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RESUMEN

Sugar beet (Beta vulgaris L.) seedlings were grown on media containing 90 to 300 mM sucrose or glucose. Compared to controls, sugar-grown plants had higher growth rate, photosynthesis, and leaf sugar levels. The steady-state level of transcripts increased significantly for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (rbcS) and the cytosolic fructose-1,6-bisphosphatase and moderately for the Rubisco large subunit (rbcL). The transcript level of sucrose phosphate synthase remained unchanged. Fructose-1,6-bisphosphatase and Rubisco activities did not change in the presence of sugars, but that of sucrose phosphate synthase increased (44 and 90% under selective and nonselective assay conditions, respectively). Accelerated leaf development was indicated by (a) autoradiograms of leaves that showed that sucrose loading occurred earlier, (b) export capacity that also occurred earlier but, after about 2 weeks, differences were not detectable, and (c) sucrose synthase activity that declined significantly. Several conclusions emerged: (a) response was nonosmotic and gene and sugar specific, (b) sugars caused accelerated leaf development and sink-to-source transition, (c) enhanced gene expression was due to advanced leaf development, and (d) whereas Rubisco and cytosolic fructose-1,6-bisphosphatase genes were sugar repressed in mature leaves of greenhouse-grown plants, they were unaffected in mature, culture-grown leaves. To our knowledge, these data provide the first evidence in higher plants that, depending on the physiological/developmental context of leaves, sugars lead to differential regulation of the same gene.

RESUMEN

Cytosolic fructose-1,6-bisphosphatase (FBPase) was purified 472-fold from sugarbeet (Beta vulgaris L.) leaves by ammonium sulfate fractionation, anion-exchange chromatography (DEAE Sepharose), cation-exchange chromatography (S-Sepharose), gel filtration (Sephacryl S-300), and hydrophobic interaction chromatography (Phenyl Sepharose). The dissociated polypeptide (molecular mass of 37 kD) was used to generate polyclonal antibodies. Western blot analysis revealed a single band that was identified as the cytosolic FBPase. Enzyme activity and protein and transcript levels were measured under various light and dark conditions in growth chamber-grown plants. FBPase protein level remained unchanged during a diurnal cycle, but enzyme activity and transcript levels were highest and lowest at the end of the light and dark periods, respectively. Light-dependent increase in the enzyme activity and transcript level was gradual, occurring several hours after the onset of light. At the end of an extended dark period (48 h), FBPase activity was negligible, protein level was unchanged, and transcript level had declined (but considerable amounts of transcript remained). Neither activity nor protein and transcript were detected in etiolated leaves. Nearly 24 h of continuous exposure to light was required before the FBPase protein and activity reached maximal levels. Unlike the chloroplastic FBPase, which is light activated (direct regulation), changes in the cytosolic FBPase activity and transcription appear to be light dependent in an indirect manner. The data provide first evidence on the coarse control of this enzyme via a light-dependent modulation of transcription and posttranslational modification.

RESUMEN

Fructose-1,6 bisphosphatase (FBPase) is a ubiquitous enzyme controlling a key reaction. In non-photosynthetic tissues, it regulates the rate of gluconeogenesis. In photosynthetic tissues, two FBPase isozymes (chloroplastic and cytosolic) play key roles in carbon assimilation and metabolism. The cytosolic FBPase is one of the regulatory enzymes in the sucrose biosynthetic pathway - its activity is regulated by both fine and coarse control mechanisms. Kinetic and allosteric properties of the plant cytosolic FBPase are remarkably similar to the mammalian and yeast FBPase, but differ greatly from those of the chloroplastic FBPase. Cytosolic FBPase is relatively conserved among various organisms both at amino acid and nucleotide sequence levels. There is slightly higher similarity between mammalian FBPase and plant cytosolic FBPase than there is between the two plant FBPases. Expression of plant cytosolic FBPase gene is developmentally regulated and appears to be coordinated with the expression of Rubisco and other carbon metabolism enzymes. Similar to the gluconeogenic FBPase, relatively rapid end product repression of FBPase gene occurs in plant. However, unlike the gluconeogenic FBPase, a concurrent decline in plant FBPase activity does not occur in response to increased end product levels. The physiological significance of FBPase gene repression, therefore, remains unclear in plants. Both expression and activity of the cytosolic FBPase are regulated by environmental factors such as light and drought conditions. Light-dependent modulation of FBPase activity in plants appears to involve some type of posttranslational modification. In addition to elucidating the exact nature of the presumed posttranslational modification, cloning of genomic and upstream sequences is needed before we fully understand the molecular regulation of the cytosolic FBPase in plants. Use of transgenic plants with altered rates of FBPase activity offers potential for enhanced crop productivity.

RESUMEN

The mechanism of sucrose transport into vacuoles isolated from leaf tissue has been studied only in barley (Hordeum vulgare) mesophyll cells. In this tissue, sucrose transport was reported to be a facilitated diffusion. We have observed a facilitated diffusion of sucrose into vacuoles isolated from this tissue. However, no pH dependence was observed. Evidence is presented indicating that the pH dependence of sucrose uptake into vacuoles may be an artifact, reflecting tonoplast instability and survival of isolated vacuoles in different buffers. Apparently vacuoles do not withstand exposure to some commonly used buffers.

RESUMEN

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl(2), and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H(+)-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H(+)-translocating ATPase located at the plasma membrane of the mesophyll cells.

RESUMEN

The objectives of this work were to determine the path of phloem unloading and if a sucrose carrier was present in young sugar beet (Beta vulgaris L.) taproots. The approach was to exploit the characteristics of the sucrose analog, 1'-fluorosucrose (F-sucrose) which is a poor substrate for acid invertase but is a substrate for sucrose synthase. Ten millimolar each of [(3)H]sucrose and [(14)C]F-sucrose were applied in a 1:1 ratio to an abraded region of an attached leaf for 6 hours. [(14)C]F-sucrose was translocated and accumulated in the roots at a higher rate than [(3)H]sucrose. This was due to [(3)H]sucrose hydrolysis along the translocation path. Presence of [(3)H]hexose and [(14)C]F-sucrose in the root apoplast suggested apoplastic sucrose unloading with its subsequent hydrolysis. Labeled F-sucrose uptake by root tissue discs exhibited biphasic kinetics and was inhibited by unlabeled sucrose, indicating that immature roots have the ability for carrier-mediated sucrose transport from the apoplast. Collectively, in vivo and in vitro data indicate that despite sucrose hydrolysis by the wall-bound invertase, sucrose hydrolysis is not entirely essential for sugar accumulation in this tissue.

RESUMEN

In isolated phloem segments of celery (Apium graveolens L.), a tissue highly specific for sucrose and mannitol uptake, glucose uptake occurs at very low rates and exhibits biphasic kinetics. Nonpenetrating inhibitors such as parachloromercuribenzene sulfonic acid did not inhibit glucose uptake. However, uptake was greatly inhibited by penetrating inhibitors such as N-ethylmaleimide and carbonylcyanide-m-chlorophenyl hydrazone. Carbonylcyanide-m-chlorophenyl hydrazone inhibition of uptake was reversed by washing and addition of thiol reagents to uptake solutions. Phlorizin, a competitive inhibitor of glucose caused moderate inhibition of uptake only after 3 hours of tissue exposure. Low pH, fusicoccin, and low turgor which enhance H(+)-sugar cotransport did not alter uptake rates. Furthermore, glucose did not induce alkalinization of the uptake media. Efflux analysis indicated that the presence of 50 millimolar unlabeled glucose in the wash media enhanced exchange of the labeled glucose across the tonoplast. Results indicate that the glucose carrier is not located at the plasmalemma but appears to be present at the membrane of an intracellular compartment, most likely the tonoplast. Carrier-mediated glucose transport in this tissue is proposed to be a facilitated diffusion.

RESUMEN

Phloem tissue isolated from celery (Apium graveolens L.) was used to investigate the regulation of sucrose uptake by turgor (manipulated by 50-400 milliosomolal solutions of polyethylene glycol) and hormones indoleacetic acid (IAA) and gibberillic acid (GA(3)). Sucrose uptake was enhanced under low cellular turgor (increase in the V(max)). Furthermore, enhancement of sucrose uptake was due to a net increase in influx rates since sucrose efflux was not affected by cell turgor. Manipulations of cell turgor had no effect on 3-O-methyl glucose uptake. When 20 millimolar buffer was present in uptake solutions, low turgor-induced effects were observed only at low pH range (4.5-5.5). However, the effect was extended to higher external pH (up to 7.5) when buffer was omitted from uptake solutions. A novel interaction between cellular turgor and hormone treatments was observed, in that GA(3) (10 micromolar) and IAA (0.1-100 micromolar) enhanced sucrose uptake only at moderate turgor levels. The hormones elicited little or no response on sucrose uptake under conditions of low or high cell turgor. Low cell turgor, IAA, GA(3), and fusicoccin caused acidification by isolated phloem segments in a buffer-free solution. It is suggested that enhanced sucrose uptake in response to low turgor and/or hormones was mediated through the plasmalemma H(+)-ATPase and most likely occurred at the site of loading.

RESUMEN

The uptake of different sugars was studied in segments of isolated phloem from petioles of celery (Apium graveolens L.) in order to determine the kinetics and specificity of phloem loading in this highly uniform conductive tissue. The uptake kinetics of sucrose and the sugar alcohol, mannitol, which are both phloem-translocated, indicated presence of a single saturable system, while uptake of non-phloem sugars (glucose and 3-O-methylglucose) exhibited biphasic kinetics with lower uptake rates than those for sucrose and mannitol. The presence of unlabeled mannitol, 3-O-methylglucose and maltose in the incubation solution did not cause inhibition of labeled-sucrose uptake, indicating high carrier specificity and lack of sucrose hydrolysis in vivo. The pH optimum for sucrose uptake was 5-6. Furthermore, a rapid and transient alkalinization of the external media by sucrose indicated a sugar/H(+)-cotransport mechanism. Dual-labeling experiments showed that sucrose influx continued at a constant rate (V max=15 µmol·h(-1)·(g FW)(-1)), whereas sucrose efflux was low and insensitive to external concentration. Therefore, the saturable uptake kinetics for sucrose did not appear to be the result of an equilibrium between rates of sucrose influx and efflux.

RESUMEN

The effect of gibberellic acid (GA(3)) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA(3) (10 micromolar) applied to the source leaf, enhanced export of [(14)C]sucrose (generated by (14)CO(2) fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA(3) was applied 24 hours prior to the (14)CO(2) pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA(3) increased the proportion of sucrose exported. After a 10-min exposure to [(14)C]GA(3), radioactivity was found only in the source leaf. Following a 2 hour exposure to [(14)C]GA(3), radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [(14)C]GA(3) in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA(3) effect in the lower stem with time after treatment. We conclude that in the short term, GA(3) enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA(3) is outside the source leaf. GA(3) accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA(3) is rapidly metabolized in the lower stem thus attenuating any GA effect.

RESUMEN

The fate of exogenously applied, labeled abscisic acid (+/-)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10(-6) molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues.

RESUMEN

The mode of abscisic acid (ABA) uptake was studied in excised leaf and root tissue discs of sugar beet (Beta vulgaris L.). Discs were incubated in buffered medium that contained 1 mm CaCl(2) and [(14)C]ABA. The sensitivity of ABA uptake to metabolic inhibitors and temperature indicated that the ABA transport system had an energy-dependent component. Energy-dependent uptake was greater in leaf than in root tissue (70% and 50%, respectively). Energy-dependent uptake by both tissues and passive uptake by root tissues were highly pH dependent. Maximal uptake was observed at pH 5.5. Leaf tissue incubated in the dark showed a 50% reduction of uptake as compared with tissue under light. The decrease was due to reduced passive uptake.The results suggest that ABA moves across membranes as the undissociated lipophilic species. As a weak acid, ABA would dissociate and accumulate in the more alkaline compartment. Therefore, the distribution of ABA within the tissue is regulated by the pH differential between any two compartments. Although diffusion may be the predominant form of transport, the uptake of ABA is dependent on metabolic energy for the establishment of a pH gradient across the membrane.

RESUMEN

Active sucrose uptake by discs of mature sugar beet (Beta vulgaris L. cv GW-D2 and USH-20) root tissue shows a biphasic dependence on external sucrose. At concentrations up to 20 millimolar sucrose, the active uptake mechanism appears to approach saturation, with an apparent K(m) of 3.6 millimolar. At higher external sucrose concentrations, a linear dependence becomes obvious indicating the probable presence of a nonsaturable, metabolically dependent uptake component. Active transport was not observed at external sucrose concentrations that caused tissue plasmolysis. Passive sucrose uptake in unplasmolyzed tissue showed a linear dependence on external sucrose concentration. The mitochondrial and/or suspected vacuolar ATPase inhibitors oligomycin, diethylstilbestrol, and N,N-dicyclohexylcarbodiimide strongly inhibited active sucrose uptake, whereas the putative plasmalemma-specific ATPase inhibitor orthovanadate was without effect.Sucrose efflux patterns from root discs indicated three distinct sucrose compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole with the vacuole being the slowest releasing compartment. The sucrose contents and volumes of the compartments indicated that sucrose uptake into the vacuole was against a concentration gradient. Combined sucrose uptake/efflux analyses indicated that sucrose uptake into the vacuole is primarily an active transport process while transport into the cytoplasm is apparently passive, at least at external sucrose concentrations above 20 millimolar. We discuss the possibility that active sucrose uptake into the vacuoles of sugar beet storage cells is rate limited by passive sucrose transport to the active uptake site.

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RESUMEN

To investigate the abscisic acid (ABA) production of tomato (Mill.) plants in response to diurnal stressful temperatures, five-week old seedlings were exposed to day/night temperatures of 10/5, 15/10, 25/15, 35/25, or 45/35 C. The daylength was 16 hours with a light intensity of approximately 400 microeinsteins per meter per second. Plant tops were sampled at 12, 24, 48, and 72 hours. Free, alkaline-hydrolyzable (conjugated), and total ABA quantities were measured using standard gas chromatographic techniques. All temperature regimes significantly increased both free and conjugated ABA levels over concentrations in control plants (25/15 C). The highest ABA levels were observed in plants exposed to the coolest temperature of 10/5 C. Since normal water potentials were obtained in plants of all treatments, the observed ABA response was not due to temperature-induced water stress. Therefore, temperature stress, like several other environmental stresses, induces the plant to produce high levels of ABA. Because of the similar involvement of ABA in temperature-induced and other environmental stresses, ABA may be a common mediator for all plant stresses.