Biochemical changes during sucrose deprivation in higher plant cells

The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell.     

Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

An experimental arrangement was described that enables nuclear magnetic resonance spectra of compressed plant cells to be recorded while circulating a medium through the sample. The system provided a convenient arrangement for monitoring by 31P NMR the behavior of plant cells over a long period of time under different conditions such as sucrose starvation. Perfusion of compressed sycamore cells with sucrose-free culture medium triggered a progressive decrease in the glucose 6-P and uridine-5'-diphosphate-alpha-D-glucose resonances over 30 h. When almost all the intracellular carbohydrate pool had disappeared the nucleotide triphosphate resonances decline progressively. These changes were accompanied by a Pi accumulation in the vacuole and a phosphorylcholine (P-choline) accumulation in the cytoplasm. The very long lag phase observed for ATP and P-choline evolution was comparable with that observed for the progressive intracellular digestion of cytoplasmic constituents (Journet, E., Bligny, R. and Douce, R. (1986) J. Biol. Chem. 261, 3193-3199). Addition of sucrose in the circulating system after a long period of sucrose starvation led to a disappearance of the cytoplasmic Pi resonance and a marked increase in that of glucose 6-P. Under these conditions the vacuolar Pi pool did not fluctuate to buffer the Pi in the cytoplasm. The results suggest that Pi which has been sequestered in the vacuole during the course of sucrose starvation is not restored to the cytoplasm for rapid metabolic processes. Furthermore, the presence of P-choline in plant cells in large excess should be considered as a good marker of membrane utilization after a long period of sucrose starvation and is very likely related to stress.     

Relationship between the cytoplasm and the vacuole phosphate pool in Acer pseudoplatanus cells

The Pi concentration of Acer pseudoplatanus cells in the two major intracellular compartments, the cytoplasm and the vacuole, has been studied using 31P NMR. For sycamore cells containing approximately 2 mM of total Pi, the cytoplasmic Pi and the vacuolar Pi concentrations were approximately 6 and 1.5 mM, respectively. When the cells were transferred to a phosphate-deficient medium, the vacuolar Pi decreased rapidly while the cytoplasmic Pi decreased slowly during the first 48 h, indicating that Pi in the cytoplasm was maintained at the expense of the vacuolar Pi. When the Pi-starved cells (i.e., those containing less than 0.5 mumol of total Pi/g wet wt) were transferred to a medium containing 300 microM Pi, Pi entered the cells rapidly and accumulated in the cytoplasm. Once the cytoplasmic Pi pool was filled, Pi was taken up in the vacuole until the vacuole Pi pool was filled. On the contrary when the non-Pi-starved cells were transferred to a phosphate-rich medium (i.e., containing 45 mM Pi), Pi entered the cells slowly by diffusion and accumulated in the vacuole but not in the cytoplasm. These results demonstrate that the Pi content of the cytoplasm is maintained at the expense of the vacuolar Pi pool when sycamore cells are transferred to either a phosphate-deficient or a phosphate-rich medium.     

An important pool of sucrose linked to starch biosynthesis is taken up by endocytosis in heterotrophic cells

We have recently shown the occurrence of endocytic sucrose uptake in heterotrophic cells. Whether this mechanism is involved in the sucrose-starch conversion process was investigated by comparing the rates of starch accumulation in sycamore cells cultured in the presence or absence of the endocytic inhibitors wortmannin and 2-(4-morpholynyl-)-8-phenyl-4H-1 benzopyran-4-1 (LY294002). These analyses revealed a two-phase process involving an initial 120 min wortmannin- and LY294002-insensitive starch accumulation period, followed by a prolonged phase that was arrested by the endocytic inhibitors. Both wortmannin and LY294002 led to a strong reduction of the intracellular levels of both sucrose and the starch precursor molecule, ADPglucose. No changes in maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism occurred in cells cultured with endocytic inhibitors. In addition, starch accumulation was unaffected by endocytic inhibitors when cells were cultured with glucose. These results provide a first indication that an important pool of sucrose incorporated into the cell is taken up by endocytosis prior to its subsequent conversion into starch in heterotrophic cells. This conclusion was substantiated further by experiments showing that sucrose-starch conversion was strongly prevented by both wortmannin and LY294002 in both potato tuber discs and developing barley endosperms.     

Is the Cytosolic Pi Concentration a Limiting Factor for Plant Cell Respiration?

The substrate-dependent O₂ uptake by sycamore (Acer pseudoplatanus L.) cell mitochondria in the presence of ADP and limiting Pi concentrations has been measured. The Pi concentration for half-maximum O₂ uptake rate was found to be in the range 20 to 50 micromolar for all the substrates tested. ³¹P NMR of intact sycamore cells indicated that the Pi concentration in the cytoplasm was in the range 5 to 6 millimolar, approximately 100-fold higher than the Pi concentration required for maximum O₂ uptake rates by isolated mitochondria. When sycamore cells were transferred to a culture medium devoid of Pi, the cytoplasmic Pi concentration decreased from 6 to less than 3 millimolar, but the intact cell respiration remained practically constant for at least 4 days. These results strongly suggest that, in vivo, the respiration rate of sycamore cells is not limited by the quantity of Pi supplied to the mitochondria.     

Altered metabolic fluxes result from shifts in metabolite levels in sucrose phosphorylase-expressing potato tubers

As reported in a previous paper (Plant, Cell and Environment 24, 357–365, 2001), introduction of sucrose phosphorylase into the cytosol of potato results in increased respiration, an inhibition of starch accumulation and decreased tuber yield. Herein a more detailed investigation into the effect of sucrose phosphorylase expression on tuber metabolism, in order to understand why storage and growth are impaired is described. (1) Although the activity of the introduced sucrose phosphorylase was low and accounted for less than 10% of that of sucrose synthase its expression led to a decrease in the activities of enzymes of starch synthesis relative to enzymes of glycolysis and relative to total amylolytic activity. (2) Incubation of tuber discs in [¹⁴C]glucose revealed that the transformants display a two‐fold increase of the unidirectional rate of sucrose breakdown. However this was largely compensated by a large stimulation of sucrose re‐synthesis and therefore the net rate of sucrose breakdown was not greatly affected. Despite this fact major shifts in tuber metabolism, including depletion of sucrose to very low levels, higher rates of glycolysis, and larger pools of amino acids were observed in these lines. (3) Expression of sucrose phosphorylase led to a decrease of the cellular ATP/ADP ratio and energy charge in intact growing tubers. It was estimated that at least 30% of the ATP formed during respiration is consumed as a result of the large acceleration of the cycle of sucrose breakdown and re‐synthesis in the transformants. Although the absolute rate of starch synthesis in short‐term labelling experiments with discs rose, starch synthesis fell relative to other fluxes including respiration, and the overall starch content of the tubers was lower than in wild‐type tubers. (4) External supply of amino acids to replace sucrose as an osmoticum led to a feed‐back inhibition of glycolysis, but did not restore allocation to starch. (5) However, an external supply of the non‐metabolizable sucrose analogue palatinose – but not sucrose itself – stimulated flux to starch in the transformants. (6) The results indicate that the impaired performance of sucrose phosphorylase‐expressing tubers is attributable to decreased levels of sucrose and increased energy consumption during sucrose futile cycling, and imply that sucrose degradation via sucrose synthase is important to maintain a relatively large sucrose pool and to minimize the ATP consumption required for normal metabolic function in the wild type.     

Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies

We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H(+)-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g(-1) cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H(+) ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H(+) extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H(+)-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H(+)-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.     

Sugar and starch changes in pea cotyledons during germination

Changes in starch and sugar contents in the cotyledons during germination have been compared in a smooth (cv. Alaska) and a wrinkled (cv. Progress) cultivar of the garden pea ( Pisum sativum L.). In both cultivars there was an initial accumulation of sucrose due to the hydrolysis of sucrosyl oligosaccharides, but galactose did not accumulate in the cotyledons. Starch mobilization in the Progress pea was linear with time and started before the rise in α-amylase (EC 3.2.1.1) activity in the cotyledons; sucrose was synthesized in the cotyledons, and their excision from the axis resulted in an additional accumulation of this sugar. In the Alaska pea, the onset of starch hydrolysis coincided with the rise in α-amylase activity; no accumulation of sucrose was found in excised cotyledons, whilst the sucrose content decreased continuously in attached cotyledons.
The same sugars were found in the cotyledons of both cultivars, suggesting a common pathway for starch breakdown. Maltose, maltotriose and linear malto-dextrins were not present and only trace amounts of glucose were detected, suggesting a degradation of starch by phosphorylase after an initial attack by α-amylase. α-Amylase activity in the cotyledons was higher in the presence of the axis, but was influenced by the water content of the cotyledons. Transient changes in α-amylase activity correlated well with changes in the rate of starch hydrolysis, but after 2–3 days starch mobilization was reduced in excised cotyledons probably due to the resynthesis of starch.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Possible role played by R1 protein in starch accumulation in bean (Phaseolus vulgaris) seedlings under phosphate deficiency

The effect of phosphate (Pi) deficiency on starch accumulation was studied in bean (Phaseolus vulgaris). After 3 weeks of Pi deprivation total Pi concentration in root and shoot was reduced by 68% and 42%, respectively; however, only shoot growth was affected. In leaves, Pi deprivation induced glucose, fructose and starch accumulation. Pi deficiency did not affect starch synthesis, but it reduced its mobilization during the dark period. At the same time, starch produced by Pi deficient plants have fewer Pi bound and was also less susceptible to beta-amylase hydrolysis. R1 protein is the protein responsible of phosphorylating C3 and C6 glucosyl residues of the polyglucan, increasing the hydration capacity and the interaction with amylolytic enzymes. Pi deprivation did not change the amount of R1 protein detected in total extracts but decreased its association with starch granules.     

Transport and Metabolism of a Sucrose Analog (1'-Fluorosucrose) into Zea mays L. Endosperm without Invertase Hydrolysis

1′-Fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L., Pioneer Hybrid 3320) kernels with the same magnitude and kinetics as sucrose. ¹⁴C-Label from [¹⁴C]FS and [¹⁴C]sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was absorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of [¹⁴C] sucrose from supplied [¹⁴C]glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggests that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded from the phloem, hexoses are not specifically needed for uptake into maize endosperm.     

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5'- and 3'-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8-(14)C]adenosine and [8-(14)C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.     

Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO₂ respired during ripening was positively correlated with sugar accumulation (R² = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO₂ was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.     

Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet: III. Diurnal Changes in Carbon Partitioning and Carbon Export

The effect of low phosphate supply (low P) was determined on the diurnal changes in the rate of carbon export, and on the contents of starch, sucrose, glucose, and fructose 2,6-bisphosphate (F2,6BP) in leaves. Low-P effects on the activities of a number of enzymes involved in starch and sucrose metabolism were also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers and the low-P treatment induced nutritionally. Low-P treatment decreased carbon export from the leaf much more than it decreased photosynthesis. At growth chamber photon flux density, low P decreased carbon export by 34% in light; in darkness, export rates fell but more so in the control so that the average rate in darkness was higher in low-P leaves. Low P increased starch, sucrose, and glucose contents per leaf area, and decreased F2, 6BP. The total extractable activities of enzymes involved in starch and sucrose synthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5-diphosphoglucose pyrophosphorylase, and sucrose-phosphate synthase. The activities of some enzymes involved in starch and sucrose breakdown were also increased by low P. We propose that plants adapt to low-P environments by increasing the total activities of several phosphatases and by increasing the concentrations of phosphate-free carbon compounds at the expense of sugar phosphates, thereby conserving Pi. The partitioning of carbon among the various carbon pools in low-P adapted leaves appears to be determined in part by the relative capacities of the enzymes for starch and sucrose metabolism.     

Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis

High-resolution 31P nuclear magnetic resonance spectroscopy and 14C fluorography have been used to identify and quantitate intermediates of the Embden-Meyerhof pathway in intact cells and cell extracts of Streptococcus lactis. Glycolysing cells contained high levels of fructose 1,6-bisphosphate (a positive effector of pyruvate kinase) but comparatively low concentrations of other glycolytic metabolites. By contrast, starved organisms contained only high levels of 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate. The concentration of Pi (a negative effector of pyruvate kinase) in starved cells was fourfold greater than that maintained by glycolysing cells. The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruvate kinase: the increase in the phosphoenolpyruvate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi (by a maltose-plus-arginine phosphate trap) caused the restoration of pyruvate kinase activity in starved cells. Time course studies showed that Pi was conserved by formation of fructose 1,6-bisphosphate during glycolysis. Conversely, during starvation high levels of Pi were generated concomitant with depletion of intracellular fructose 1,6-bisphosphate. The concentrations of Pi and fructose 1,6-bisphosphate present in starved and glycolysing cells of S. lactis varied inversely. The activity of pyruvate kinase in the growing cell may be modulated by the relative concentrations of the two antagonistic effectors.     

Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.     

Increased levels of adenine nucleotides modify the interaction between starch synthesis and respiration when adenine is supplied to discs from growing potato tubers

To investigate the importance of the overall size of the total adenine nucleotide pool for the regulation of primary metabolism in growing potato tubers, freshly cut discs were provided with zero or 2 mM adenine in the presence of 1 or 100 mM [U-¹⁴C]glucose or 100 mM [U-¹⁴C]sucrose in the presence and absence of 20 mM orthophosphate (Pi). Adenine led to a 150–250% increase of the total adenine nucleotide pool, which included an increase of ADP, a larger increase of ATP and an increase of the ATP:ADP ratio. There was a 50–100% increase of ADP-glucose (ADPGlc), and starch synthesis was stimulated. Respiratory oxygen uptake was stimulated, and the levels of glycerate-3-phosphate, phosphoenolpyruvate and α-ketoglutarate decreased. The response to adenine was not modified by Pi. It is proposed that increased ATP stimulates ADPGlc pyrophosphorylase, leading to a higher rate of starch synthesis. The impact on starch synthesis is constrained, however, because increased ADP can lead to a stimulation of respiration and decline of glycerate-3-phosphate, which will inhibit ADPGlc pyrophosphorylase. The quantitative impact depends on the conditions. In the presence of 1 mM glucose, the levels of phosphorylated intermediates and the rate of starch synthesis were low. Adenine led to a relatively large stimulation of respiration, but only a small stimulation of starch synthesis. In the presence of 100 mM glucose, discs contained high levels of phosphorylated intermediates, low ATP:ADP ratios (<3) and low rates of starch synthesis (<20% of the metabolised glucose). Adenine led to marked increase of ATP and 2- to 4-fold stimulation of starch synthesis. Discs incubated with 100 mM sucrose already had high ATP:ADP ratios (>8) and high rates of starch synthesis (>50% of the metabolised sucrose). Adenine led to a further increase, but the stimulation was less marked than in high glucose. These results have implications for the function of nucleotide cofactors in segregating sucrose mobilisation and respiration, and the need for energy conservation during sugar-starch conversions. Received: 9 February 2000 / Accepted: 9 June 2000     

Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.     

Control of phosphate transport across the plasma membrane of Chara corallina

This paper examines the control of phosphate uptake into Chara corallina. Influxes of inorganic phosphate (Pi) into isolated single internodal cells were measured with ³²Pi. Pretreatment of cells without Pi for up to 10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm and the vacuole remained quite constant. When cells were pre-treated with 0.1 mM Pi, the subsequent influx of Pi was low. Under these conditions the Pi concentrations in the cytoplasm was almost the same as that of Pi-starved cells, but vacuolar Pi increased with time. Transfer of cells from medium containing 0.1 mM Pi to Pi-free medium induced an increase of Pi influx within 3 d irrespective of the concentration of Pi in the vacuole.During Pi starvation, neither the membrane potential nor the cytoplasmic pH changed. Manipulation of the cytoplasmic pH by weak acids or ammonium decreased the Pi influx slightly.Pi efflux was also measured, using cells loaded with ³²Pi. Addition of a low concentration of Pi in the rinsing medium rapidly and temporarily induced an increase in the efflux.The results show that Pi influx is controlled by factors other than simple feedback from cytoplasmic or vacuolar Pi concentrations or thermodynamic driving forces for H⁺-coupled Pi uptake. It is suggested that uptake of Pi is controlled via the concentration of Pi in the external medium through induction or repression of two types of plasma membrane Pi transporters.Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation