Exploring the metabolic and physiological roles of HQT in S. lycopersicum by gene editing

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation

Influencia de la radiación UV-B, sobre la biosíntesis de antocianinas en frutos de manzana (Malus domestica Borkh) CV.”ANNA” para condiciones de trópico alto en Boyacá Colombia

1 recurso en línea (73 páginas) : figuras, tablas.En la manzana roja, la coloración de la piel y las características fisicoquímicas, son factores importantes que determinan la calidad de los frutos; estos parámetros tienen un control de orden genético pero influenciado por condiciones ambientales en etapa de maduración de frutos. El color rojo está ligado a la biosíntesis de antocianinas, que son un grupo de pigmentos de color rojo, hidrosolubles, producto del metabolismo secundario mediado por luz UV, Y ampliamente distribuidos en el reino vegetal. En el presente estudio se determinó el IUV, se cuantifico antocianinas totales y se analizaron SST en frutos maduros de manzana cv “ANNA” cultivados en diferentes altitudes en localidades de Boyacá: Soracá a 2820 m.s.n.m, Tuta a 2640m.s.n.m y Nuevo Colon a 2450 m.s.n.m. Los resultados mostraron que en la localidad a mayor altitud (Soracá), el valor promedio del IUV fue mayor (14.9) y la acumulación de antocianinas totales igualmente fue el más alto (25.4 mg/100g). Se ha encontrado una correlaciòn positiva entre la intensidad de la radiación UV, altitud y concentración de antocianinas totales, mientras que para los SST no se encontraron diferencias significativas.Bibliografía y webgrafía: páginas 61-70MaestríaMagíster en Ciencias Biológica

A noncanonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat

Plants produce an array of natural products with important ecological functions. These compounds are often decorated with oligosaccharide groups that influence bioactivity, but the biosynthesis of such sugar chains is not well understood. Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits, as exemplified by avenacins, antimicrobial defense compounds produced by oats. Avenacins have a branched trisaccharide moiety consisting of L-arabinose linked to 2 D-glucose molecules that is critical for antifungal activity. Plant natural product glycosylation is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). We previously characterized the arabinosyltransferase that initiates the avenacin sugar chain; however, the enzymes that add the 2 remaining D-glucose molecules have remained elusive. Here we characterize the enzymes that catalyze these last 2 glucosylation steps. AsUGT91G16 is a classical cytosolic UGT that adds a 1,2-linked D-glucose molecule to L-arabinose. Unexpectedly, the enzyme that adds the final 1,4-linked D-glucose (AsTG1) is not a UGT, but rather a sugar transferase belonging to Glycosyl Hydrolase family 1 (GH1). Unlike classical UGTs, AsTG1 is vacuolar. Analysis of oat mutants reveals that AsTG1 corresponds to Sad3, a previously uncharacterized locus shown by mutation to be required for avenacin biosynthesis. AsTG1 and AsUGT91G16 form part of the avenacin biosynthetic gene cluster. Our demonstration that a vacuolar transglucosidase family member plays a critical role in triterpene biosynthesis highlights the importance of considering other classes of carbohydrate-active enzymes in addition to UGTs as candidates when elucidating pathways for the biosynthesis of glycosylated natural products in plants

Natural Blues: Structure Meets Function in Anthocyanins

Choices of blue food colourants are extremely limited, with only two options in the USA, synthetic blue no. 1 and no. 2, and a third available in Europe, patent blue V. The food industry is investing heavily in finding naturally derived replacements, with limited success to date. Here, we review the complex and multifold mechanisms whereby blue pigmentation by anthocyanins is achieved in nature. Our aim is to explain how structure determines the functionality of anthocyanin pigments, particularly their colour and their stability. Where possible, we describe the impact of progressive decorations on colour and stability, drawn from extensive but diverse physico-chemical studies. We also consider briefly how this understanding could be harnessed to develop blue food colourants on the basis of the understanding of how anthocyanins create blues in nature

Voids in network-forming liquids and their influence on the structure and dynamics

A Voronoi analysis is applied to clarify the origin of certain unusual physical properties of three representative network-forming ionic liquids: SiO2, ZnCl2, and BeCl2. In each of these fluids, the local structure under ambient pressure comprises a tetrahedral unit of anions around cations which link together to form a network. The Voronoi analysis is used to characterize the empty space (voids) within the network. Despite sharing the same local tetrahedral arrangement, the network properties of the three liquids differ markedly on an intermediate length scale and this leads to significant differences in the properties of the voids. It is shown how the void analysis helps to interpret the anomalous behaviour of the diffusivity in SiO2, which over a certain range of density increases with increasing density, and to clarify the origin of the intermediate range order in the atomic positions, as seen in the much discussed first sharp diffraction peak (or pirepeak) in diffraction data

The structural features of thousands of T-DNA insertion sites are consistent with a double-strand break repair based insertion mechanism

Kleinbölting N, Huep G, Appelhagen I, Viehöver P, Li Y, Weisshaar B. The structural features of thousands of T-DNA insertion sites are consistent with a double-strand break repair based insertion mechanism. Molecular Plant. 2015;8(11):1651-1664.Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host's DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34 000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double-strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments

Thermodynamically Stable Cationic Dimers in Carboxyl-Functionalized Ionic Liquids: The Paradoxical Case of “Anti-Electrostatic” Hydrogen Bonding

We show that carboxyl-functionalized ionic liquids (ILs) form doubly hydrogen-bonded cationic dimers (c+=c+) despite the repulsive forces between ions of like charge and competing hydrogen bonds between cation and anion (c+–a−). This structural motif as known for formic acid, the archetype of double hydrogen bridges, is present in the solid state of the IL 1−(carboxymethyl)pyridinium bis(trifluoromethylsulfonyl)imide [HOOC−CH2−py][NTf2]. By means of quantum chemical calculations, we explored different hydrogen-bonded isomers of neutral (HOOC–(CH2)n–py+)2(NTf2−)2, single-charged (HOOC–(CH2)n–py+)2(NTf2−), and double-charged (HOOC– (CH2)n−py+)2 complexes for demonstrating the paradoxical case of “anti-electrostatic” hydrogen bonding (AEHB) between ions of like charge. For the pure doubly hydrogen-bonded cationic dimers (HOOC– (CH2)n−py+)2, we report robust kinetic stability for n = 1–4. At n = 5, hydrogen bonding and dispersion fully compensate for the repulsive Coulomb forces between the cations, allowing for the quantification of the two equivalent hydrogen bonds and dispersion interaction in the order of 58.5 and 11 kJmol−1, respectively. For n = 6–8, we calculated negative free energies for temperatures below 47, 80, and 114 K, respectively. Quantum cluster equilibrium (QCE) theory predicts the equilibria between cationic monomers and dimers by considering the intermolecular interaction between the species, leading to thermodynamic stability at even higher temperatures. We rationalize the H-bond characteristics of the cationic dimers by the natural bond orbital (NBO) approach, emphasizing the strong correlation between NBO-based and spectroscopic descriptors, such as NMR chemical shifts and vibrational frequencies

TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds

Appelhagen I, Lu G-H, Huep G, Schmelzer E, Weisshaar B, Sagasser M. TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds. The Plant Journal. 2011;67(3):406-419.Summary Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP-type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA-specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development