Molecular Basis for Chemical Evolution of Flavones to Flavonols and Anthocyanins in Land Plants

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3β-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of (2S)-flavanones to dihydroflavonols, but its origin and evolution remain elusive. Here, we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium (Physcomitrella) patens and Selaginella moellendorffii, suggesting that they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem.

The success of land plants in the colonization of and adaptation to terrestrial ecosystems has been particularly attributed to the emergence and evolution of a unique metabolic capacity that synthesizes diverse specialized metabolites, including flavonoids, a highly polymorphic class of polyphenols (Weng and Chapple, 2010). The flavonoid metabolites have been classified into several subgroups, namely flavanones, dihydroflavonols, flavones, flavonols, flavan-3,4-diols, flavan-3-ols, and anthocyanins, based on their oxidation status and substitution patterns of the core skeleton (Winkel-Shirley, 2001; Martens et al., 2010). Along with the evolution of land plants, different classes of flavonoids emerged (Koes et al., 1994). The basal land plants liverworts produce chalcones, flavanones, and flavones; whereas lycophytes gained the ability to produce proanthocyanidins (Markham, 1984; Koes et al., 1994). Furthermore, both pteridophyta and gymnosperms, while dominated with flavone production, began to produce flavonols (Markham, 1984; Koes et al., 1994). Finally, flavonols and anthocyanins are well represented in angiosperms. Flavonols, which bear a 3-hydroxyl group in the core structure, have been exploited as effective photoprotectants against UV-B radiation (Solovchenko and Schmitz-Eiberger, 2003), as signal providers to symbionts (Hungria et al., 1991), as regulators of the transport of phytohormones (Peer and Murphy, 2007), and as determinants of conditional male fertility (Muhlemann et al., 2018). Anthocyanins, derived from dihydroflavonol, are important for sexual reproduction, acting as attractants for insect pollinators and for animal dispersers of seed (Shimada et al., 2005). It is obvious that a clear chemical evolution trace from chalcones, flavanones, and flavones to flavonols and anthocyanins, occurs across plant phyla. However, the molecular basis for such a chemical evolution remains mysterious.The biosynthesis of flavones and flavonols requires chemical conversion of a common precursor, (2S)-flavanone, and is catalyzed by flavone synthase I (FNS I) and flavanone 3β-hydroxylases (F3Hs), respectively. Both enzymes as well as flavonol synthase (FLS) and anthocyanidin synthase (ANS) belong to a larger enzyme family, the 2-oxoglutarate-dependent dioxygenases (2-ODDs; Farrow and Facchini, 2014). FNS I converts (2S)-flavanone to flavone via desaturation of carbon 2 and 3 of the heterologous ring of flavanone (Gebhardt et al., 2005, 2007), while F3H catalyzes the conversion of (2S)-flavanone to (2R,3R)-dihydroflavonol by hydroxylation of the C-3β position (Supplemental Fig. S1). Subsequently, FLS converts (2R,3R)-dihydroflavonols to their corresponding flavonols, and ANS catalyzes the nonpigmented leucoanthocyanidins (leucopelargonidin, leucocyanidin, and leucodelphinidin) to the pigmented anthocyanidins (pelargonidin, cyanidin, and delphinidin, respectively; Supplemental Fig. S1). These four classes of 2-ODD enzymes phylogenetically form two distinct subgroups, one consisting of F3H and FNS I and the other consisting of FLS and ANS. FNS I and F3H both use flavanone as substrate and exhibit, in general, a relatively narrow substrate specificity (Turnbull et al., 2000; Martens et al., 2003), whereas ANS and FLS display some degree of promiscuity in their substrate preferences and catalytic activities. For example, Arabidopsis (Arabidopsis thaliana) FLS1 is not only capable of converting dihydroflavonols to their corresponding flavonols but also mediates the oxidation of 2S-flavanone (naringenin) to both dihydrokaempferol enantiomers, an activity normally associated with F3H (Prescott et al., 2002). While F3Hs are ubiquitous in vascular plants, FNS Is appear to be confined to the Apiaceae family as well as a few non-Apiaceae species such as rice (Oryza sativa; Lee et al., 2008), maize (Zea mays), and Arabidopsis (Falcone Ferreyra et al., 2015). Prior to the discovery of FNS Is in those non-Apiaceae species, it was assumed that the gene encoding FNS I arose from duplication and mutation of F3H (Martens et al., 2001, 2003; Gebhardt et al., 2005, 2007). However, the FNS Is revealed in both Z. mays and Arabidopsis show very poor sequence similarity with those present in Apiaceae species, which suggests that the evolution of the FNS Is was not as clear-cut as was originally believed. It is likely that the evolution of FNS occurred several times independently. In several cereal crops, such as Z. mays, O. sativa, and wheat (Triticum aestivum), flavones are the major flavonoid substances, which protect the plants during pathogen attack and under biotic or abiotic stress conditions (Righini et al., 2019).Previously, we found that the liverwort Plagiochasma appendiculatum FNS I (which should change to PaFNS I/F2H, according to the function) converted flavanone to 2-hydroxyflavanone and flavone (Han et al., 2014). The dual FNS I and F2H activities of PaFNS I/F2H, together with the fact that its amino acid sequence shares a higher identity with F3Hs than with FNS Is, implicates an evolutionary connection between liverwort FNS Is and seed plant F3Hs. On the other hand, previous in silico analysis failed in identifying any F3H sequences in either the bryophyte Physcomitrium (Physcomitrella) patens or the lycophyte Selaginella moellendorffii, even though both species produce dihydroflavonol-derived metabolites. To identify when and how F3H emerged and evolved to produce a vast variety of flavonoid metabolites, we systematically identified FNS I and F3H homologous sequences from species of different phyla, including liverworts, P. patens, S. moellendorffii, gymnosperms, and angiosperms. Subsequent biochemical characterization revealed that the functionally promiscuous FNS Is widely emerged in the liverworts, which evolved into a dual-function enzyme with both FNS I and F3H activities in both P. patens and S. moellendorffii. Further evolution led to the emergence of F3H with a minor level of FNS I activity in gymnosperm species, while those generated by angiosperm species showed a more specific F3H activity.     

Most and Least Preferred Colours Differ According to Object Context: New Insights from an Unrestricted Colour Range

Humans like some colours and dislike others, but which particular colours and why remains to be understood. Empirical studies on colour preferences generally targeted most preferred colours, but rarely least preferred (disliked) colours. In addition, findings are often based on general colour preferences leaving open the question whether results generalise to specific objects. Here, 88 participants selected the colours they preferred most and least for three context conditions (general, interior walls, t-shirt) using a high-precision colour picker. Participants also indicated whether they associated their colour choice to a valenced object or concept. The chosen colours varied widely between individuals and contexts and so did the reasons for their choices. Consistent patterns also emerged, as most preferred colours in general were more chromatic, while for walls they were lighter and for t-shirts they were darker and less chromatic compared to least preferred colours. This meant that general colour preferences could not explain object specific colour preferences. Measures of the selection process further revealed that, compared to most preferred colours, least preferred colours were chosen more quickly and were less often linked to valenced objects or concepts. The high intra- and inter-individual variability in this and previous reports furthers our understanding that colour preferences are determined by subjective experiences and that most and least preferred colours are not processed equally.     

The Relation of Signal Transduction to the Sensitivity and Dynamic Range of Bacterial Chemotaxis

Complex networks of interacting molecular components of living cells are responsible for many important processes, such as signal processing and transduction. An important challenge is to understand how the individual properties of these molecular interactions and biochemical transformations determine the system-level properties of biological functions. Here, we address the issue of the accuracy of signal transduction performed by a bacterial chemotaxis system. The chemotaxis sensitivity of bacteria to a chemoattractant gradient has been measured experimentally from bacterial aggregation in a chemoattractant-containing capillary. The observed precision of the chemotaxis depended on environmental conditions such as the concentration and molecular makeup of the chemoattractant. In a quantitative model, we derived the chemotactic response function, which is essential to describing the signal transduction process involved in bacterial chemotaxis. In the presence of a gradient, an analytical solution is derived that reveals connections between the chemotaxis sensitivity and the characteristics of the signaling system, such as reaction rates. These biochemical parameters are integrated into two system-level parameters: one characterizes the efficiency of gradient sensing, and the other is related to the dynamic range of chemotaxis. Thus, our approach explains how a particular signal transduction property affects the system-level performance of bacterial chemotaxis. We further show that the two parameters can be derived from published experimental data from a capillary assay, which successfully characterizes the performance of bacterial chemotaxis.     

Soluble Combinatorial Libraries: Extending the Range and Repertoire of Chemical Diversity

The generation and use of libraries made up of millions of chemical entities and the ability to identify the active compound in such libraries are at the forefront of a revolution in drug discovery and basic research. Such libraries, when made up of peptide sequences, offer a fundamental, practical advance in the study of interactions between peptides and their biochemical or pharmacological targets. The utility of soluble peptide libraries ranging from three to eight amino acids in length, and made up of mixtures from 361 tripeptides to 200 billion decapeptides, is described. These are readily usable in virtually all in vitro (and even in vivo) assay systems. The examples presented illustrate the utility of soluble peptide libraries for the study of antibody/antigen interactions, the identification of highly active opioid peptides in receptor binding studies using crude rat brain homogenates, and in vivo studies, in which the peptide mixtures making up the library are administered intravenously to determine peptide sequences that affect heart rate and blood pressure. A new class of library is also described, termed a modified peptide library, which is used to determine potent anti-Staphylococcus aureus compounds.     

Modern Approaches to Chemical Modification of Proteins in Biological Tissues: Consequences and Application

Products made of biomaterials, such as heart valve prostheses, vascular grafts, and patches for vascular and intracardiac plastics, are currently used in cardiovascular surgery. The biological tissue used for prosthetics is the alternation of transverse and longitudinal layers of collagen fibers consisting of type I collagen (75%), elastin (<5%), cell elements, as well as glycoproteins, glycosaminoglycans, and other components of the cell matrix. Chemical modifications of components of a biological tissue allow for retention of its natural architectonics and stability of collagen structure over time, while simultaneously increasing the collagen resistance to enzymatic and mechanical destruction and preventing cellular and immune effects on the part of the recipient organism. Proteins in biological tissues are chemically modified (preserved) by the formation of intramolecular and intermolecular cross-links between the amino groups of amino acid residues in collagen molecules. However, cross-linking increases the calcification of biomaterial, making the tissue more rigid and leading to the rupture of the valve flaps, stenosis (reduced clearance), or insufficiency (a decrease in the closure function) of the heart valves. Calcification can also result from specific physiological features of recipient (the patient who received the artificial organ), the nature of the preserving agent, components of the dead cells, defects of collagen structure, cavities in tissues, and the presence of lipids, elastin fibers, glycosaminoglycans, and so on. The factors that induce calcification of the materials used for prosthetic repair and the corresponding methods for its prevention are reviewed. All methods are conventionally divided into three groups: chemical pretreatment of tissues, modification of the preservation method, and posttreatment of preserved tissues with chemical agents. The mechanisms of the processes underlying the effect of chemical agents on the structures of biological tissues are described. The results of their use in clinical practice and prospects for methods still under development and in preclinical trials are discussed, as well as the reasons why some methods have failed. The advantages and disadvantages of various types of treatments are considered. Variants of new methods for chemical modification of biological materials potentially effective in reducing the risk of calcification are proposed.     

Wet Chemical Colorimetric Estimation of CO: An Update on the Reduced Silver Sol Spectral and Kinetic Transformations to Silver nanoparticle

Measurements of ppm (v/v) level CO_g concentration is conveniently performed by its preconcentration in alkaline absorber solution of Ag⁺-(4)- HCO₂-C₆H₄-SO₂NH₂ complex, followed by a spectral measurement of the reduced silver sol. In this study, the transitory nature of this latter species and its subsequent real-time transformation to silver nanoparticle are presented. These results were based on spectral measurements made under varying concentrations of alkali, (4)-HCO₂-C₆H₄-SO₂NH₂, and Ag⁺ in the absorber solution, and in the presence of a wide range of sampled CO_g concentration. The initially created light yellow colored sol with its broad absorption profile peaking at 380 nm and absorption coefficient 3500?±?300 cm^?1 M^?1 (related to the amount of sampled [CO_g] as standardized by gas chromatographic analysis) changed into the characteristic yellow orange nanoparticle with its plasmon band peak absorption at 425 nm and absorption coefficient 6350?±?300 cm^?1 M^?1. Under different sampling conditions, the respective first-order conversion rates varied between 0.03 and 0.15 h^?1, whereas simultaneous dynamic light scattering measurements revealed steady growth of the averaged particle size ranging from 60 to 300 nm.     

The Mobility of Manganese in the Wheat Plant: II. Redistribution in Relation to Concentration and Chemical State

Wheat plants were grown in solution culture at two levels ofmanganese supply until the second leaves were fully expanded.The supply of manganese was then curtailed and symptoms of deficiencyappeared in subsequent growth. Although redistribution of manganeseat rates dependent upon initial content occurred in both high-and low-manganese plants, first and second leaves always retainedsufficient for normal function, and remained healthy. In a secondexperiment no redistribution from the second leaves was observedunder similar conditions of supply and demand. When second leaves were killed and placed in water, up to 80per cent, of the manganese diffused out, but extraction of similarleaves with 80 per cent, methanol removed only 15—20 percent, of that present. These results suggest that the lack ofmobility shown by the element is not due to precipitation orcomplex formation within the tissue, although at high levelsof manganese supply the solubility of manganese silicate mightbe exceeded, and some precipitation may occur.     

藏红花凝集素分子化学修饰与其活性的关系

对甘露糖专一性结合藏红花凝集素 (Crocussativuslectin ,CSL)分子进行化学修饰 ,测定酿酒酵母 (S .cerevisiae)凝集活性和寡糖专一性结合活性的变化 .实验结果表明 ,Cys的修饰与活性无关 ,Arg、Tyr和His的修饰降低了CSL分子的酵母凝集活性和寡糖结合活性 ,但对CSL的CD光谱无显著影响 ,表明其为凝集素的活性氨基酸残基 .Glu和Asp的化学修饰可使CSL的凝集活性大幅度降低 ,与特异性寡糖的亲和力增大 ,CD光谱变化明显 ,提示CSL分子中的Glu和Asp对其空间结构影响较大 ,氨基酸羧基的修饰导致CSL构象改变 ,蛋白与寡糖的结合位点暴露 ,可有效结合的位点数增加