Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using β-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K_m for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.     

烤烟根系分泌物中单糖的组分含量及其相关分析

以4个不同烤烟品种为研究材料,采用盆栽试验,运用高效毛细管区带电泳法测定各品种根际土、非根际土、根系及叶片中的单糖组分及含量,并分析其相关关系,探究根系分泌物中糖类的分泌特性。结果表明：在各样品中,共检出木糖、葡萄糖、半乳糖、核糖、阿拉伯糖和鼠李糖6种糖;不同品种根际土、非根际土、根系及叶片中检出的糖组分及含量均存在差异;同一品种中,叶片最高,根系次之,根际土和非根际土最低;相关性分析表明,木糖、阿拉伯糖、葡萄糖、鼠李糖和半乳糖总量在根际土、非根际土、叶片和根系间呈正相关关系,各单糖组分间均呈正相关关系,部分组分呈显著或极显著相关关系。研究表明,不同烤烟品种根系分泌这些单糖存在品种差异,且根系分泌单糖可能是一个沿浓度梯度的扩散过程。     

Sugar transporters in higher plants--a diversity of roles and complex regulation

Sugar-transport proteins play a crucial role in the cell-to-cell and long-distance distribution of sugars throughout the plant. In the past decade, genes encoding sugar transporters (or carriers) have been identified, functionally expressed in heterologous systems, and studied with respect to their spatial and temporal expression. Higher plants possess two distinct families of sugar carriers: the disaccharide transporters that primarily catalyse sucrose transport and the monosaccharide transporters that mediate the transport of a variable range of monosaccharides. The tissue and cellular expression pattern of the respective genes indicates their specific and sometimes unique physiological tasks. Some play a purely nutritional role and supply sugars to cells for growth and development, whereas others are involved in generating osmotic gradients required to drive mass flow or movement. Intriguingly, some carriers might be involved in signalling. Various levels of control regulate these sugar transporters during plant development and when the normal environment is perturbed. This article focuses on members of the monosaccharide transporter and disaccharide transporter families, providing details about their structure, function and regulation. The tissue and cellular distribution of these sugar transporters suggests that they have interesting physiological roles.     

Effect of monomeric and oligomeric sugar osmolytes on ΔGD, the Gibbs energy of stabilization of the protein at different pH values: Is the sum effect of monosaccharide individually additive in a mixture?

Thermal denaturation curves of ribonuclease-A were measured by monitoring changes in the far-UV circular dichroism (CD) spectra in the presence of different concentrations of six sugars (glucose, fructose, galactose, sucrose, raffinose and stachyose) and mixture of monosaccharide constituents of each oligosaccharide at various pH values in the range of 6.0-2.0. These measurements gave values of T(m) (midpoint of denaturation), DeltaH(m) (enthalpy change at T(m)), DeltaC(p) (constant-pressure heat capacity change) under a given solvent condition. Using these values of DeltaH(m), T(m) and DeltaC(p) in appropriate thermodynamic relations, thermodynamic parameters at 25 degrees C, namely, DeltaG(D)(o) (Gibbs energy change), DeltaH(D)(o) (enthalpy change), and DeltaS(D)(o) (entropy change) were determined at a given pH and concentration of each sugar (including its mixture of monosaccharide constituents). Our main conclusions are: (i) each sugar stabilizes the protein in terms of T(m) and DeltaG(D)(o), and this stabilization is under enthalpic control, (ii) the protein stabilization by the oligosaccharide is significantly less than that by the equimolar concentration of the constituent monosaccharides, and (iii) the stabilization by monosaccharides in a mixture is fully additive. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of protein in their native and denatured states are not perturbed on the addition of sugars.     

Mannose efflux from the cells: a potential source of mannose in blood

All mammals have 50-100 μM mannose in their blood. However, the source of the dynamic pool of mannose in blood is unknown. Most of it is thought to be derived from glucose in the cells. We studied mannose uptake and release by various cell types. Interestingly, our results show that mannose taken up by the cells through transporters is handled differently from the mannose released within the cells due to glycan processing of protein-bound oligosaccharides. Although more than 95% of incoming mannose is catabolized, most of the mannose released by intracellular processing is expelled from the cells as free mannose predominantly via a nocodazole-sensitive sugar transporter. Under physiological conditions, incoming mannose is more accessible to hexokinase, whereas mannose released within the cells is protected from HK and therefore has a different fate. Our data also suggest that generation of free mannose due to the processing of glycoconjugates composed of glucose-derived mannose and its efflux from the cells can account for most of the mannose found in blood and its steady state maintenance.     

Sugars en route to the roots. Transport,metabolism and storage within plant roots and towards microorganisms of the rhizosphere

In plants, the root is a typical sink organ that relies exclusively on the import of sugar from the aerial parts. Sucrose is delivered by the phloem to the most distant root tips and, en route to the tip, is used by the different root tissues for metabolism and storage. Besides, a certain portion of this carbon is exuded in the rhizosphere, supplied to beneficial microorganisms and diverted by parasitic microbes. The transport of sugars toward these numerous sinks either occurs symplastically through cell connections (plasmodesmata) or is apoplastically mediated through membrane transporters (MST, mononsaccharide tranporters, SUT/SUC, H+/sucrose transporters and SWEET, Sugar will eventually be exported transporters) that control monosaccharide and sucrose fluxes. Here, we review recent progresses on carbon partitioning within and outside roots, discussing membrane transporters involved in plant responses to biotic and abiotic factors.     

Regulation of water balance in mangroves

Background Mangroves are a group of highly salt-tolerant woody plants. The high water use efficiency of mangroves under saline conditions suggests that regulation of water transport is a crucial component of their salinity tolerance.Scope This review focuses on the processes that contribute to the ability of mangroves to maintain water uptake and limit water loss to the soil and the atmosphere under saline conditions, from micro to macro scales. These processes include: (1) efficient filtering of the incoming water to exclude salt; (2) maintenance of internal osmotic potentials lower than that of the rhizosphere; (3) water-saving properties; and (4) efficient exploitation of less-saline water sources when these become available.Conclusions Mangroves are inherently plastic and can change their structure at the root, leaf and stand levels in response to salinity in order to exclude salt from the xylem stream, maintain leaf hydraulic conductance, avoid cavitation and regulate water loss (e.g. suberization of roots and alterations of leaf size, succulence and angle, hydraulic anatomy and biomass partitioning). However, much is still unknown about the regulation of water uptake in mangroves, such as how they sense and respond to heterogeneity in root zone salinity, the extent to which they utilize non-stomatally derived CO₂ as a water-saving measure and whether they can exploit atmospheric water sources.     

Analysis of transport activity of Arabidopsis sugar alcohol permease homolog AtPLT5

The transporters responsible for sugar uptake into non-photosynthetic sink tissues in plants, such as roots and flowers, have not been fully identified and analyzed. Plants encode around 100 putative sugar transporters within the major facilitator superfamily, yet only a few have been studied. Here we report the analysis of a sugar alcohol permease homolog (AtPLT5, At3g18830) from Arabidopsis. A wide range of sugars including hexoses, pentoses, tetroses, a sugar acid, and sugar alcohols but not disaccharides induced inward currents in oocytes expressing AtPLT5. AtPLT5 expression also resulted in 14C-labeled substrate uptake in oocytes, indicating that AtPLT5 encodes an ion-coupled uptake transporter. K(0.5) values for glucose and sorbitol were highly dependent on external pH. Expression of AtPLT5 was found primarily in sink tissues: in the elongation zone of roots, in the inflorescence stem, and several floral structures, especially in the floral abscission zone. Expression was induced by mechanical wounding and insect feeding. Analysis of transport properties and expression in Arabidopsis indicate that AtPLT5 functions to transport a wide range of sugars into specific sink tissues in the plant.     

Different Sugar Kinases Are Involved in the Sugar Sensing of Galdieria sulphuraria

The unicellular acidophilic red alga Galdieria sulphuraria is a facultative heterotroph with a complex uptake system for sugars and polyols, consisting of at least 14 transporters. Upon transfer to heterotrophic conditions, these transporters were induced simultaneously. Once induced, transporters for common hexoses and pentoses are apparently not down-regulated under heterotrophic conditions. Uptake of deoxysugars (FUC/Rha), however, was repressed by substrates metabolized via gluco-, galacto-, glycero-, or hexokinase, whereas substrates phosphorylated by xylulokinase had no effect. This indicates that several sugar kinases play a key role in sugar sensing. In contrast, polyol transporters were repressed only by glucose or its analogs but not by other sugars. This repression does not involve the activity of kinases. Most likely this type of glucose sensing is independent of metabolism and takes place prior to or during uptake. In its natural environment, these two different sensing mechanisms would enable the alga to utilize a mixture of different substrates in a most economic way by repressing dispensible transporters.     

Structural Basis for Outer Membrane Sugar Uptake in Pseudomonads

Substrate-specific outer membrane channels of Gram-negative bacteria mediate uptake of many small molecules, including carbohydrates. The mechanism of sugar uptake by enterobacterial channels, such as Escherichia coli LamB (maltoporin), has been characterized in great detail. In pseudomonads and related organisms, sugar uptake is not mediated by LamB but by OprB channels. Beyond the notion that OprB channels seem to prefer monosaccharides as substrates, very little is known about OprB-mediated sugar uptake. Here I report the X-ray crystal structure of an OprB channel from Pseudomonas putida F1. The structure shows that OprB forms a monomeric, 16-stranded β-barrel with a constriction formed by extracellular loops L2 and L3. The side chains of two highly conserved arginine residues (Arg⁸³ and Arg¹¹⁰) and a conserved glutamate (Glu¹⁰⁶) line the channel constriction and interact with a bound glucose molecule. Liposome swelling uptake assays show a strong preference for monosaccharide transport over disaccharides. Moreover, substrates with a net negative charge are disfavored by the channel, probably due to the negatively charged character of the constriction. The architecture of the eyelet and the absence of a greasy slide provide an explanation for the observed specificity of OprB for monosaccharides rather than the oligosaccharides preferred by LamB and related enterobacterial channels.     

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.     

Reprogramming of sugar transport pathways in Escherichia coli using a permeabilized SecY protein-translocation channel

In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications.     

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.