Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue.

Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Beta vulgaris L. ssp. conditiva) root vacuoles. Positive hybridoma clones and sub-clones were identified by prescreening using an enzyme-linked immunosorbent assay (ELISA) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited sucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110- to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(IgM)-specific ELISAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [14C]-sucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier.

Amplification and cloning of sugarcane sucrose synthase cDNA by anchored PCR.

We have used a strategy based on the polymerase chain reaction (PCR) to amplify and construct full-length sucrose synthase (SS) cDNA of sugarcane. Two SS-specific internal primers were synthesized based on their complementarity to published consensus sequences of the SS gene of maize and wheat. Amplification of full-length cDNA was achieved by an anchored PCR method utilizing primers which extend to 5' and 3' ends of specific cDNA. In the first step, a homopolymeric oligo(dC) tail was added to the 3' end of single-stranded cDNAs. The two SS cDNAs were amplified, one with a 5' end (SSp1) and the other with a 3' end (SSp2) using one internal SS primer and the other anchored end primer. Finally, overlapping fragments were identified by restriction mapping, and the non-overlapping fragments were excised and religated to reconstruct full-length cDNA. Partial sequences of the reconstructed cDNAs (SS-5' and SS-3') were compared with the published SS sequences to confirm that the amplified DNA was a copy of the SS transcript.

High Performance Liquid Chromatography-Based Reevaluation of Disaccharides Produced upon Incubation of Sugarcane Vacuoles with UDP-Glucose.

A reanalysis of products formed after short-term incubation of sugarcane (Saccharum spp. hybrid cv H50-7209) vacuole preparations with uridine diphosphate [(14)C]glucose was performed. The results indicated that the ethanol-soluble substance previously identified as sucrose did not elute with sucrose when subjected to high performance liquid chromatography but had the same retention time as a disaccharide tentatively identified as laminaribiose.

UDP-Glucose-Dependent Sucrose Translocation in Tonoplast Vesicles from Stalk Tissue of Sugarcane.

Tonoplast vesicles isolated from stalk parenchyma tissue of sugarcane plants transport sucrose via a uridine diphosphate glucose (UDPGlc)-dependent group translocator. No sucrose transport via an ATP-dependent system could be detected. The products of UDPGlc uptake in the vesicles were sucrose and sucrose phosphate which, upon hydrolysis with alkaline phosphatase and invertase, showed that both hexose moieties are derived from UDPGlc.

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction, oxygen uptake and pH change.

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the ß-configurated pyridine nucleotide and the α-configurated form. Since NADH-linked dehydrogenases usually do not work with α-NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.

A group translocator for sucrose assimilation in tonoplast vesicles of sugarcane cells.

Existence of a group translocator for sucrose transfer into vacuoles of sugarcane (Saccharum sp.) cells has been further confirmed by the use of tonoplast vesicles isolated from intact vacuoles. The group translocator depends on external UDP-Glucose (Glc) and, via a series of enzymic reactions within the tonoplast, sucrose phosphate and sucrose are deposited inside the vesicles. Fructose-6-phosphate was not required for UDP-Glc uptake, nor was it taken up. None of the other sugar phosphates tested were taken up nor were the nucleotide sugars, UDP-Galactose and ADP-Glc. The uptake of UDP-Glc was concentration-dependent with a K(m) of 1.2 millimolar and a V(max) of 83.3 nanomoles per minute per milligram protein. The optimum pH for UDP-Glc uptake was 7.0. Uptake of UDP-Glc was inhibited by para-chloromercuribenzene-sulfonic acid, UDP, and GDP; carbonyl cyanide m-chlorophenylhydrazone inhibited to a lesser extent.

Evidence for a uridine-5'-diphosphate-glucose-protectedp-chloromercuribenzene sulfonic acid-binding site in sugarcane vacuoles.

The uptake of uridine-5'-diphosphate (UDP) glucose into vacuoles isolated fromSaccharum sp. cells was fully inhibited by pretreatment with 50 µMp-chloromercuribenzenesulfonic acid (PCMBS) and was not affected by N-ethylmaleimide up to a concentration of 5 mM. The addition of 10 mM UDP-glucose during the pretreatment partially protected the uptake mechanism from PCMBS inhibition, while the presence of adenosine-5'-diphosphate (ADP) glucose or of various hexose-phosphates had no protective effect. Parallel experiments on the binding of [(203)Hg]PCMBS to the vacuoles showed that UDP-glucose and UDP added at 10 mM concentrations caused a 40% decrease in the binding of PCMBS while ADP-glucose did not inhibit the binding. The results indicate the presence in a previously proposed group translocator of at least one site that can bind UDP-glucose. This site, which is blocked by PCMBS, interacts with the nucleotide moiety of UDP-glucose.

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet.

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[(14)C]glucose in inhibited by high concentrations of osmoticum.

Group translocation as a mechanism for sucrose transfer into vacuoles from sugarcane cells.

Isolated vacuoles from sugarcane cells took up uridine diphosphate glucose (UDP-Glc) from the surrounding medium at a rapid rate. After a 7-min incubation of vacuoles with UDP-[14C]Glc, sucrose and sucrose phosphate were identified in the vacuole extract. UDP-Glc in the incubation medium was converted to hexose phosphates, sucrose, and glucose, with very little UDP-Glc remaining. Fructose 6-phosphate was not required for UDP-Glc uptake nor was [(14)C]fructose 6-phosphate taken up even in the presence of UDP-Glc. Glucose 6-phosphate and glucose 1-phosphate also were not taken up into vacuoles. UDP-Glc uptake showed saturation kinetics with a K(m) of 0.7 mM and a V(max) of 11.1 nmol/min per 10(6) vacuoles. The optimum pH for UDP-Glc uptake was between 6.5 and 7.0. Uptake of UDP-Glc could be inhibited by p-chloromercuriphenylsulfonic acid, UDP, and GDP, and to a lesser extent by carbonyl cyanide m-chlorophenylhydrazone. The UDP-Glc binding site was specific for UDP-Glc; adenosine diphosphate glucose was not taken up, and guanosine diphosphate glucose did not compete with UDP-Glc for the binding site. The results suggest that sucrose transfer into vacuoles from sugarcane is via a group translocation mechanism, probably involving five tonoplast-bound enzymes.

Evidence for a plasmalemma redox system in sugarcane.

A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O(2) consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K(+), 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH.

Developmental and Biochemical Characteristics of Cold-treated Anthers of Saccharum spontaneum.

The role of cold in stimulating androgenic haploid development was evaluated by assessing morphological and biochemical changes occurring in Saccharum spontaneum anthers during 3 weeks of incubation of panicle sections at 10 °C. During incubation, anthers increased in size; and although many microspores lost viability, those that survived proceeded to divide symmetrically rather than asymmetrically as found in normal microsporogenesis. Anthers which contained dividing microspores differed biochemically from anthers containing non-viable microspores. Anthers initially with high reducing sugars and total free amino acids and with high amylase activity were most likely to produce dividing microspores. Amide metabolism during incubation correlated with microspore development. In all anthers amides increased during cold incubation. The anthers most likely to contain non-viable microspores had much higher asparagine content relative to glutamine content.

Plant regeneration via somatic embryogenesis in sugarcane.

Plants, regenerated from callus cultures of sugarcane (Saccharum officinarum L.) clone IJ76-316, originated through somatic embryogenesis. Callus cultures were established from primordial leaves and apical meristems on Murashige and Skoog medium (MS) supplemented with 3 mg 1(-1) 2,4-dichlorophenoxy acetic acid and 100 ml 1(-1) coconut water (MSC3). Nodular calli formed within 2 weeks of culture. Calli were maintained on MSC3 medium by transfer every 3 to 4 weeks. Somatic embryogenesis occurred after 10 weeks culture of callus on MSC3 medium. Somatic embryogenesis was also observed in cell suspension cultures initiated from calli maintained on MSC3 and then cultured in half strength MS liquid medium supplemented with 0.5 mg 1(-1) 2,4-D. Somatic embryos produced coleoptiles and shoots 2 to 4 weeks after transfer to MS medium supplemented with 100 ml 1(-1) coconut water (MSC), and produced complete plantlets within 4 weeks of further culture on half-strengh MS medium (half-MS) with 30 g 1(-1) sucrose. Calli grown on MSC3 medium, when transferred to half-MS medium containing 15 g 1(-1) sucrose, produced tiny plantlets, circa 4-10 mm, without forming coleoptiles, suggesting precocious germination of somatic embryos. The regenerates included morphological variants.

Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION.

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.

Vacuoles from Sugarcane Suspension Cultures : II. CHARACTERIZATION OF SUGAR UPTAKE.

Vacuoles, isolated from sugarcane (Saccharum sp.) cells, took up 3-O methylglucose and sucrose and the evidence suggests specific transport systems for these sugars. There was no evidence of sugar efflux from preloaded vacuoles. Vacuoles in situ accumulated 3-O methylglucose, sucrose, glucose, and fructose, as shown by incubation of protoplasts with labeled sugar and subsequent analysis of vacuolar and cytoplasmic radio-activity. During the initial minutes of incubation, the amount and concentration of labeled sugar was higher in the cytoplasm than in the vacuole, but subsequently there was active uptake and accumulation into the vacuole. The rate of hexose transfer into the vacuole in situ approached that of hexose uptake by isolated vacuoles; however, the rate of sucrose uptake by isolated vacuoles was below the in situ rate. The site of sucrose synthesis was in the cytoplasm.

Vacuoles from Sugarcane Suspension Cultures : III. PROTONMOTIVE POTENTIAL DIFFERENCE.

The electrochemical proton gradient across the tonoplast of isolated (Saccharum sp.) vacuoles and vacuoles in situ was measured. The isolated vacuoles show no significant protonmotive potential difference, the pH gradient of 0.8 (inside acid) was balanced by a membrane potential of about -80 mv (inside negative). From pH and uncoupler insensitivity and K(+) sensitivity, it was concluded that the experimentally caused K(+) gradient created the electric potential.Qualitatively different results were obtained on vacuoles in situ: the pH gradient is greater (1.3), the membrane potential positive inside. Uncoupler sensitivity is evidence for an enzyme system transducing protons inward as a cause for the considerable protonmotive force at the tonoplast in situ.

Mechanism of uptake of L-arginine by sugar-cane cells.

Suspension cells of sugar cane were used as a model system for cells of higher plants to study the mechanism of L-arginine uptake. The uptake system is specific for the L-arginine molecule in the fully ionized state, i.e. delta-guanidino group and alpha-amino group positively charged and carboxyl group negatively charged. This was concluded because the Km value for uptake increased strongly for: (a) L-arginine analogues which lack the charged carboxyl group (L-arginine methyl ester, agmatin); (b) L-arginine analogues, which lack the charged alpha-amino group (L-arginine acid, gamma-guanidinobutyric acid); (c) L-arginine analogues, which lack the charged delta-guanidino group or gamma-guanidinoxy group (L-citrulline, L-canavanine at neutral and alkaline pH-values). The importance of the positive charge of the delta-guanidino group or gamma-guanidinoxy group was further documented by Km values for L-arginine and L-canavanine at different pH values. Only at pH values where the gamma-guanidinoxy group is protonated, was there an effective uptake of L-canavanine and effective competition of L-canavanine with L-arginine. The length of the L-arginine molecule was less important: slightly larger (L-homoarginine) or shorter analogues (L-lysine) were taken up rather well. A spatial rearrangement at the alpha-carbon (D-ariginine) was, however, not tolerated. The uptake of L-arginine proceeds by electrogenic uniport, there is no evidence for symport or antiport of another molecule (though L-canavanine uptake at neutral pH value causes a transient alkalinization of the suspension medium). Charge equilibration is brought about by efflux of protons and potassium ions.

The mechanism of sugar uptake by sugarcane suspension cells.

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. - (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). - (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the Km value was higher (70 µM) than for net influx (24 µM); the exchange efflux is proposed to be mediated by the same transport system with a Km value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. - (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H(+) per sugar; during the onset of hexose transport there was a K(+) exit of 0.94 K(+) per sugar for charge compensation. (It was assumed that the "real" stoichiometries are 1 H(+) and 1 K(+) per sugar.) The Km values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. - (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. - (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.

Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture.

Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose.