenic (50-70% of total arsenic present in leaves depending on arsenic species application) from non-ground intact leaves with deionized water in comparison with the buffer (20-40% of total arsenic) is ascribed to osmotic effects. The arsenic species analysis revealed a cleavage of the amino groups from the phenyl ring for plants treated with aminophenylarsonic acids. A further important metabolic effect consisted in the production of inorganic arsenate and arsenite from the phenylated arsonic acid groups.