The Health Promoting Bioactivities of Lactuca sativa can be Enhanced by Genetic Modulation of Plant Secondary Metabolites.
ABSTRACT: Plant secondary metabolites are protective dietary constituents and rol genes evidently increase the synthesis of these versatile phytochemicals. This study subjected a globally important vegetable, lettuce (Lactuca sativa) to a combination of untargeted metabolomics (LC-QTof-MS) and in vitro bioactivity assays. Specifically, we examined the differences between untransformed cultured lettuce (UnT), lettuce transformed with either rolABC (RA) or rolC (RC) and commercially grown (COM) lettuce. Of the 5333 metabolite features aligned, deconvoluted and quantified 3637, 1792 and 3737 significantly differed in RA, RC and COM, respectively, compared with UnT. In all cases the number of downregulated metabolites exceeded the number increased. In vitro bioactivity assays showed that RA and RC (but not COM) significantly improved the ability of L. sativa to inhibit ?-glucosidase, inhibit dipeptidyl peptidase-4 (DPP-4) and stimulate GLP-1 secretion. We putatively identified 76 lettuce metabolites (sesquiterpene lactones, non-phenolic and phenolic compounds) some of which were altered by several thousand percent in RA and RC. Ferulic acid levels increased 3033-9777%, aminooxononanoic acid increased 1141-1803% and 2,3,5,4'tetrahydroxystilbene-2-O-?-d-glucoside increased 40,272-48,008%. Compound activities were confirmed using commercially obtained standards. In conclusion, rol gene transformation significantly alters the metabolome of L.sativa and enhances its antidiabetic properties. There is considerable potential to exploit rol genes to modulate secondary metabolite production for the development of novel functional foods. This investigation serves as a new paradigm whereby genetic manipulation, metabolomic analysis and bioactivity techniques can be combined to enable the discovery of novel natural bioactives and determine the functional significance of plant metabolites.
Project description:In vitro suspension culture techniques are cost effective for large-scale production of secondary metabolites. In the present study, firstly, suspension cultures of untransformed Lactuca sativa were prepared using different hormonal combinations and were subjected to different pH, temperature and salt concentrations. Maximum biomass was obtained for suspensions supplemented with 1.5 mg/L BAP and 0.1 mg/L NAA, at pH 5.8, temperature 28 °C and 0 mM NaCl concentration. Using these parameters, suspensions were produced for rol ABC- and rol C-transformed lines of L. sativa. All the transgenic lines showed prominent increase in fresh weight (FW) and dry weight (DW) with maximum values for rol ABC2 line producing 169.8 mg/mL FW and 25.3 mg/mL DW. The exudates of transformed and untransformed plants were tested for the antioxidant activity and in vivo assays on rats. Maximum phenolic content (261 ?g/mL) and flavonoid content (637.6 ?g/mL) were obtained for rol C1 transgenic line. Total antioxidant capacity was found maximum (1451.7 ?g/mL) for untransformed lettuce, whereas rol C1 showed maximum total reducing power activity (637.6 ?g/mL). In DPPH assay, maximum activity (104.7 ?g/mL) was shown by rol ABC3 line. In rats analgesic assay, maximum activity (74.9%) was shown by rol C2. Line rol C1 showed maximum anti-inflammatory activity (69.2%) and maximum antidepressant activity (minimum immobility time of 55 s). Maximum anticoagulant activity was observed for rol ABC2 with maximum clotting time of 130 s. The present study could help in using lettuce suspension culture as platform for the enhanced production of important metabolites.
Project description:Encapsulated bioactive agents applied to the Lactuca sativa L. present an innovative approach to stimulate the production of plant secondary metabolites increasing its nutritive value. Calcium and copper ions were encapsulated in biopolymeric microparticles (microspheres and microcapsules) either as single agents or in combination with biocontrol agents, Trichoderma viride spores, a fungal plant growth mediator. Both, calcium and copper ions are directly involved in the synthesis of plant secondary metabolites and alongside, Trichoderma viride can provide indirect stimulation and higher uptake of nutrients. All treatments with microparticles had a positive effect on the enhancement of plant secondary metabolites content in Lactuca sativa L. The highest increase of chlorophylls, antioxidant activity and phenolic was obtained by calcium-based microparticles in both, conventionally and hydroponically grown lettuces. Non-encapsulated fungus Trichoderma viride enhanced the synthesis of plant secondary metabolites only in hydroponics cultivation signifying the importance of its encapsulation. Encapsulation proved to be simple, sustainable and environmentally favorable for the production of lettuce with increased nutritional quality, which is lettuce fortified with important bioactive compounds.
Project description:Lettuce (Lactuca sativa L.) is a widely consumed horticultural species. Its significance lies in a high polyphenolic compound content, including phenolic acids and flavonols. In this work, we have probed the ability of Rhizobium laguerreae HUTR05 to promote lettuce growth, under in vitro and greenhouse conditions (both non-saline and saline conditions). This strain has shown several in vitro plant growth promotion mechanisms, as well as capacity to colonize lettuce seedlings roots. We have analyzed the effect of the rhizobacterium inoculation on mineral and bioactive compounds in lettuce, under greenhouse conditions, and found a rise in the content of certain phenolic acids and flavonoids, such as derivatives of caffeoyl acid and quercetin. The genome analysis of the strain has shown the presence of genes related to plant growth-promoting rhizobacteria (PGPR) mechanisms, defense from saline stress, and phenolic compound metabolism (such as naringenin-chalcone synthase or phenylalanine aminotransferase).
Project description:The present study aimed to compare polyphenols among red lettuce cultivars and identify suitable cultivars for the development and utilization of healthy vegetables. Polyphenols, mineral elements, and antioxidant activity were analyzed in the leaves of six red pigmented lettuce (Lactuca sativa L.) cultivars; thereafter, we assessed the anti-tumor effects of cultivar B-2, which displayed the highest antioxidant activity. Quadrupole-Orbitrap mass spectrometry analysis revealed four classes of polyphenols in these cultivars. The composition and contents of these metabolites varied significantly among cultivars and primarily depended on leaf color. The B-2 cultivar had the highest antioxidant potential than others because it contained the highest levels of polyphenols, especially anthocyanin, flavone, and phenolic acid; furthermore, this cultivar displayed anti-tumor effects against the human lung adenocarcinoma cell line A549, human hepatoma cell line Bel7402, human cancer colorectal adenoma cell line HCT-8, and HT-29 human colon cancer cell line. Hence, the new red-leaf lettuce cultivar B-2 has a distinct metabolite profile, with high potential for development and utilization of natural phytochemical and mineral resources in lettuces and can be used as a nutrient-dense food product.
Project description:Retinol (ROL), the alcohol form of vitamin A, is known to control cell fate decision of various types of stem cells in the form of its active metabolite, retinoic acid (RA). However, little is known about whether ROL has regulatory effects on colonic stem cells. We examined in this study the effect of ROL on the growth of murine normal colonic cells cultured as organoids. As genes involved in RA synthesis from ROL were differentially expressed along the length of the colon, we tested the effect of ROL on proximal and distal colon organoids separately. We found that organoid forming efficiency and the expression level of Lgr5, a marker gene for colonic stem cells were significantly enhanced by ROL in the proximal colon organoids, but not in the distal ones. Interestingly, neither retinaldehyde (RAL), an intermediate product of the ROL-RA pathway, nor RA exhibited growth promoting effects on the proximal colon organoids, suggesting that ROL-dependent growth enhancement in organoids involves an RA-independent mechanism. This was confirmed by the observation that an inhibitor for RA-mediated gene transcription did not abrogate the effect of ROL on organoids. This novel role of ROL in stem cell maintenance in the proximal colon provides insights into the mechanism of region-specific regulation for colonic stem cell maintenance.
Project description:Vitamin A (VA), all-trans-retinol (at-ROL), and its derivative, all-trans-retinoic acid (at-RA), are required for neuron development. The effects of these retinoids are dependent upon the nutritional status of the rat and tissue-specific dynamics of retinoid access and utilization. The purpose of this study was to determine the status of at-ROL and at-RA in the peripheral olfactory organ of postnatal rats fed a normal diet and rats fed a VA-deficient (VAD) diet. Extracted retinoids were analyzed by HPLC. Resolved sample peaks were identified by comparing their elution times and spectra with those of authentic standards. Mean at-RA and at-ROL concentrations of 23 pmol/g olfactory tissue and 0.13 nmol/g, respectively, were recovered from olfactory tissue. The ratio of at-RA:at-ROL in olfactory was approximately 2 times that in testis and 200 times that in liver. at-ROL was depleted from the liver and olfactory organ of rats fed a VAD diet from birth to 70 d of age. Surprisingly, at-RA was still present in olfactory tissue from these rats. At 90 d of age, the VAD rats were frankly deficient and at-RA was no longer detectable in olfactory tissue. The comparatively high ratio of at-RA:at-ROL in the peripheral olfactory organ and the persistence of at-RA in at-ROL-depleted tissues strongly suggests that maintenance of local stores of at-RA is functionally relevant in this tissue.
Project description:A diet rich in phytonutrients from fruit and vegetables has been acknowledged to afford protection against a range of human diseases, but many of the most popular vegetables are low in phytonutrients. Wild relatives of crops may contain allelic variation for genes determining the concentrations of these beneficial phytonutrients, and therefore understanding the genetic basis of this variation is important for breeding efforts to enhance nutritional quality. In this study, lettuce recombinant inbred lines, generated from a cross between wild and cultivated lettuce (Lactuca serriola and Lactuca sativa, respectively), were analysed for antioxidant (AO) potential and important phytonutrients including carotenoids, chlorophyll and phenolic compounds. When grown in two environments, 96 quantitative trait loci (QTL) were identified for these nutritional traits: 4 for AO potential, 2 for carotenoid content, 3 for total chlorophyll content and 87 for individual phenolic compounds (two per compound on average). Most often, the L. serriola alleles conferred an increase in total AOs and metabolites. Candidate genes underlying these QTL were identified by BLASTn searches; in several cases, these had functions suggesting involvement in phytonutrient biosynthetic pathways. Analysis of a QTL on linkage group 3, which accounted for >30% of the variation in AO potential, revealed several candidate genes encoding multiple MYB transcription factors which regulate flavonoid biosynthesis and flavanone 3-hydroxylase, an enzyme involved in the biosynthesis of the flavonoids quercetin and kaempferol, which are known to have powerful AO activity. Follow-up quantitative RT-PCR of these candidates revealed that 5 out of 10 genes investigated were significantly differentially expressed between the wild and cultivated parents, providing further evidence of their potential involvement in determining the contrasting phenotypes. These results offer exciting opportunities to improve the nutritional content and health benefits of lettuce through marker-assisted breeding.
Project description:We previously demonstrated that 4-oxoretinol (4-oxo-ROL) activated retinoic acid receptors (RARs) in F9 stem cells. We showed that 4-oxo-ROL inhibited the proliferation of normal human mammary epithelial cells (HMECs). To understand the mechanisms by which 4-oxo-ROL regulates HMEC growth we examined gene expression profiles following 4-oxo-ROL or all-trans retinoic acid (tRA). We also compared growth inhibition by tRA, 4-oxo-ROL, or 4-oxo-RA. All three retinoids inhibited HMEC proliferation. Gene expression analyses indicated that 4-oxo-ROL and tRA modulated gene expression in closely related pathways. The expression of many genes, e.g. ATP-binding cassette G1 (ABCG1); adrenergic receptorbeta2 (ADRB2); ras-related C3 botulinum toxin substrate (RAC2); and short-chain dehydrogenase/reductase 1 gene (SDR1) was changed after 4-oxo-ROL or tRA. Metabolism of these retinoids was analyzed by high-performance liquid chromatography (HPLC). In 1 microM tRA treated HMECs all of the tRA was found intracellularly, and tRA was the predominant intracellular retinoid. In 1 microM 4-oxo-ROL treated HMECs most 4-oxo-ROL was esterified to 4-oxoretinyl esters, no tRA was detected, and 4-oxo-ROL and 4-oxo-RA were observed intracellularly. In 1 microM 4-oxoretinoic acid (4-oxo-RA) treated HMECs little intracellular 4-oxo-RA was detected; most 4-oxo-RA was in the medium. Our results indicate that: (a) 4-oxo-ROL regulates gene expression and inhibits proliferation of HMECs; (b) 4-oxo-ROL and tRA regulate some of the same genes; (c) more tRA is found in cells, as compared to 4-oxoretinoic acid, when each drug is added at the same concentration in the medium; and (d) the mechanism by which 4-oxo-ROL exerts its biological activity does not involve intracellular tRA production.
Project description:Considering that functional components of plant foods are mainly secondary-metabolism products, we investigated the shaping of health-promoting compounds in hydroponically grown butterhead lettuce (Lactuca sativa L. var. capitata) as a function of the strength of the nutrient solution utilized. To this aim, untargeted metabolomics profiling, in vitro antioxidant capacity (total phenolics, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays), and the inhibition of selected enzyme activities were investigated in two butterhead lettuce cultivars with different pigmentation, i.e., green and red Salanova. Full-strength nutrition, together with half- and quarter-strength solutions of macronutrients, was tested. Our results indicate that by reducing the nutrients strength, we could elicit a distinctive shaping of the phenolic profile of lettuce. It is noteworthy that only specific classes of phenolics (namely, lignans and phenolic acids, followed by flavones and anthocyanins) were modulated by the induction of nutritional eustress (fold-change values in the range between -5 and +11). This indicates that specific responses, rather than a generalized induction of phenolic compounds, could be observed. Nonetheless, a genotype-dependent response could be observed, with the red cultivar being much more responsive to nutritional deprivation than the green Salanova lettuce. Indeed, analysis of variance (ANOVA) confirmed a genotype x nutrition interaction in red Salanova (p < 0.001). As a consequence of the changes in phenolic composition, also the antioxidant capacity (p < 0.001) and amylase inhibition (p < 0.001) properties were affected by the growing conditions. However, the effect on cholinesterase and tyrosinase inhibition was poorly affected by the nutritional strength. Provided that yields are not compromised, the application of a controlled nutritional eustress in hydroponically cultivated lettuce may represent a valuable strategy to produce food with tailored functional features in a sustainable manner.
Project description:Lettuce is an important leafy vegetable that represents a significant dietary source of antioxidants and bioactive compounds. However, the levels of metabolites in different lettuce cultivars are poorly characterized. In this study, we used combined GC?×?GC-TOF/MS and UPLC-IMS-QTOF/MS to detect and relatively quantify metabolites in 30 lettuce cultivars representing large genetic diversity. Comparison with online databases, the published literature, standards as well using collision cross-section values enabled putative identification of 171 metabolites. Sixteen of these 171 metabolites (including phenolic acid derivatives, glycosylated flavonoids, and one iridoid) were present at significantly different levels in leaf and head type lettuces, which suggested the significant metabolomic variations between the leaf and head types of lettuce are related to secondary metabolism. A combination of the results and metabolic network analysis techniques suggested that leaf and head type lettuces contain not only different levels of metabolites but also have significant variations in the corresponding associated metabolic networks. The novel lettuce metabolite library and novel non-targeted metabolomics strategy devised in this study could be used to further characterize metabolic variations between lettuce cultivars or other plants. Moreover, the findings of this study provide important insight into metabolic adaptations due to natural and human selection, which could stimulate further research to potentially improve lettuce quality, yield, and nutritional value.