Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.     

Genome-wide annotation and functional identification of aphid GLUT-like sugar transporters

Background

Phloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity.

Results

In this study, using a de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9 (ApST1, ApST3, ApST4 and ApST9) as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters.

Conclusions

The gut enhanced expression of ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-647) contains supplementary material, which is available to authorized users.     

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.     

Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.     

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.     

Characterization of <Emphasis Type="Italic">SWEET</Emphasis> family members from loquat and their responses to exogenous induction

The plant SWEET family is a sugar transporter family that plays a significant role in plant development. Here, seven loquat SWEET family members were identified by RNA-seq. These were designated as EjSWEET1, EjSWEET2a, EjSWEET2b, EjSWEET2c, EjSWEET4, EjSWEET15, and EjSWEET17. Phylogenetic and predictive functional annotation analyses suggest that the loquat SWEETs are classified as having sucrose, glucose and fructose transportation features. The in vivo responses of loquat SWEETs to exogenous sugar or NaCl was investigated by applying high concentrations of sugar or salt to 7-month-old loquat seedlings cultured in a nutrient medium. The results showed that most loquat SWEET genes can respond to exogenous applications of sucrose, glucose, fructose and salt. The response of EjSWEET1 to exogenous fructose was faster than the others, indicating that EjSWEET1 is more sensitive to exogenous fructose compared with other loquat SWEETs. EjSWEET15 can be induced by sucrose, but is suppressed by glucose. This indicates its possible role in sucrose transporting. The response of loquat SWEETs to NaCl showed broadly similar patterns compared to sugars. However, after a longer time of NaCl treatment, most loquat SWEETs are upregulated, especially EjSWEET15. This indicates its long-term response to high salinity.     

Identification of QTLs Associated with Conversion of Sucrose to Hexose in Mature Fruit of Japanese Pear

Sweetness is the most important trait for fruit breeding and is fundamentally determined by both total and individual sugar contents. We analyzed the contents of sucrose, fructose, glucose, and sorbitol in mature fruit in an F₁ population derived from crossing modern Japanese pear cultivar ‘Akizuki’ and breeding line ‘373-55’. A genetic linkage map was constructed using simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP). We identified two regions associated with individual sugar contents on linkage group (LG) 1 and LG 7. The percentages of the variance in sucrose, fructose, and glucose explained by the quantitative trait loci (QTLs) were 26.6, 15.9, and 18.5%, respectively, for the region on LG 1, and 22.2, 20.0, and 9.5%, respectively, for the region on LG 7. In both regions, genotypes associated with increases in sucrose were associated with decreases in both fructose and glucose. The 1.5-LOD support intervals of the QTLs on LGs 1 and 7 include SSRs within the regions flanking acid invertase genes PPAIV3 and PPAIV1, respectively. Because acid invertase is a key enzyme in the conversion of sucrose to hexose, these are promising candidates for genes underlying those QTLs and controlling individual sugar contents. We also found one region on LG 11 that explained 21.4% of the variation in total sugar content but was not significantly associated with variation for individual sugars. The information obtained in this study will accelerate research and breeding programs to improve fruit sweetness.     

Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high ¹⁴C‐fructose and ¹⁴C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

Has Arabidopsis SWEETIE protein a role in sugar flux and utilization?

In plants, sugars affect growth and development and play an important role in the intricate machinery of signal transduction. Understanding the mechanisms behind the flux of sugar in the plant is of central interest. We recently characterized an Arabidopsis mutant: sweetie, which is defective in the control of growth and development, sterile, shows premature senescence and affects sugar metabolism. Our microarray analysis showed that 15 genes annotated as sugar transporter related proteins were found to be upregulated in sweetie while one sugar transporter gene was found to be downregulated. Most of them are unspecified sugar transporters but four genes have been annotated as monosaccharide transporters and one has been annotated as a disaccharide transporter. Moreover, as computer analyses predicted that SWEETIE might be a membrane protein and might have a function of glycosyl transferase, our data suggest that SWEETIE could be involved in the general control of sugar flux and modulates many important processes such as morphogenesis, flowering, stress responses and senescence.Key words: Arabidopsis thaliana, sweetie mutant, microarray, sugar flux, sugar transport     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

The CsSUT1 promoter from Citrus sinensis confers sink-specific expression of a downstream reporter gene in transgenic Arabidopsis

Membrane-localized H⁺-symporting sucrose transporters (SUC or SUT proteins) are involved in sucrose loading into the phloem of source tissues and sucrose uptake into sink tissues, which are essential events in the growth and development of higher plants. While many of these sucrose transporters are localized in the phloem, others function in sink tissues. In an attempt to gain insight into which class the CsSUT1 gene from Citrus sinensis falls, we isolated a 1537-bp upstream region of this gene (CsSUT1p), inserted it upstream of the ??-glucuronidase (GUS) reporter gene and transformed the resulting vector into Arabidopsis thaliana. Histochemical and semi-quantitative RT-PCR analyses indicated that the CsSUT1p conferred GUS expression in floral tissues and the roots of young seedlings, but not above ground vegetative tissues. In flowers, GUS expression was noted in young floral buds, as well as immature stamens and carpels. Deletion analyses indicated that a ?1052 to ?1 fragment (relative to the translational start codon at +1) of the CsSUT1p, but not a ?496 to ?1 fragment, was able to drive the same pattern of expression of a downstream reporter gene in transgenic Arabidopsis. Taken together, these results suggest that the CsSUT1 gene, like numerous SUC/SUT genes from other plant species, may play a role in the uptake of sucrose into sink tissues.     

Potassium enhances the sugar assimilation in leaves and fruit by regulating the expression of key genes involved in sugar metabolism of Asian pears

Potassium (K) plays an important role in fruit quality, and is well known as the most important quality element. A field experiment was conducted with four K levels of 0 (control), 150 (K150), 300 (K300), 450 (K450) kg K₂O ha^?1 in 2014–2015. The aim was to elucidate the roles of K in fruit growth, and the mechanism of K in regulating sugar metabolism between the leaves and fruit of Asian pear (Pyrus L.). The results showed that the K concentration and accumulation in leaves and fruit with the net photosynthetic rate and SPAD value of leaves were found to increase with the increase of K application rates. Increasing K application rates also led to promote the effectiveness of accumulation of glucose, fructose, sorbitol, and sucrose in fruit. During the early fruit development stage, the increase of all soluble sugars in leaves was correlated with the up-regulation expression of gene AIV and S6PDH. Furthermore, with fruit development, the expression of AIV1, SPS1 and SUS, S6PDH and SDH3 involved in sugar metabolism in leaves were up-regulated by increasing the K application rates, resulting in higher accumulation of soluble sugars in leaves. Interestingly, at the fruit maturity stage the expression of SUT in leaves, and SPS1, SUS and SUT in fruit was significantly up-regulated, leading to higher sucrose accumulation in fruit. Thus, K-promoted sugar accumulation of the leaves and fruit might result from up-regulated expression levels of key genes involved in sugar metabolism by K in leaves and fruit.     

Sucrose Metabolism in Netted Muskmelon Fruit during Development

Sugar content and composition are major criteria used in judging the quality of netted muskmelon (Cucumis melo L. var reticulatus) fruit. Sugar composition and four enzymes of sucrose metabolism were determined in `Magnum 45' muskmelon fruit at 10-day intervals beginning 10 days after pollination (DAP) until full-slip (35 DAP). Sugar content increased in both outer (green) mesocarp and inner (orange) mesocarp between 20 and 30 DAP. The major proportion of total increase in sugar was attributed to sucrose accumulation. The large increase in sucrose relative to glucose and fructose was accompanied by a dramatic decrease in acid invertase activity, which was highest in both tissues at 10 and 20 DAP, and increases in sucrose phosphate synthase (SPS) and sucrose synthase activities. The green tissue had a lower proportion of total sugar as sucrose, greater invertase activity, and less SPS activity than the orange tissue. Changes in relative sucrose content were highly correlated with changes in enzyme activity. The results strongly suggest that increases in the proportion of sucrose found in melon fruit were associated with a decline in acid invertase activity and an increase in SPS activity approximately 10 days before full-slip. Therefore, these enzymes apparently play a key role in determining sugar composition and the quality of muskmelon fruit.     

Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza

Arbuscular mycorrhizal fungi enhance CO₂ assimilation of their hosts which ensure the demand for carbohydrates of these obligate biotrophic microorganisms. Photosynthetic parameters were measured in tomato colonised or not by the arbuscular mycorrhizal fungus Glomus mosseae. In addition, carbohydrate contents and mRNA accumulation of three sucrose transporter genes were analysed. Mycorrhizal plants showed increased opening of stomata and assimilated significant more CO₂. A higher proportion of the absorbed light was used for photochemical processes, while non-photochemical quenching and the content of photoprotective pigments were lower. Analysis of sugar contents showed no significant differences in leaves but enhanced levels of sucrose and fructose in roots, while glucose amounts stayed constant. The three sucrose transporter encoding genes of tomato SlSUT1, SlSUT2 and SlSUT4 were up-regulated providing transport capacities to transfer sucrose into the roots. It is proposed that a significant proportion of sugars is used by the mycorrhizal fungus, because only amounts of fructose were increased, while levels of glucose, which is mainly transferred towards the fungus, were nearly constant.     

Herbivory-induced glucose transporter gene expression in the brown planthopper,Nilaparvata lugens

Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.     

Effects of ATP and cAMP on salt and sugar (responses of chemosensilla in the blowfly)

• 1.1. The addition of cAMP to stimulating solutions of NaCl, fructose (furanose sugar), sucrose, or glucose (pyranose sugars) decreases the responsiveness of labellar chemosensilla in Phormia.
• 2.2. The addition of ATP, while decreasing the responsiveness to NaCl or fructose enhances the responsiveness to glucose and sucrose.
• 3.3. The inhibiting effect of ATP on NaCl or fructose responses is suppressed by GDPßS, an inhibitor of adenylate cyclase (and thus of cAMP synthesis); moreover GDPßS further enhances the increase in response due to ATP when added to the sucrose or glucose solutions.
• 4.4. Results suggest a possible involvement of cAMP and ATP in the taste reception mechanism in the blowfly.

     

罗汉果果肉中糖类物质组成与含量分析

罗汉果果实中富含糖分,糖类物质的组成及其含量对果实的内在品质有重要影响,然而多年来对其品质的研究多集中在罗汉果苷上,果实中可溶性糖种类与含量迄今尚未见有系统地报道。该研究以干燥的罗汉果果实为材料,采用PMP柱前衍生化一高效液相色谱紫外检测法、高效液相色谱示差折光检测法分别检测果肉中可溶性糖的种类与含量,并进行方法学考察。结果表明:PMP柱前衍生化一高效液相色谱紫外检测法只能检出罗汉果果实中存在的2种还原性醛糖——葡萄糖、甘露糖;而高效液相色谱示差折光检测法则可一次性检出葡萄糖、果糖、蔗糖、棉籽糖、多糖5种糖分。与柱前衍生化法相比,高效液相色谱示差折光检测法更适合用来全面分析罗汉果果实中糖分的种类和含量。不同罗汉果品种果实中糖的组分一致,但含量有显著差别。另外,样品的干燥方式会影响果实中的总糖及各组分的相对含量。冻干果肉中蔗糖和葡萄糖相对含量最高,烘干则导致蔗糖和葡萄糖下降,果糖与多糖相对含量增加。