Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Effect of Abscisic Acid on Sorbitol Uptake in Growing Apple Fruits

Levels of abscisic acid (ABA) in the fruit flesh of developingapples (cv. Golden Delicious) were measured by electron capturegas chromatography. ABA content of the tissue, calculated ona fresh weight basis, decreased at a constant rate from 200µg g^–1 in young fruit to 10 µg g^–1 inolder fruit and then increased when the ripening process commenced.On a whole fruit basis, the ABA level increased during the initialphase of fruit growth, remained constant during the linear growthphase and increased again when fruits started to ripen. During fruit development the ABA content correlated with therate of sorbitol uptake, when measured after discs of fruittissue were incubated in [¹⁴C]sorbitol. Sorbitol uptake washigh during the initial growth phase and declined at a constantrate during fruit development. ABA present in the incubation medium stimulated sorbitol uptakeinto fruit tissue at concentrations higher than 10^–8 M,whereas indolyl-3-acetic acid had no effect on uptake. When comparing sorbitol uptake in different zones of young fruit,it was found that uptake was higher in discs of outer fruitlayers than in discs from inner fruit zones. Key words: Pyrus malus, Apple, Abscisic acid, Sorbitol     

Movement of Abscisic Acid Across the Plasmalemma and the Tonoplast of Guard Cells of Valerianella locusta

Uptake experiments and efflux compartmental analyses of abscisic acid (ABA) with acid treated epidermal peels of Valerianella locusta were performed to elucidate the mechanisms of transport of ABA across the plasmalemma and tonoplast of guard cells. ABA uptake across the plasmalemma is linearly correlated with external ABA concentration in the incubation medium. Under alkaline conditions ABA-uptake was not significantly above background, indicating that ABA uptake occurs mainly by diffusion of undissociated ABAH as the most permeable species, which is trapped afterwards in the alkaline cytosol as impermeable ABA^?. Efflux analysis of ABA revealed a saturable component of ABA transfer across the tonoplast. A Woolf-Augustinsson-Hofstee analysis suggested the existence of two transport systems for ABA at the tonoplast. The high affinity transport system had a K_M of 0.21 mol m^?3 and a V_max 85.8 amol ABA cell^?1 h^?1. Using the data of the uptake and efflux experiments we calculated the permeability coefficients of ABA for the plasmalemma and the tonoplast of guard cells, which are 2.46 10^?7 m s–¹ and 1.26 10^?8m s^?1, respectively. The distribution of the pH-probe (¹⁴C)-DMO between medium, cytosol and vacuole was investigated and used to calculate cytosolic and vacuolar pH. The vacuolar pH is too low to explain the high vacuolar ABA concentration by trapping of ABA^?, whereas the cytosol is sufficiently alkaline to act as an efficient anion trap. Therefore we conclude that ABA transport across the guard cell tonoplast is catalyzed by a saturable uptake component.     

Stimulation of the Uptake of Sorbitol into Vacuoles from Apple Fruit Flesh by Abscisic Add and into Protoplasts by Indoleacetic Acid

The uptake of sorbitol into vacuoles from immature flesh ofapple fruit (Maluspumila Mill, var domestica Schneid.) was facilitatedby 10^–6 M ABA, while such uptake into protoplasts wasnot stimulated. By contrast, the application of 10^–5 MIAA facilitated uptake of sorbitol into protoplasts but didnot significantly into vacuoles. (Received July 17, 1990; Accepted December 25, 1990)     

Bioregulator enhancement of sink activity in sugar beet

The sink mobilizing abillity is partially determined by sugar uptake rates of storage cells. Two synthetic growth regulators (Pix and BAS 106W) were tested for their effect on sucrose uptake in root tissue discs or glucose uptake in cell cultures of sugar beet. In tissue discs, uptake at the plasmalemma was determined by incubating the discs for 1 h in the presence of 5 mM sucrose and at the tonoplast for 4 h in the presence of 40 mM sucrose. Cell cultures were incubated for 1 h in the presence of 1 mM glucose. Pix (10 mg l^–1) caused a 20% stimulation of active sucrose uptake at the plasmalemma. Active sucrose uptake at the tonoplast was increased 67% by 100 mg l^–1 Pix. No effect of BAS 106W was observed on sucrose uptake in tissue discs. In cell cultures, a 65% enhancement of active glucose uptake was observed with both Pix and BAs 106W. When the bioregulators were applied to the root medium of seedlings, Pix but not BAS 106W resulted in increased root/shoot ratio, translocation of ¹⁴C-assimilates, and allocation of more biomass to the root sink. The data suggested that sugar transport and translocation may be used as biochemical criteria for rapid screening of effective yield enhancing bioregulators.     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Role of ABA in stamen and pistil development in the normal and solanifolia mutant of tomato (Lycopersicon esculentum)

Summary The role of abscisic acid (ABA) in stamen and pistil development of the normal and solanifolia (sf/sf) mutant of tomato (Lycopersicon esculentum Mill.) was analyzed. The solanifolia mutant produces flowers with separate floral organs, unlike the fused organs of normal flowers, and has greater number of carpels and locules per ovary than the normal. Applications of 10^–5 M ABA to normal floral buds produced flowers with separate stamens, but higher concentrations (10^–4 M ABA) resulted in the complete suppression of stamen growth or stamens that were devoid of anthers. ABA at both 10^–4 and 10^–5 M also induced an increase in the number of carpels and locules in normal flowers, but not in mutant ones. Analysis of endogenous ABA by a radioimmunoassay revealed that the pistils of mutant flowers contained a significantly higher level of ABA than those of normal flowers, but there was no difference in the ABA content of the stamens. The non-fusion of the stamens and the high number of carpels and locules in solanifolia mutant flowers may be explained by the high level of ABA in the floral apex during the initiation and development of carpels.     

Inducible effects of abscisic acid on sporophyte discs from Laminaria japonica Areschoug (Laminariales, Phaeophyceae)

The effects of exogenous abscisic acid (ABA;10^–7–10^–5 M), a knownplantgrowth regulator, on reproduction and growth were investigated by culturingdiscs from sporophytes of Laminaria japonica Areschoug.ABAplays a role in triggering sorus formation, and it was found that sorusformation of discs was fastest in 10^–5 M ABA. Theapplication of 10^–5 M ABA to culturing discs alsosuppressed the expansion of surface area. ABA contents in sorus and vegetativeparts of the sporophyte were determined by bioassay. The mean ABA content insorus parts obtained from sporophytes was 0.222 ± 0.053g equivalent-ABA g wet weight^–1, which wasabout five times higher than the content found in vegetative parts (0.048± 0.009 g equivalent-ABA g wetweight^–1). Taken together these results suggest that sorusdevelopment requires an elevated level of ABA and is associated with decreasingvegetative growth and that the ABA level of the sporophyte may play a crucialrole in reproduction.     

ATP-Promoted Sorbitol Transport into Vacuoles Isolated from Apple Fruit

Sorbitol was transported actively into vacuoles isolated fromapple (Malus pumilla Mill, var domestica Schneid.) fruit flesh.The uptake was stimulated up to twofold by the addition of ATP,and the ATP dependent uptake showed a saturation curve as tothe substrate concentration. The optimum uptake of sorbitolwas pursued in the acidic range of pH 5 to 6. The K_m value forthe ATP dependent sorbitol uptake was about 5 mM. Sorbitol uptake was clearly inhibited by PCMB and uncouplers(CCCP and DCCD), and to a lesser extent by orthovanadate, butonly slightly by oligomycin. K⁺ stimulated sorbitol uptake.Sorbitol was converted to other sugars (glucose) only very slowlywhen transported across the tonoplast. This suggests that sorbitolis transported into vacuoles by a carrier mediated transportsystem coupled with H⁺- ATPase, localized on the tonoplast.Sucrose uptake into the vacuoles was also enhanced by ATP. (Received May 31, 1986; Accepted March 2, 1987)     

灵武长枣果实质膜和液泡膜H~+-ATPase和H~+-PPase活性与果实糖分积累

以不同发育时期灵武长枣(Ziziphus jujuba cv.Lingwuchangzao)的果实为材料,通过测定与分析果肉组织中细胞质膜、液泡膜H+-ATPase和H+-PPase活性、果实糖分含量变化,研究了灵武长枣果实质膜、液泡膜H+-ATPase和H+-PPase活性与糖积累特性的关系。结果表明:(1)果实第二次快速生长期之前主要积累葡萄糖和果糖,之后果实迅速积累蔗糖,葡萄糖和果糖含量则逐渐下降,成熟期果实主要积累蔗糖。(2)在果实发育的缓慢生长期S1,质膜H+-ATPase活性最低;第一次快速生长期,质膜H+-ATPase活性最高;缓慢生长期S2,其活性降低;第二次快速生长期,质膜H+-ATPase活性升至次高;完熟期,质膜H+-ATPase活性下降幅度较大。(3)在果实发育过程中,液泡膜H+-ATPase和H+-PPase活性的变化趋势相似。缓慢生长期S1,液泡膜H+-ATPase和H+-PPase活性较低;从缓慢生长期S1至第一次快速生长期缓慢下降至最低;从第一次快速生长期开始,液泡膜H+-ATPase和H+-PPase活性呈现为逐渐增高的变化趋势;除第二次快速生长期以外,液泡膜H+-PPase活性始终高于H+-ATPase。由此推测,质膜H+-ATPase和液泡膜H+-ATPase、H+-PPase对灵武长枣果实糖分的跨膜次级转运起到重要的调控作用。     

14C-Studies on Apple Trees. V. Translocation of Labelled Compounds from Leaves to Fruit and their Conversion within the Fruit

Following application of ¹⁴CO₂ to fruit spur leaves, the majority of the ¹⁴C absorbed is transfered to the fruit on the same spur, and the total content of ¹⁴C within the leaf-fruit system as a whole remains virtually constant with time. The considerable reduction in activity in the leaves is accounted for mainly by a decrease in the amount of ¹⁴C-sorbitol, although relatively speaking the decrease in ¹⁴C-sucrose is also considerable. The major part of the activity of the sugar fraction in the conducting tissues between blade and fruit (petiol, spur) is found in sorbitol. Immediately following uptake of ¹⁴C yia the leaves a large part of the activity of the sugar fraction in the fruit is found in sorbitol; but this activity is rapidly reduced, accompanied by an increase in sucrose activity, and over longer periods of time increases in particular in glucose and fruclose activity, and in that of methanol insoluble compounds. The changes in activity distribution in the fruit vary with the variety of fruit and the dates within the growing season. By injecting labelled sorbitol directly into the fruit sorbitol is converted into sucrose, glucose and fructose, while injection of labelled sucrose, glucose and fructose has yielded proof of interconversions between these compounds but no measurable amounts of surbitol. After application of ¹⁴CO₂ directly to the outer skin of the fruit considerably less of the activity is found in sorbitol than is the case in leaves following exposure to ¹⁴CO₂. A minor, but significant, translocation of ¹⁴C away from the fruit was found to take place following the application of labelled ¹⁴C compounds to the fruit. The smallness of the respiratory loss of ¹⁴C in the leaf-fruit system is discussed. It is concluded that in apple trees considerable translocation occurs in the form of sorbitol which in the fruits rapidly converted into other compounds.     

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.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Characterization of Hexose Transporter for Facilitated Diffusion of the Tonoplast Vesicles from Pear Fruit

Tonoplast vesicles were prepared from the flesh tissue of maturepear fruit. Sugar uptakes into the vesicles determined by twodifferent methods, the membrane and the gel filtration methods,were quite similar. The uptake was highest for glucose and subsequently,in order, for fructose, sucrose and sorbitol. It was not stimulatedby addition of ATP, although the vesicles could create a protongradient. However, the uptakes were significantly inhibitedby p-chloromercuribenzene sulphonate (PCMBS, SH-reagent andinhibitor of sugar transporter). Further, the PCMBS-sensitiveuptakes of glucose and fructose saturated with their increasedconcentrations. Thus, these PCMBS-sensitive uptakes are mediatedby the transporter of facilitated diffusion. The uptakes ofglucose or fructose each had two K_m values. K_m values for glucosewere 0.35 and 18 mM, and those for fructose were 1.6 and 25raM. The uptake of 0.2 mM glucose was inhibited by 2 mM fructoseand that of 2 mM fructose was inhibited by 2 mM glucose, butneither was inhibited by sucrose or sorbitol. O-methyl-glucose(OMG) also inhibited both the glucose and fructose uptakes.Therefore, the same transporter may mediate both glucose andfructose uptakes at lower concentrations; this hexose transportsystem differed from the sucrose and sorbitol transport systems. ¹Research Fellow of the Japan Society for the Promotion of Science. ²Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan.     

Highly-efficient somatic embryogenesis from cell suspension cultures of phalaenopsis orchids by adjusting carbohydrate sources

Summary The influences of various carbohydrate sources, dried yeast (DY), and 6-benzylaminopurine (BA) were estimated on growth and development of shoot tip-derived suspension cells of phalaenopsis orchid. Among the carbohydrates tested on Doriataenopsis cultured on gelled medium, glucose at 58.4 mM gave the highest efficiency of protocorm-like body (PLB) formation. Maltose and sorbitol only induced PLB formation without callus proliferation. Sucrose induced comparable callus proliferation to glucose but without PLB formation. In contrast, fructose resulted in half the amount of callus proliferation as occurred with glucose. Lactose was an inadequate carbon source as neither PLB formation nor callus proliferation occurred. DY enhanced cell proliferation at 0.1–1gl⁻¹ but inhibited both cell proliferation and PLB formation at 10gl⁻¹. Low BA (0.4 μM) slightly increased callus proliferation but inhibited PLB formation. Only one treatment, sucrose and 1 gl⁻¹ DY, yielded a small number of plants. For suspension cultures of Phalaenopsis Snow Parade and P. Wedding Promenade, PLB formation was most efficiently induced by sucrose at 29.2 mM for P. Snow Parade and 14.6 mM glucose for P. Wedding Promenade. Histological observation revealed that cells in suspension culture developed into plants through the same developmental proess as germinating seeds.     

八个贵州地方桃品种果实甜酸风味品质分析

为了评价贵州地方桃品种果实的甜酸风味品质,以主栽桃品种‘燕红’作对照,采用高效液相色谱法测定了贵州8个地方桃品种的果肉糖酸组分及含量。结果表明:(1)8个地方桃品种果肉中糖主要由蔗糖、葡萄糖、果糖和山梨醇组成,其中蔗糖的平均含量最高(55.62 mg/g),约占总糖的71.30%,但变异系数仅为17.92%;8个地方桃品种中,‘米桃’的葡萄糖与果糖含量差异较大,其果糖/葡萄糖的比值为1.21,而其它7个品种的葡萄糖与果糖含量相近。(2)8个地方桃品种果肉中有机酸主要由苹果酸、柠檬酸、奎宁酸和莽草酸组成,其中苹果酸含量最高,约占总酸的60.61%,但在‘白花桃’果实中有机酸含量以奎宁酸为主。(3)对8个地方桃品种果实的糖酸组分进行主成分分析发现,苹果酸含量和山梨醇含量的载荷系数分别为0.910和0.897,说明它们是影响果实甜酸风味的主导因素,且对改善果实的甜酸风味品质具有重要作用。8个贵州地方桃果实的甜酸风味分别为:‘黄腊桃’为甜,‘血桃’、‘青桃’、‘镇远桃’、‘红枫桃’为酸甜,‘白花桃’、‘西桃’和‘米桃’为酸。     

Transport Properties of the Tomato Fruit Tonoplast : II. Citrate Transport

Citrate transport across the membrane of tomato fruit tonoplast vesicles was investigated. In the tonoplast vesicles, [¹⁴C]methylamine uptake was stimulated 10-fold by MgATP and strongly inhibited by NO₃⁻. Under identical experimental conditions, [¹⁴C]citrate uptake was inhibited by 5 millimolar free Mg²⁺, and this inhibition was reversed in the presence of ATP, presumably by ATP chelation of free Mg²⁺. No evidence was obtained in support of energy-linked ATP stimulation of citrate uptake. Citrate uptake showed saturation kinetics, and was inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid and by other organic acids. The pH-dependence of uptake suggested that citrate³⁻ was the transported species. Our results indicate that citrate transport across the tomato fruit tonoplast occurs by facilitated diffusion of citrate³⁻. The carrier shares some features in common with anion channels in that it is relatively nonspecific for organic acids and is inhibitable by 4,4′-diisothyocyano-2,2′-stilbenedisulfonic acid.     

<Emphasis Type="Italic">Narcissus</Emphasis> bulblet formation <Emphasis Type="Italic">in vitro</Emphasis>: effects of carbohydrate type and osmolarity of the culture medium

Shoot clump cultures of Narcissus cultivars St. Keverne and Hawera were used to investigate the effects of culture medium carbon supply, type of carbohydrate and osmolarity on in vitro bulblet development. Increasing the medium osmolarity using mannitol or sorbitol, which did not act as substrates for growth, failed to stimulate bulblet formation with either cultivar. An exception to this was a relatively small increase in total bulblet dry weight per culture, in the cultivar Hawera only, caused by adding 30 g l ^–1 sorbitol in combination with 30 g l^–1 sucrose. Simultaneously increasing the medium osmolarity and carbon supply using the metabolisable carbohydrate sources, sucrose, glucose, fructose or an equimolar mixture of glucose and fructose stimulated bulblet production, total dry matter accumulation and partitioning into bulblets. At comparable levels of carbon supply up to 19.0 g l^–1, bulblet development of both cultivars was similar with monosaccharide and sucrose media. This indicates that substrate supply is more important for bulblet development than osmolarity of the culture medium. The cultivar Hawera also showed similar responses to monosaccharide and sucrose media supplying 37.9 g C l^–1, despite the high osmolarity of monosaccharide media (c. 650 m Osm kg^–1, equivalent to –1.6 MPa, compared to 380 m Osm kg^–1 for sucrose medium). However in St. Keverne total dry matter accumulation and dry weight per bulblet were further stimulated only by increasing the sucrose supply from 19.0 to 37.9 g C l^–1, not by increasing the monosaccharide supply. Implications of the findings for Narcissus micropropagation are discussed.     

Response surface optimization for the continuous glucose isomerization process

Production of fructose via a continuous glucose isomerization process was optimized using response surface methodology. Glucose isomerization was performed using immobilized glucose isomerase in a flow-through tubular reactor. Process factors eg pH (7.0–7.8), temperature (50–60°C), flow rate (5–17 ml min^–1) and glucose content (30–50% w/w) of the feedstock solution were simultaneously tested according to a central composite experimental design. Measured responses such as % isomerization, and fructose yield (gh^–1) has an excellent correlation with tested factors. The highest desirability,D, (geometric mean of % isomerization and fructose yield) was obtained when the feedstock (56–60°C) had 34–36% glucose, a pH of 7.4–7.8 and was pumped at 15 ml min^–1.