Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato

Phenylpropanoids comprise an important class of plant secondary metabolites. A number of transcription factors have been used to upregulate-specific branches of phenylpropanoid metabolism, but by far the most effective has been the fruit-specific expression of AtMYB12 in tomato, which resulted in as much as 10% of fruit dry weight accumulating as flavonols and hydroxycinnamates. We show that AtMYB12 not only increases the demand of flavonoid biosynthesis but also increases the supply of carbon from primary metabolism, energy and reducing power, which may fuel the shikimate and phenylalanine biosynthetic pathways to supply more aromatic amino acids for secondary metabolism. AtMYB12 directly binds promoters of genes encoding enzymes of primary metabolism. The enhanced supply of precursors, energy and reducing power achieved by AtMYB12 expression can be harnessed to engineer high levels of novel phenylpropanoids in tomato fruit, offering an effective production system for bioactives and other high value ingredients

Tomatoes, lycopene-containing foods and cancer risk

Tomatoes, lycopene-containing foods and cancer ris

Combined Dietary Anthocyanins, Flavonols, and Stilbenoids Alleviate Inflammatory Bowel Disease Symptoms in Mice

Dietary polyphenols are associated with a wide range of health benefits, protecting against chronic diseases and promoting healthy aging. Dietary polyphenols offer a complementary approach to the treatment of inflammatory bowel diseases (IBDs), a group of common chronic intestinal inflammation syndromes for which there is no cure. Tomato is widely consumed but its content of polyphenols is low. We developed a tomato variety, Bronze, enriched in three distinct classes of polyphenols: flavonols, anthocyanins, and stilbenoids. Using Bronze tomatoes as a dietary supplement as well as Indigo (high anthocyanins and flavonols), ResTom (high stilbenoids) and wild-type tomatoes, we examined the effects of the different polyphenols on the host gut microbiota, inflammatory responses, and the symptoms of chronic IBD, in a mouse model. Bronze tomatoes significantly impacted the symptoms of IBD. A similar result was observed using diets supplemented with red grape skin containing flavonols, anthocyanins, and stilbenoids, suggesting that effective protection is provided by different classes of polyphenols acting synergistically

Role of transcriptional regulation in the evolution of plant phenotype: A dynamic systems approach

© 2015 Wiley Periodicals, Inc. A growing body of evidence suggests that alterations in transcriptional regulation of genes involved in modulating development are an important part of phenotypic evolution, and this can be documented among species and within populations. While the effects of differential transcriptional regulation in organismal development have been preferentially studied in animal systems, this phenomenon has also been addressed in plants. In this review, we summarize evidence for cis-regulatory mutations, trans-regulatory changes and epigenetic modifications as molecular events underlying important phenotypic alterations, and thus shaping the evolution of plant development. We postulate that a mechanistic understanding of why such molecular alterations have a key role in development, morphology and evolution will have to rely on dynamic models of complex regulatory networks that consider the concerted action of genetic and nongenetic components, and that also incorporate the restrictions underlying the genotype to phenotype mapping process.CONACyT 180098, 180380, 167705, 152649 and PAPIIT UNAM IN203214-3, IN203113-3, IN203814-3. BFU2012–34821 (MINECO) to C.G. and an institutional grant from Fundación Ramón Aceres to CBMSOPeer Reviewe

Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction–ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named “GB parts” and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4–5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.Sarrion-Perdigones, A.; Palací, J.; Granell Richart, A.; Orzáez Calatayud, DV. (2014). Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy. Methods in Molecular Biology. 1116:133-151. doi:10.1007/978-1-62703-764-8_10S1331511116Haseloff J, Ajioka J (2009) Synthetic biology, history, challenges and prospects. J R Soc Interface 6(Suppl 4):S389–S391Check E (2005) Synthetic biology, designs on life. Nature 438:417–418Kosuri S, Eroshenko N, LeProust EM et al (2010) Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28:1295–1299Ellis T, Adie T, Baldwin GS (2011) DNA assembly for synthetic biology, from parts to pathways and beyond. Integr Biol 3:109–118Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345Gibson DG, Glass JI, Lartigue C et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56Sarrion-Perdigones A, Falconi EE, Zandalinas SI et al (2011) GoldenBraid, an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6:e21622Sarrion-Perdigones A, Vilar-Vazquez M et al (2013) GoldenBraid2.0, A comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631Engler C, Gruetzner R, Kandzia R (2009) Golden gate shuffling, a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647Bracha-Drori K, Shichrur K, Katz A et al (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:419–427Smaczniak C, Immink RG, Muino JM et al (2012) Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc Natl Acad Sci U S A 109:1560–1565de Folter S, Immink RG, Kieffer M et al (2005) Comprehensive interaction map of the Arabidopsis MADS Box transcription factors. Plant Cell 17:1424–1433Lorenz WW, McCann RO, Longiaru M et al (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci U S A 88:4438–4442Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96: 14147–14152Hellens RP, Edwards EA, Leyland NR et al (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832Butelli E, Titta L, Giorgio M et al (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26: 1301–1308Kapila J, DeRycke R, VanMontagu M et al (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–10

Painted flowers: Eluta generates pigment patterning in Antirrhinum

*In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum /Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. *We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the non-suppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and by promoter activation/repression assays. *Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential co-factors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with greatest repression on those parts of the petals where Eluta is most highly expressed. *Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties. <br/

A bronze-tomato enriched diet affects the intestinal microbiome under homeostatic and inflammatory conditions

Inflammatory bowel diseases (IBD) are debilitating chronic inflammatory disorders that develop as a result of a defective immune response toward intestinal bacteria. Intestinal dysbiosis is associated with the onset of IBD and has been reported to persist even in patients in deep remission. We investigated the possibility of a dietary-induced switch to the gut microbiota composition using Winnie mice as a model of spontaneous ulcerative colitis and chow enriched with 1% Bronze tomato. We used the near isogenic tomato line strategy to investigate the effects of a diet enriched in polyphenols administered to mild but established chronic intestinal inflammation. The Bronze-enriched chow administered for two weeks was not able to produce any macroscopic effect on the IBD symptoms, although, at molecular level there was a significant induction of anti-inflammatory genes and intracellular staining of T cells revealed a mild decrease in IL17A and IFNγ production. Analysis of the microbial composition revealed that two weeks of Bronze enriched diet was sufficient to perturb the microbial composition of Winnie and control mice, suggesting that polyphenol-enriched diets may create unfavorable conditions for distinct bacterial species. In conclusion, dietary regimes enriched in polyphenols may efficiently support IBD remission affecting the intestinal dysbiosis