Search

Functional innovation at the protein level is a key source of evolutionary novelties. The constraints on functional innovations are likely to be highly specific in different proteins, which are shaped by their unique histories and the extent of global epistasis that arises from their structures and biochemistries. These contextual nuances in the sequence–function relationship have implications both for a basic understanding of the evolutionary process and for engineering proteins with desirable properties. Here, we have investigated the molecular basis of novel function in a model member of an ancient, conserved, and biotechnologically relevant protein family. These Major Facilitator Superfamily sugar porters are a functionally diverse group of proteins that are thought to be highly plastic and evolvable. By dissecting a recent evolutionary innovation in an α-glucoside transporter from the yeast Saccharomyces eubayanus , we show that the ability to transport a novel substrate requires high-order interactions between many protein regions and numerous specific residues proximal to the transport channel. To reconcile the functional diversity of this family with the constrained evolution of this model protein, we generated new, state-of-the-art genome annotations for 332 Saccharomycotina yeast species spanning ∼400 My of evolution. By integrating phylogenetic and phenotypic analyses across these species, we show that the model yeast α-glucoside transporters likely evolved from a multifunctional ancestor and became subfunctionalized. The accumulation of additive and epistatic substitutions likely entrenched this subfunction, which made the simultaneous acquisition of multiple interacting substitutions the only reasonably accessible path to novelty. [ABSTRACT FROM AUTHOR]

The genetic basis of phenotypic variation is a long-standing concern of evolutionary biology. Coloration has proven to be a visual, easily quantifiable, and highly tractable system for genetic analysis and is an ever-evolving focus of biological research. Compared with the homogenized brown-yellow cocoons of wild silkworms, the cocoons of domestic silkworms are spectacularly diverse in color, such as white, green, and yellow-red; this provides an outstanding model for exploring the phenotypic diversification and biological coloration. Herein, the molecular mechanism underlying silkworm green cocoon formation was investigated, which was not fully understood. We demonstrated that five of the seven members of a sugar transporter gene cluster were specifically duplicated in the Bombycidae and evolved new spatial expression patterns predominantly expressed in silk glands, accompanying complementary temporal expression; they synergistically facilitate the uptake of flavonoids, thus determining the green cocoon. Subsequently, polymorphic cocoon coloring landscape involving multiple loci and the evolution of cocoon color from wild to domestic silkworms were analyzed based on the pan-genome sequencing data. It was found that cocoon coloration involved epistatic interaction between loci; all the identified cocoon color-related loci existed in wild silkworms; the genetic segregation, recombination, and variation of these loci shaped the multicolored cocoons of domestic silkworms. This study revealed a new mechanism for flavonoids-based biological coloration that highlights the crucial role of gene duplication followed by functional diversification in acquiring new genetic functions; furthermore, the results in this work provide insight into phenotypic innovation during domestication. [ABSTRACT FROM AUTHOR]

We previously demonstrated that alterations in sugar partitioning affect the expression of genes involved in hormone biosynthesis and responses, including BRANCHED1 (BRC1), resulting in enhanced shoot branching in transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). The exogenous treatment of wild-type Arabidopsis plants with sugars showed the same transcript characteristics, indicating that sugars act as a signal for branching. We also found that the reductions induced in BRC1 expression levels in wild-type plants by the sugar treatments were suppressed in the knockout mutant of sugar transporter 1 (stp1-1). Intracellular sugar contents were similar in stp1-1 and wild-type plants following the sugar treatments, suggesting that STP1 acts as a factor for the regulation of shoot branching depending on extracellular sugar contents. Abbreviations: BRC1: BRABCHED1; FBP/SBPase: fructose-1,6-/sedoheptulose-1,7-bisphosphatase; Glc: glucose; HXK: hexokinase; SnRK1.1/AKIN10: SNF1-RELATED PROTEIN KINASE 1.1; Suc: sucrose; SnRK1: sucrose non-fermenting 1-related protein kinase; STP: sugar transporter protein [ABSTRACT FROM AUTHOR]

The yeast S. cerevisiae senses glucose through Snf3 and Rgt2, transmembrane proteins that generate an intracellular signal in response to glucose that leads to inhibition of the Rgt1 transcriptional repressor and consequently to derepression of HXT genes encoding glucose transporters. Snf3 and Rgt2 are thought to be glucose receptors because they are similar to glucose transporters. In contrast to glucose transporters, they have unusually long C-terminal tails that bind to Mth1 and Std1, paralogous proteins that regulate function of the Rgt1 transcription factor. We show that the C-terminal tail of Rgt2 is not responsible for its inability to transport glucose. To gain insight into how the glucose sensors generate an intracellular signal, we identified RGT2 mutations that cause constitutive signal generation. Most of the mutations alter evolutionarily-conserved amino acids in the transmembrane spanning regions of Rgt2 that are predicted to be involved in maintaining an outward-facing conformation or to be in the substrate binding site. Our analysis of these mutations suggests they cause Rgt2 to adopt inward-facing or occluded conformations that generate the glucose signal. These results support the idea that Rgt2 and Snf3 are glucose receptors that signal in response to binding of extracellular glucose and inform the basis of their signaling. [ABSTRACT FROM AUTHOR]

Fruits are an important part of the human diet and sugar content is a major criterion used to evaluate fruit quality. Melon fruit accumulate high sugar concentrations during their development; however, the mechanism through which these sugars are transported into the vacuoles of fruit cells for storage remains unclear. In this study, three tonoplast sugar transporters (TSTs), CmTST1, CmTST2, and CmTST3, were isolated from melon plants. Analysis of subcellular localization revealed that all these proteins were targeted to the tonoplast, and evaluation of spatial expression and promoter-GUS activity indicated that they had different expression patterns in the plant. RT-PCR and qRT-PCR results indicated that CmTST2 exhibited the highest expression level among the TST isoforms during melon fruit development. Histochemical and immunohistochemistry localization experiments were performed to identify the tissue- and cell-type localization of CmTST2 in the fruit, and the results indicated that both its transcription and translation were in the mesocarp and vascular cells. Overexpressing the CmTST2 gene in strawberry fruit and cucumber plants by transient expression and stable transformation, respectively, both increased sucrose, fructose, and glucose accumulation in the fruit. The results indicate that CmTST2 plays an important role in sugar accumulation in melon fruit. [ABSTRACT FROM AUTHOR]

This study investigated the role of the sugar transporter OsSWEET11 during the early stage of rice caryopsis development using b-glucoronidase (GUS) to represent its expression, together with clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR- Cas9)-mediated knockout, cross-fertilization and RNA sequencing (RNA-seq) analyses. The results showed that OsSWEET11 was expressed strongly in developing caryopsis, particularly in the ovular vascular trace, nucellar epidermis and cross cells. The knockout of OsSWEET11 significantly decreased the sucrose concentration in the mutant embryo sacs and led to defective grain filling compared with that of the wild-type (WT) plant. Moreover, the expression of 2,549 genes in the mutant caryopsis was affected. The grain weight and seed setting percentage were also decreased in the mutants. The cross-fertilization of the mutant and WT rice revealed that the mutated maternal donor induced defective grain filling. These results strongly suggested that OsSWEET11 played an important role in sucrose release from maternal tissue to the maternal-filial interface during the early stage of caryopsis development. It might also induce sucrose release from the ovular vascular trace and cross cells of developing caryopsis. These findings bridge the gap in the understanding of post-phloem sugar transport during the early stage of rice caryopsis development. [ABSTRACT FROM AUTHOR]

d-xylose andl-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. AlthoughPichia stipitisCBS 6054 grows usingd-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependentd-arabinose reductase from cells grown ond-arabinose, and found that the enzyme was identical to the knownd-xylose reductase (XR). The enzyme activity of XR withd-arabinose was previously reported to be only 1% that withd-xylose. Thekcat/Kmvalue withd-arabinose (1.27 min−1 mM−1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those withl-arabinose andd-xylose, respectively. Among the 34 putative sugar transporters fromP. stipitis, only seven genes exhibited uptake ability not only ford-arabinose, but also ford-glucose and other pentose sugars includingd-xylose andl-arabinose inSaccharomyces cerevisiae. d-Xylose reductase, encoded byXYL1gene, plays as an aldose reductase involved not only ind-xylose andl-arabinose but alsod-arabinose metabolism. [ABSTRACT FROM PUBLISHER]

Transcriptional repression of STP1 is rapidly induced by phosphorylatable sugars through an HXK1-independent signalling pathway involving the participation of sugar-responsive cis elements localized in the promoter.Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H+/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5′ regulatory region allowed us to delimit a region of 309bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified. [ABSTRACT FROM AUTHOR]

Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H+/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5′ regulatory region allowed us to delimit a region of 309 bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified. [ABSTRACT FROM AUTHOR]

The mobility of sugars between source and sink tissues in plants depends on sugar transport proteins. Studying the corresponding genes allows the manipulation of the sink strength of developing fruits, thereby improving fruit quality for human consumption. Tomato (Solanum lycopersicum) is both a major horticultural crop and a model for the development of fleshy fruits. In this article we provide a comprehensive inventory of tomato sugar transporters, including the SUCROSE TRANSPORTER family, the SUGAR TRANSPORTER PROTEIN family, the SUGAR FACILITATOR PROTEIN family, the POLYOL/MONOSACCHARIDE TRANSPORTER family, the INOSITOL TRANSPORTER family, the PLASTIDIC GLUCOSE TRANSLOCATOR family, the TONOPLAST MONOSACCHARIDE TRANSPORTER family and the VACUOLAR GLUCOSE TRANSPORTER family. Expressed sequence tag (EST) sequencing and phylogenetic analyses established a nomenclature for all analyzed tomato sugar transporters. In total we identified 52 genes in tomato putatively encoding sugar transporters. The expression of 29 sugar transporter genes in vegetative tissues and during fruit development was analyzed. Several sugar transporter genes were expressed in a tissue- or developmental stage-specific manner. This information will be helpful to better understand source to sink movement of photoassimilates in tomato. Identification of fruit-specific sugar transporters might be a first step to find novel genes contributing to tomato fruit sugar accumulation. [ABSTRACT FROM PUBLISHER]

Genetic evidence indicates that only the β-anomer of galactose is transported to Kluyveromyces lactis cells by galactose/glucose transporter Hgt1p, and that aldose-1-epimerase encoded by GAL10 is a prerequisite for growth on galactose. Minor aldose-1-epimerases other than Gal10p also exist in K. lactis. Using a mutant defective in both aldose-1-epimerases, we show by solid-state nuclear magnetic resonance spectroscopy that only β-anomer is transported in the cell and stays without or with a slow rate of conversion to α-anomer. Signals due to intracellular β-galactose appeared at two positions, both of which were shifted towards higher magnetic fields than that of β-galactose in aqueous solution, suggesting that incorporated galactose binds to cellular components, probably proteins. [ABSTRACT FROM AUTHOR]

The article presents a study which aims to characterize a bacterial laminaribiose phosphorylase (LBCbac). The study used high-performance anion-exchange chromatography to quantify soluble sugars in the preparation of the laminarioligosaccharide (LO) mixture. The results found that the presence of LO-specific adenosine triphosphate (ATP)-binding casette (ABC) transporter suggests that the role of LBCbac is to use laminaribiose obtained outside the cell.

Aldose-1-epimerase or mutarotase (EC 5.1.3.3) catalyzes interconversion of α/β-anomers of aldoses, such as glucose and galactose, and is distributed in a wide variety of organisms from bacteria to humans. Nevertheless, the physiological role of this enzyme has been elusive in most cases, because the α-form of aldoses in the solid state spontaneously converts to the β-form in an aqueous solution until an equilibrium of α : β=36.5 : 63.5 is reached. A gene named GAL10 encodes this enzyme in yeast. Here, we show that the GAL10-encoded mutarotase is necessary for utilization of galactose in the milk yeast Kluyveromyces lactis, and that this condition is presumably created by the presence of the β-specific galactose transporter, which excludes the α-anomer from the α/β-mixture in the medium at the cell surface. Thus, we found that a mutarotase-deficient mutant of K. lactis failed to grow on medium, in which galactose was the sole carbon source, but, surprisingly, that the growth failure is suppressed by concomitant expression of the Saccharomyces cerevisiae-derived galactose transporter Gal2p, but not by that of the K. lactis galactose transporter Hgt1p. We also suggest the existence of another mutarotase in K. lactis, whose physiological role remains unknown, however. [ABSTRACT FROM AUTHOR]

The plant-parasitic nematode Heterodera schachtii stimulates plant root cells to form syncytial feeding structures which synthesize all nutrients required for successful nematode development. Cellular re-arrangements and modified metabolism of the syncytia are accompanied by massive intra- and intercellular solute allocations. In this study the expression of all genes annotated as sugar transporters in the Arabidopsis Membrane Protein Library was investigated by Affymetrix gene chip analysis in young and fully developed syncytia compared with non-infected Arabidopsis thaliana roots. The expression of three highly up-regulated (STP12, MEX1, and GTP2) and three highly down-regulated genes (SFP1, STP7, and STP4) was analysed by quantitative RT-PCR (qRT-PCR). The most up-regulated gene (STP12) was chosen for further in-depth studies using in situ RT-PCR and a nematode development assay with a T-DNA insertion line revealing a significant reduction of male nematode development. The specific role of STP12 expression in syncytia of male juveniles compared with those of female juveniles was further shown by qRT-PCR. In order to provide evidence for sugar transporter activity across the plasma membrane of syncytia, fluorescence-labelled glucose was used and membrane potential recordings following the application of several sugars were performed. Analyses of soluble sugar pools revealed a highly specific composition in syncytia. The presented work demonstrates that sugar transporters are specifically expressed and active in syncytia, indicating a profound role in inter- and intracelluar transport processes. [ABSTRACT FROM PUBLISHER]

Saccharomyces cerevisiae uses glucose preferentially to any other carbon source and this preferential use is ensured by control mechanisms triggered by glucose. The consensus is that inactivation of sugar transporters other than glucose transporters is one of these mechanisms. This inactivation is called catabolite inactivation because of its apparent analogy with the catabolite inactivation of gluconeogenic enzymes. Recently, doubt has been cast on the role of the inactivation of the sugar transporters in controlling the use of glucose because this inactivation neither is specifically triggered by glucose nor specifically affects non-glucose sugar transporters. Based on the fact that this inactivation has been almost exclusively investigated using nitrogen-starved cells, it has been proposed that it might be due to the stimulation of the protein turnover that follows nitrogen starvation. The results obtained in this work support this possibility since they show that the presence of a nitrogen source in the medium strongly inhibited the inactivation. It is concluded that, in growing yeast cells, the contribution of the inactivation by glucose of the non-glucose sugar transporters to the preferential use of glucose is much lower than generally believed. [ABSTRACT FROM AUTHOR]

Processed lignocellulosic biomass is a source of mixed sugars that can be used for microbial fermentation into fuels or higher value products, like chemicals. Previously, the yeast Saccharomyces cerevisiae was engineered to utilize its cellodextrins through the heterologous expression of sugar transporters together with an intracellular expressed β-glucosidase. In this study, we screened a selection of eight (putative) cellodextrin transporters from different yeast and fungal hosts in order to extend the catalogue of available cellobiose transporters for cellobiose fermentation in S. cerevisiae. We confirmed that several in silico predicted cellodextrin transporters from Aspergillus niger were capable of transporting cellobiose with low affinity. In addition, we found a novel cellobiose transporter from the yeast Lipomyces starkeyi , encoded by the gene Ls120451. This transporter allowed efficient growth on cellobiose, while it also grew on glucose and lactose, but not cellotriose nor cellotetraose. We characterized the transporter more in-depth together with the transporter CdtG from Penicillium oxalicum. CdtG showed to be slightly more efficient in cellobiose consumption than Ls120451 at concentrations below 1.0 g/L. Ls120451 was more efficient in cellobiose consumption at higher concentrations and strains expressing this transporter grew slightly slower, but produced up to 30% more ethanol than CdtG. [ABSTRACT FROM AUTHOR]