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.     

The cellular pathway of postphloem sugar transport in developing tomato fruit

The cellular pathway of postphloem sugar transport was elucidated in the outer pericarp of tomato (Lycopersicon esculentum Mill cv. Floradade) fruit at 13–14 and 23–25 days after anthesis (DAA). These developmental stages are characterized by phloem-imported sugars being accumulated as starch and hexose, respectively. The symplasmic tracer, 5(6)-carboxyfluorescein, loaded into the storage parenchyma cells of pericarp discs, moved readily in the younger fruit but was immobile in fruit at 23–25 DAA. Symplasmic mobility of [¹⁴C]glucose was found to be identical to 5(6)-carboxyfluorescein. For the older fruit, the pericarp apoplasm was shown to be freely permeable to the apoplasmic tracer, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate. Indeed, the transport capacity of the pericarp apoplasm was such that the steady-state rate of in-vitro glucose uptake by pericarp discs accounted fully for the estimated rate of in-vivo glucose accumulation. For fruit at 23–25 DAA, the inhibitory effects of the sulfhydryl group modifier, p-chloromer-curibenzenesulfonic acid (PCMBS), on [¹⁴C]glucose and [¹⁴C]fructose uptake by the pericarp discs depended on the osmolality of the external solution. The inhibition was most pronounced for pericarp discs enriched in storage parenchyma. Consistent with the PCMBS study, strong fluorescent signals were exhibited by the storage parenchyma cells of pericarp discs exposed to the membrane-impermeable thiol-binding fluorochrome, mono-bromotrimethylammoniobimane. The fluorescent weak acid, sulphorhodamine G, was accumulated preferentially by the storage parenchyma cells. Accumulation of sulphorhodamine G was halted by the ATPase inhibitor erythrosin B, suggesting the presence of a plasma-membrane-bound H⁺-ATPase. A linkage between the putative H⁺-ATPase activity and hexose transport was demonstrated by an erythrosin-B inhibition of [¹⁴C]glucose and [¹⁴C]fructose uptake. In contrast, comparable evidence for an energy-coupled hexose porter could not be found in the pericarp of younger fruit at 13–14 DAA. Overall, the data are interpreted to indicate that: (i) The postphloem cellular pathway in the outer fruit pericarp shifts from the symplasm during starch accumulation (13–14 DAA) to the apoplasm for rapid hexose accumulation (23–25 DAA). (ii) An energy-coupled plasma-membrane hexose carrier is expressed specifically in storage parenchyma cells at the latter stage of fruit development.     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

Increased hexose transport in the roots of tomato plants submitted to prolonged hypoxia

We investigated the effects of prolonged hypoxia on the sugar uptake in tomato (Solanum lycopersicum L. var. MP-1) roots. Hydroponic cultures of whole tomato plants were submitted to hypoxic treatment for 1 week, and the roots were analyzed for sugar concentrations, hexose uptake and hexose transporter expression level. Contrary to what has been observed after anoxic shock or short-term hypoxic treatment, we show that sugar concentrations increase and hexose uptake is up-regulated in the roots after 1 week of hypoxic treatment. Increased hexose transport is concomitant with the induction of the hexose transporter gene LeHT2. These responses may be due either to a direct effect of low O₂ supply, or to a secondary effect associated with the increase in sugar concentrations, which, typically, develops in most hypoxic plants.     

Mechanisms Involved in Calcium Deficiency Development in Tomato Fruit in Response to Gibberellins

Although gibberellins (GAs) have been shown to induce development of the physiological disorder blossom-end rot (BER) in tomato fruit (Solanum lycopersicum), the mechanisms involved remain largely unexplored. BER is believed to result from calcium (Ca) deficiency, but the relationship between Ca content and BER incidence is not strong. Our objectives were to better understand how GAs and a GA biosynthesis inhibitor affect BER development in tomato fruit. Tomato plants of two BER-susceptible cultivars, ‘Ace 55 (Vf)’ and ‘AB2,’ were grown in a greenhouse environment and subjected to Ca-deficiency conditions. Plants were treated weekly during fruit growth and development with 300 mg L^?1 GA₄₊₇, 300 mg L^?1 prohexadione-calcium (Apogee^®, a GA biosynthesis inhibitor), or water beginning 1 day after flower pollination. GA₄₊₇ treatment induced an increase in BER incidence in both cultivars up to 100%, whereas ‘Ace 55 (Vf)’ and ‘AB2’ plants treated with Apogee did not show BER incidence. The number of functional xylem vessels was higher in the placental and pericarp tissue of tomato fruit treated with Apogee at the early stages of fruit growth. Treatment with Apogee also increased fruit pericarp Ca concentration. GA₄₊₇ treatment enhanced the expression of the putative CAX and Ca-ATPase genes, that code for proteins involved in Ca movement into storage organelles. The lowest water-soluble apoplastic Ca concentration and the highest membrane leakage values were observed in the pericarp of GA₄₊₇-treated fruit. These results suggest that GAs consistently reduced fruit Ca uptake and water-soluble apoplastic Ca concentration, leading to leakier plasma membranes and an increase in BER development in fruit tissue of both tomato cultivars.     

Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs

Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.     

Effect of carbon source and sucrose concentration on growth and hexose accumulation of grape berries cultured in vitro

Using in vitro culture of isolated small berries of Vitis vinifera L. cv. Sultana, it was possible to study the effect of different carbon sources and sucrose concentration on fruit growth, hexose accumulation and soluble invertase activity during the first stage of berry development by eliminating the source tissue. Berries cultured in vitro lack stage III of berry development which is characterised by massive accumulation of water and sugars, and thereby berries reached only 30% of the weight of those grown in the plant. Sucrose and glucose were both good carbon sources for berry growth, while fructose was not as good. Berry growth, hexose accumulation and invertase activity increased as sucrose concentration increased up to 15% in the medium. Furthermore, the onset of hexose accumulation in cultured berries depended on the concentration of sucrose in the medium, starting earlier at higher concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild‐type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose : sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.     

宁夏枸杞果实韧皮部及其周围细胞超微结构研究

应用透射电镜技术研究了宁夏枸杞果实韧皮部细胞的超微结构变化。结果表明:(1)随着枸杞果实的发育成熟,果实维管组织中的韧皮部筛分子筛域逐渐变宽,筛孔大而多,通过筛孔的物质运输十分活跃;筛分子和伴胞间有胞间连丝联系,伴胞属传递细胞类型,与其相邻韧皮薄壁细胞和果肉薄壁细胞连接处的细胞界面发生质膜内突,整个筛分子/伴胞复合体与韧皮薄壁细胞之间形成共质体隔离,韧皮部糖分的卸载方式主要以质外体途径进行。(2)韧皮薄壁细胞间的胞间连丝较多,而韧皮薄壁细胞与果肉薄壁细胞的胞间连丝相对较少,但果肉薄壁细胞间几乎无胞间连丝;果肉薄壁细胞之间胞间隙较大,细胞壁和质膜内突间形成较大的质外体空间,为质外体的糖分运输创造了条件。(3)筛管、伴胞、韧皮薄壁细胞和果肉薄壁细胞中丰富的囊泡以及活跃的囊泡运输现象,暗示囊泡也参与了果实糖分的运输过程。研究推测,枸杞果实韧皮部同化物的卸载方式以及卸载后的同化物运输主要以质外体途径为主。     

Photosynthetic activity of the calyx, green shoulder, pericarp, and locular parenchyma of tomato fruit

Photosynthesis of tomato fruit was studied using green fruit from six heritage cultivars of Lycopersicon esculentum Mill. and one of Lycopersicon pimpinellifolium. Chlorophyll concentrations in the green shoulder, pericarp and locular parenchyma of the fruit were determined and the apparent photosynthetic electron transport activity (ETR) and chlorophyll fluorescence quenching characteristics of these tissues and the calyx were compared. In all cultivars, green shoulder formation, apparent as intense pigmentation of the proximal pericarp shoulder, was positively related to the degree of shading of the fruit during development. Appearing as a photosynthetic adaptive trait for increasing the photoautotrophic capacity of fruit grown under low light, the green shoulder contained 17-57% of the total pericarp chlorophyll content. The pericarp below the green shoulder had lower chlorophyll a+b. At a photon flux density (PFD) of 1200 mol m^-2 s^-1, different fruit tissues were found to have different levels of ETR. In 'Yellow Pear', the upper surface of the calyx had an ETR of 154 mol m^-2 s^-1, while the lower surface had an ETR of 88 mol m^-2 s^-1. On the green shoulder, ETR was 203 mol m^-2 s^-1, whereas in the pericarp distal to the green shoulder, ETR was 97 mol m^-2 s^-1. In the locular parenchyma, ETR was 66 mol m^-2 s^-1. This trend towards a lower ETR in distal and internal fruit tissues appeared to indicate a shift towards a more shade-type photosynthesis. Concomitant with this shift were changes in chlorophyll fluorescence quenching characteristics. Generally when tissues displayed reduced levels of ETR they also displayed a faster decrease in the photochemical quenching coefficient qp and a more rapid diversion of absorbed photon energy to non-photosynthetic activity found in the calyx, green shoulder, pericarp, and locular parenchyma suggest that all of these tissues have significant roles in CO2 scavenging and the provision of carbon assimilates. The potential role of fruit photosynthesis in influencing the fruit acid to sugar ratio and hence fruit quality is discussed.     

Sink Metabolism in Tomato Fruit : II. Phloem Unloading and Sugar Uptake

Analysis of [³H]-(fructosyl)-sucrose translocation in tomato (Lycopersicon esculentum Mill.) indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast followed by extracellular hydrolysis. Apoplastic sucrose, glucose, and fructose concentrations were estimated as 1 to 7, 12 to 49, and 8 to 63 millimolar, respectively in the tomato fruit pericarp tissue. Hexose concentrations were at least four-fold greater than sucrose at all developmental stages. Short-term uptake of [¹⁴C]sucrose, -glucose, and -fructose in tomato pericarp disks showed first order kinetics over the physiologically relevant concentration range. The uptake rate of [¹⁴C]-(glucosyl)-1′-fluorosucrose was identical to the rate of [¹⁴C]sucrose uptake, suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Together these results suggest that although sucrose is at least partially hydrolyzed in the apoplast, sucrose may enter the metabolic carbohydrate pool directly. In addition, sugar uptake across the plasma membrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato fruit is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast.     

Effects of the Lycopersicon chmielewskii sucrose accumulator gene (sucr) on fruit yield and quality parameters following introgression into tomato

A gene controlling fruit sucrose accumulation, sucr, was introgressed from the wild tomato species Lycopersicon chmielewskii into the genetic background of a hexose-accumulating cultivated tomato, L. esculentum. During introgression, the size of the L. chmielewskii chromosomal segment containing sucr was reduced by selection for recombination between RFLP markers for the sucr gene and flanking loci. The effects of sucr on soluble solids content, fruit size, yield and other fruit parameters were studied in the genetic background of the processing tomato cultivar Huntl00. In a segregating BC₅F₂ generation, the smallest introgression containing sucr-associated markers was necessary and sufficient to confer high-level sucrose accumulation, the effects of which were completely recessive. Fruit of sucr/sucr genotypes were smaller than those of +/sucr or +/+ genotypes at all stages of development. The timing of sugar accumulation and total sugar concentration were unaffected by sugar composition. No differences in total fruit biomass (fresh weight of red and green fruit) at harvest were observed between the genotypes, and sucrose accumulators produced greater numbers of fruit than hexose accumulators in one family. However, the proportion of ripe fruit at harvest, and hence yield of ripe fruit, as well as average ripe fruit weight and seed set were reduced in sucr/sucr genotypes. Sucrose accumulation was also associated with increased soluble solids content, consistency, serum viscosity, predicted paste yield and acidity, and decreased color rating. In the first backcross to L. chmielewskii, hexose accumulators (+/sucr) had larger fruit than sucrose accumulators (sucr/sucr), while no difference in soluble solids was detected.     

Cell turgor changes associated with ripening in tomato pericarp tissue

The pressure microprobe was used to determine whether the turgor pressure in tomato (Lycopersicon esculentum Mill., variety “Castelmart”) pericarp cells changed during fruit ripening. The turgor pressure of cells located 200 to 500 micrometers below the fruit epidermis was uniform within the same tissue (typically ± 0.02 megapascals), and the highest turgors observed (<0.2 megapascals) were much less than expected, based on tissue osmotic potential (−0.6 to −0.7 megapascals). These low turgor values may indicate the presence of apoplastic solutes. In both intact fruit and cultured discs of pericarp tissue, a small increase in turgor preceded the onset of ripening, and a decrease in turgor occurred during ripening. Differences in the turgor of individual intact fruit occurred 2 to 4 days before parallel differences in their ripening behavior were apparent, indicating that changes in turgor may reflect physiological changes at the cell level that precede expression of ripening at the tissue level.     

不同产地气象因子对宁夏枸杞果实糖分积累的影响

以‘宁杞1号’枸杞为材料,采用高效液相色谱技术,测定不同产地(宁夏银川、甘肃白银和青海德令哈)不同发育时期宁夏枸杞果实中的糖分(果糖、葡萄糖、蔗糖和淀粉)含量,同时对不同产地果实发育期间的气象因子进行监测,探讨主要气象因子对枸杞果实糖分积累的影响。结果表明:(1)在宁夏枸杞果实发育期内,不同产区平均温度和昼夜温差均为银川白银德令哈的变化趋势,平均光照强度为银川白银德令哈。(2)3个产区‘宁杞1号’枸杞的果实发育过程中均以积累己糖(葡萄糖和果糖)为主,蔗糖和淀粉含量很少;成熟果实中己糖含量为德令哈银川白银。(3)影响‘宁杞1号’枸杞果实己糖积累的气象因子在银川产区主要是平均温度、白天均温和平均温差,在白银产区主要是平均温差,白天温度和平均光强也有一定影响,而在德令哈产区主要是平均温差、平均光强和白天均温。研究认为,宁夏枸杞果实发育期间银川产区较高的平均温度和昼夜温差与德令哈产区较强的平均光强和较长的光照时数均有利于枸杞果实己糖积累,而白银产区较大的相对湿度和较弱的光强均成为枸杞果实糖分积累的主要限制因子。     

The hexose transporter family of Saccharomyces cerevisiae

Saccharomyces cerevisiae accomplishes high rates of hexose transport. The kinetics of hexose transport are complex. The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome. Twenty hexose transporter genes exist in S. cerevisiae. Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome. This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode. Information about the expression and function of the transporters is presented. Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web. Received: 24 June 1996 / Accepted: 29 July 1996     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Tissue and Cellular Localization of Proteinase Inhibitors I and II in the Fruit of the Wild Tomato, Lycopersicon peruvianum (L.) Mill

The cellular and subcellular localization of proteinase inhibitor I and inhibitor II proteins in the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill., LA 107 was determined by immunoanalysis of tissue blots and protein-A gold immunocytochemistry. Tissue blot analysis showed that the proteinase inhibitor I proteins were located throughout the fruit tissue, with the exception of the seeds. Light microscopy, using immunocytochemical labeling, indicated that all the parenchyma cells of the pericarp contained inhibitor I and II proteins in dense vacuolar protein aggregates that were not membrane bound. The size, number, and morphology of the aggregates within individual cells varied greatly. The funiculus, ovule, and early embryonic tissues were devoid of inhibitor I and II. Immunocytochemical analysis using transmission electron microscopy confirmed that the proteinase inhibitor I proteins were principally located and stored in protein aggregates within the vacuole of the fruit parenchyma cells. Some cytoplasmic protein-A gold immunolabeling of inhibitor I proteins was evident, which may be related to the synthesis and intermediate transport steps preceding storage of the inhibitor I proteins in the vacuoles.     

果实中糖的运输、代谢与积累及其调控

叶片光合产物向果实运输的主要形态是蔗糖,但在木本蔷薇科果树中,光合产物的主要运输形态为山梨醇.糖从质外体空间跨膜运入共质体的过程由糖运输蛋白介导,而糖运输蛋白的基因表达伴随着果实糖的积累而增强.蔗糖代谢酶参与了细胞内外4个与糖运输有关的无效循环.己糖代谢抑制是果实糖快速积累的前提.在木本蔷薇科果实中,蔗糖代谢酶活力仍非常活跃,表明蔗糖可能与山梨醇在果实生长发育中都起重要的作用.糖作为信号分子,调节了承担糖运输与代谢的基因的表达.自然环境因子和栽培措施能有效调控糖运输、代谢与积累.反义抑制Ivr基因表达能提高番茄果实含糖量的实验结果表明遗传工程调控糖积累的潜力.阐明糖信号与其它信号互作对糖运输与代谢的调控机制是今后研究的重点.