Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.     

Sucrose transport from source to sink seeds in rice

In many higher plants, sucrose is loaded as a major carbon photoassimiliate into the phloem apoplastically by sucrose transporters (SUTs) and unloaded in sink tissues, where it serves as a storage material, carbohydrate backbone, and energy source. In sink tissues, a proportion of sucrose molecules are converted by cell wall invertases (CINs) into hexose that is imported into cells by monosaccharide transporters (MSTs). Thus, in developing seeds, co-ordinated regulation of SUTs, CINs, and MSTs is crucial in carbon distribution. Here, we summarize current efforts on the identification of SUTs, CINs, and MSTs in rice.     

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.     

Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus

Sucrose transporters in the SUT family are important for phloem loading and sucrose uptake into sink tissues. The recent localization of type III SUTs AtSUT4 and HvSUT2 to the vacuole membrane suggests that SUTs also function in vacuolar sucrose transport. The transport mechanism of type III SUTs has not been analyzed in detail. LjSUT4, a type III sucrose transporter homolog from Lotus japonicus, is expressed in nodules and its transport activity has not been previously investigated. In this report, LjSUT4 was expressed in Xenopus oocytes and its transport activity assayed by two-electrode voltage clamping. LjSUT4 transported a range of glucosides including sucrose, salicin, helicin, maltose, sucralose and both alpha- and beta-linked synthetic phenyl glucosides. In contrast to other sucrose transporters, LjSUT4 did not transport the plant glucosides arbutin, fraxin and esculin. LjSUT4 showed a low affinity for sucrose (K (0.5) = 16 mM at pH 5.3). In addition to inward currents induced by sucrose, other evidence also indicated that LjSUT4 is a proton-coupled symporter: (14)C-sucrose uptake into LjSUT4-expressing oocytes was inhibited by CCCP and sucrose induced membrane depolarization in LjSUT4-expressing oocytes. A GFP-fusion of LjSUT4 localized to the vacuole membrane in Arabidopsis thaliana and in the roots and nodules of Medicago truncatula. Based on these results we propose that LjSUT4 functions in the proton-coupled uptake of sucrose and possibly other glucosides into the cytoplasm from the vacuole.     

Framework for hybrid life cycle inventory databases: a case study on the Building for Environmental and Economic Sustainability (BEES) database

Purpose

In an effort to develop a whole building Life Cycle Assessment (LCA) tool, National Institute of Standards and Technology (NIST) is transforming new bottom-up Building for Environmental and Economic Sustainability (BEES) data into a hybrid database in which the strengths of both bottom-up and top-down approaches can be combined. The objective of this paper is to describe the framework and the process under which the hybrid BEES database is being built, with an emphasis on its accounting structure. This paper can support other efforts to build hybrid Life Cycle Inventory (LCI) databases.

Methods

The BEES hybridization utilizes the most detailed supply and use tables (SUTs)??known as item-level data??focusing particularly on the construction sectors. First, the partial SUTs at the item level are constructed and connected to standard SUTs that describe the rest of the economy, which is then followed by balancing and ??redefinition.?? Second, item-level environmental data are compiled and then also balanced and redefined, which completes the compilation of the bi-resolution SUTs with environmental data. Third, the bi-resolution SUTs are integrated with the BEES data that have been converted into matrix form. Because the completely rolled out BEES technology matrix involves a significant number of products, the integration prioritizes the product groups that are potentially the most significant contributors to the LCIA results for buildings.

Results

This step-by-step procedure will enable the creation of a hybridized BEES database, combining the strengths of both the bottom-up, process-based data and the top-down, input-output data with enhanced resolution. The benefit of hybridization at the database level??as opposed to at the individual LCA study level??is that whole-building LCA users can adopt the hybrid BEES approach, with its benefit of a more complete system definition, without the training or effort that would be required to construct a hybrid system from scratch. In addition, reformulation of new BEES data into a matrix structure better facilitates the parametric LCA application that is central to NIST??s vision to develop a tool for assessing the sustainability performance of energy technologies and systems in an integrated building design context.

Conclusions

There are currently a number of initiatives being organized to implement a hybrid approach at the LCI database level. In laying out the methodological framework for efficiently transforming an existing LCI database into a hybrid database, this paper can support future development of hybrid LCI databases.     

植物体内蔗糖转运蛋白的功能和调控

蔗糖转运蛋白（sucrose transporter,SUT）负责蔗糖的跨膜运输,在韧皮部介导的源-库蔗糖运输和为库组织供应蔗糖的生理活动中起关键作用。本文介绍植物体内蔗糖转运蛋白基因家族、细胞定位与功能调节以及高等植物的蔗糖感受机制的研究进展。     

Chloroplast phylogenomics of the New World grape species (Vitis,Vitaceae)

Vitis L. (the grape genus) is the economically most important fruit crop, as the source of grapes and wine. Phylogenetic relationships within the genus have been highly controversial. Herein, we employ sequence data from whole plastomes to attempt to enhance Vitis phylogenetic resolution. The results support the New World Vitis subgenus Vitis as monophyletic. Within the clade, V. californica is sister to the remaining New World Vitis subgenus Vitis. Furthermore, within subgenus Vitis, a Eurasian clade is robustly supported and is sister to the New World clade. The clade of Vitis vinifera ssp. vinifera and V. vinifera ssp. sylvestris is sister to the core Asian clade of Vitis. Several widespread species in North America are found to be non‐monophyletic in the plastome tree, for example, the broadly defined Vitis cinerea and V. aestivalis each needs to be split into several species. The non‐monophyly of some species may also be due to common occurrences of hybridizations in North American Vitis. The classification of North American Vitis by Munson into nine series is discussed based on the phylogenetic results. Analyses of divergence times and lineage diversification support a rapid radiation of Vitis in North America beginning in the Neogene.     

高等植物蔗糖转运的分子调控

在高等植物中,蔗糖的合成、运输与分配是一个复杂的过程。蔗糖由源到库的运输不仅与植物的生长发育相关,还受到植物体内的激素水平以及外界环境条件变化等因素的影响。蔗糖转运蛋白介导了蔗糖在植物韧皮部的装载、运输和卸载,在某些库中的蔗糖转运和库组织分配的分子调控中起有重要的生理作用。此外,简要介绍了笔者实验室在橡胶树蔗糖转运蛋白基因研究方面的最新进展。     

刺葡萄种内遗传多样性研究进展

刺萄萄主要分布在我国长江中下游及以南地区,是中国野生萄萄中果粒最大的一个种,其果实高抗炭疽病和白腐病,在湖南、江西、福建、贵州等省的部分地区一直作为鲜食和酿酒品种栽培利用。刺葡萄种内遗传多样性较为丰富,为了充分挖掘利用刺葡萄资源,提高刺葡萄资源的利用效率,本文从刺葡萄的物候学、花器类型、形态性状、果实理化性状、花粉形态、DNA遗传水平和抗性等方面进行种内多样性的研究和综述,以期为刺葡萄种质资源的保护、鉴定评价和发掘利用奠定基础。     

贵州喀斯特山区野生葡萄实生苗抗旱机理研究

以4份2生年喀斯特山区野生葡萄实生苗为试材,通过塑料大棚盆栽试验研究了自然干旱胁迫下野生葡萄幼苗的外观形态特性和生理指标变化规律,探讨其抗旱性及其抗旱机理.结果表明;(1)4份野生葡萄的抗旱性存在明显差异,以葛藟葡萄'小七孔-3'的抗旱性最强,毛葡萄'天河潭-2'的最差.(2)干旱胁迫期间,'小七孔-3'的1级旱害症状出现时间比'天河潭-2'晚12d,且旱害指数始终小于其他3份种质.(3)抗旱性强的野生葡萄叶片相对含水量、可溶性蛋白含量高于抗旱性差的株系,而其叶片水分饱和亏、细胞膜透性、丙二醛(MDA)含量变化则相反,且材料间差异随着干旱程度的增强而增加;(4)野生葡萄叶片相对含水量与其旱害指数间呈极显著负相关,而其水分饱和亏、相对电导率、丙二醛含量与旱害指数间呈极显著正相关;细胞膜脂过氧化产物丙二醛含量增加是导致葡萄旱害的首要因素.研究发现,在干旱胁迫下,抗旱性强的野生葡萄叶片膜系统稳定,水分散失较少,细胞电解质外渗较低,渗透调节物质可溶性蛋白含量增加,从而维持了正常的生理代谢活动.     

Molecular characterization and expression patterns of sucrose transport-related genes in sweet sorghum under defoliation

Sucrose is the principal form of photosynthesis products, and long-distance transport of sucrose requires sucrose transporters (SUTs) to perform loading and unloading functions. SUTs play an important role in plant growth, development and reproduction. In this study, five unique sucrose transporter (SbSUT) genes that contain full-length cDNA sequences were cloned from sweet sorghum, and these SbSUT genes were clustered into four different clades: SUT1, SUT3, SUT4 and SUT5. Heterologous expression of SbSUTs in yeast demonstrated that they were functional sucrose transporters. Tissue-specific expression profiles showed that sorghum SUT genes had different tissue-specific expression patterns, suggesting that sorghum SUT genes may play an important role in plant growth and developmental processes. After defoliation, expression patterns of SbSUT1, SbSUT2 and SbSUT4 were different in leaf sheaths, leaves and roots. Taken together, the results indicate that the above mentioned five unique sucrose transporter genes may play important roles in performing sucrose loading and unloading functions and that they exhibit different expression in response to leaf blade removal.     

贵州葡萄属野生种光合特性的研究

为掌握野生葡萄种间和种内不同单株间的光合特性差异,利用Li-6400便携式光合测定系统测定了7种野生葡萄25个单株的光合参数.结果表明:野生葡萄种间光合特性差异明显,美丽葡萄净光合速率最高,其次是刺葡萄,腺枝葡萄的净光合速率最低.野生葡萄种内不同单株间的光合指标多样性丰富,单株间光合特性差异最大的是葛藟葡萄;种内不同单株间原产地气候条件相似,其光合特性差异较小,而原产地气候差异大的单株光合特性差异也大;野生葡萄的光合速率调控机制多样,非气孔因素调控在野生葡萄种群内非常普遍.     

Genetic complementation analysis of rice sucrose transporter genes in Arabidopsis <Emphasis Type="Italic">SUC2</Emphasis> mutant <Emphasis Type="Italic">atsuc2</Emphasis>

Sucrose transporters (SUTs) play a critical role on the phloem plasma membrane in loading sucrose into the phloem of source leaves for long-distance transport to sink organs. Rice has a small gene family of five SUTs, Oryza sativa SUT1 (OsSUT1) to OsSUT5. To identify rice SUTs that function as phloem loaders, we adopted a growth restoration assay of the severe growth retardation phenotype of atsuc2, a mutant of the best-characterized Arabidopsis phloem loader AtSUC2, by introducing OsSUTs. The rice SUT genes were expressed by two different promoters, the native phloem-specific promoter of AtSUC2 (pAtSUC2) and the constitutive Cauliflower Mosaic Virus 35S (pCaMV35S) promoter. Of all the transgenic atsuc2 plants, only pAtSUC2: OsSUT1 complemented the atsuc2 mutant phenotype in a comparable manner to wild type (WT), and consistent levels of soluble sugars and starch were recovered compared to those of WT. This suggests that OsSUT1 is a functional ortholog of the Arabidopsis AtSUC2 and functions as an apoplastic phloem loader. In addition, ossut1 mutants were produced via anther culture and their primary carbohydrate levels and growth phenotypes were indistinguishable from those of WT. This suggests that the rice phloem loader OsSUT1 function may not be essential for rice vegetative growth under normal conditions.     

Chloroplast microsatellite polymorphisms in Vitis species.

The use of consensus chloroplast microsatellites primers for dicotyledonous chloroplast genomes revealed the existence of intra and interspecific length variation within the genus Vitis. Three chloroplast microsatellite loci were found to be polymorphic in samples of Vitis vinifera, Vitis berlandieri, Vitis riparia, and Vitis rupestris out of a total of 10 consensus primer pairs tested. These polymorphisms were always due to a variable number of mononucleotide residues within A and (or) T stretches in the amplified regions. Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplast in V. vinifera and to characterise the chloroplast haplotypes present in wine grape cultivars of this species grown in Spain and Greece. The different distribution of haplotype frequencies in the two ends of the Mediterranean growth area suggests the existence of independent domestication events for grapevine.     

Molecular phylogeny of the genus Vitis (Vitaceae) based on plastid markers

? Premise of the study: This work represents the first molecular phylogeny of the economically important genus Vitis, an important genetic resource for breeding in grapevine, Vitis vinifera. ? Methods: A molecular phylogeny of Vitis using a combined data set of three noncoding regions of the plastid DNA genome was constructed from 47 accessions covering 30 species of Vitis. The data for the trnL-F marker were combined with previously published data across the Vitaceae. ? Key results: The molecular phylogeny demonstrated monophyly of the genus Vitis. Based on the combined analysis of three genes, Vitis is split into three clades that mirror the continental distribution of these accessions. The diversity is highest in the Asian clade, but the general genetic distances across taxa from different continents are relatively small. ? Conclusions: The findings support a relatively recent and intense gene flow between East Asia and North America and the possible impact of hybridization on the evolution of the genus Vitis. Taxon identity in important stock collections should be screened carefully because roughly 10% of the accessions analyzed in the present study had been misidentified.