Action Mechanism of Ethylene in the Control of Sugar Translocation in Relation to Rice Coleoptile Growth I. Sucrose Metabolism

The fate of ¹⁴C-glucose fed through scutella of rice (Oryzasativa L. cv. Sasanishiki) seedling explants was investigatedin relation to ethylene action on sugar translocation to growingcoleoptiles and leaves. In the scutellum, sucrose, UDPglucoseand F6P were rapidly labeled, and sucrose-phosphate synthaseactivity was higher than sucrose synthase activity. Radioactivesucrose soon appeared in both coleoptiles and leaves, and increasedrapidly. Its specific activity in both tissues became almostequal to that in the scutella. The specific activities of ¹⁴C-glucosein both coleoptiles and leaves changed almost in parallel tothose of ¹⁴C-fructose. These results suggest that sucrose wassynthesized in the scutellum and exported to the coleoptileand leaf, where it was cleaved to glucose and fructose. Ethylene slightly increased the specific activities of ¹⁴C-sucrosein all tissues, but markedly increased those of ^l4C-glucoseand -fructose only in the coleoptile. We assume that the ethyleneenhancement of sucrose transport from scutellum to the coleoptileresults from the activation of sucrose unloading in the growingcoleoptile where imported sucrose is cleaved into glucose andfructose. (Received May 25, 1987; Accepted October 30, 1987)     

Import of Sucrose and its Partitioning in the Synthesis of Galactomannan and Raffinose-oligosaccharides in the Developing Guar (Cyamopsis tetragonolobus) Seed

Import of sucrose and its transformation to galactomannan andraffinose-oligosaccharides have been studied in the developingguar seed. The amount of galactomannan gradually increased withthe ageing of the seed. During the entire period of pod development,sucrose constituted the major portion of the free sugars inthe seed (both endosperm and cotyledons) as well as in the podwall. Besides myo-inositol, the free sugars detected in thedeveloping endosperm and cotyledons were glucose, fructose,raffinose and stachyose. Some compounds, possibly glycosides(RG values higher than that of fructose), were also detectedin the endosperm. In the later stages of seed development, therelative proportion of raffinose in the free sugars increased,reaching 50% of the total free sugars in 77-d-old cotyledons.With pod maturity, the activities of soluble acid and boundacid invertases in the pod wall increased manifold with a concomitantdecline in the non-reducing sugar content. These enzymes seemto be involved in the mobilization of sucrose from this fruitingstructure into the seed. An increased synthesis of raffinose-oligosaccharidesboth in the endosperm and cotyledons was associated with highactivities of soluble acid invertase (pH 4.8) and sucrose-UDPglucosyl transferase in these tissues. Feeding uniformly labelled¹⁴C-sugars to the detached intact pods as well as to the isolatedendosperm and cotyledons resulted in labelling of all endogenousfree sugars and galactomannan. The uptake and incorporationinto galactomannan of ¹⁴C was stimulated by Co²⁺, Mn²⁺ and Mg²⁺.Except for mannose, a major proportion of the ¹⁴C from glucose,fructose and sucrose appeared in sucrose in both endosperm andcotyledons indicating a fast reconstitution of sucrose in situ.Based on the present results, a possible mode of transformationof sucrose to galactomannan and raffinose-oligosaccharides hasbeen proposed. Key words: Sucrose, galactomannan, raffinose-oligosaccharides, invertase, sucrose-UDP glucosyl transferase, ¹⁴C-incorporation, guar seed     

Sucrose transport and hexose release in the maize scutellum

Since hexoses readily diffuse from maize scutellum cells, it should be possible to detect them if they are produced during sucrose transport at the tonoplast or the plasmalemma. To test this idea, scutellum slices were placed in dinitrophenol (DNP) (which inhibits hexose utilization while greatly increasing utilization of vacuolar sucrose), and the utilization, uptake and leakage of sugars were measured. Only negligible amounts of hexose appeared in the DNP solution during a 5-hr incubation during which the slices metabolized 72μmol of sucrose. Glucose and fructose, added at a concentration of 2 mM, were taken up by the slices at rates 33% and 14% (respectively) of the rate of vacuolar sucrose utilization. It is suggested, therefore, that sucrose transport at the tonoplast does not release free hexose into the cytoplasm. Sucrose transport at the plasmalemma was studied using DNP- and mannose-treated slices. During incubation of these slices in sucrose, the disappearance of sucrose resulted in the appearance of significant quantities of glucose and fructose in the bathing solution. Evidence is presented that sucrose is split into glucose and fructose during transport across the plasmalemma. It is concluded that free hexose is not normally a product of this splitting but is a result of an uncoupling in the transport system caused by the DNP or mannose treatments.     

Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used ¹³C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of ¹³C between carbons atoms 1 and 6 of [1-¹³C] or [6-¹³C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the ¹³C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the ¹³C sugars were supplied in vivo to the intact plant. The ¹³C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of ¹³C in all glucosyl carbons was quantified by ¹³C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background ¹³C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.     

Metabolism of C-Maltose in Avena fatua Seeds During Germination

Non-dormant and dormant seeds of Avena fatua metabolize ¹⁴C-maltose in different ways: in non-dormant seeds, ¹⁴C-maltose administered to the endosperm is readily converted to sucrose in the scutellum and translocated to the embryo; in dormant seeds, little sucrose is synthesized from ¹⁴C-maltose, and maltose and glucose tend to accumulate in the endosperm. It is suggested that biosynthesis of sucrose is essential for effective transport of the endosperm reserve to the embryonic axis in germinating seeds.     

Glucose control of sucrose synthase in the maize scutellum

The in vivo amounts of UDPG, UTP, UDP and UMP, metabolites known to influence the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS), were measured throughout 5 hr incubations of scutellum slices in fructose or water, i.e. under conditions of sucrose synthesis or breakdown. Cytosolic concentrations were estimated assuming that these metabolites were confined to the cytosol. Within the estimated in vivo concentration ranges, UDPG, UTP and UDP had little effect on the in vitro SS activity, but glucose (100 mM) inhibited SS in the synthesis direction by 63–70% and in the breakdown direction by 86–93%. Glucose inhibition of SS was considerably less when saturating levels of substrates were used. Sucrose did not inhibit SS. It is concluded that during germination the glucose produced from starch breakdown in the maize endosperm enters the scutellum and inhibits SS, preventing a futile cycle and limiting SS participation in sucrose synthesis.     

Mobilization of Endosperm Reserves and Uptake of Sucrose and Valine by the Cotyledons of Euphorbia lathyris L.

Upon germination, the endosperm triacylglycerols and proteinswere converted to sucrose and amino acids. During early postgerminativegrowth, the rate of sucrose and amino acid production exceededthe rate of uptake by the cotyledons. As a result, the levelsof total amino acid and sucrose in the endosperm increased;maximum levels were reached at 7 d and 10 d after imbibition(DAI), respectively. Intact seedlings were used to measure thedevelopment of valine, arginine, glutamic acid, and sucroseuptake rate throughout the course of endosperm depletion. Maximumamino acid uptake rates were measured at around 9 DAI, the highestuptake rate for sucrose was obtained at 12 DAI (just beforedepletion of the endosperm). The daily increase of sucrose andamino acid uptake could be manipulated, by replacing the endospermwith a pre-incubation solution during 1 d. The increase in sucroseuptake in vitro was equal to that measured with intact seedlingswhen the cotyledons were pre-incubated in 10 mol m^–3 sucrose.Higher sucrose concentrations reduced the increase of sucroseuptake; at 300 mol m^–3 sucrose (corresponding to the meanendosperm sucrose concentration) sucrose uptake after pre-incubationwas even lower than before. This reduction was largely counteractedwhen the pre-incubation solution was supplemented with minerals.The development of the valine uptake was hardly affected bysucrose, but was inhibited by several amino acids. Key words: Euphorbia lathyris seedling, sucrose uptake, amino acid uptake, reserve mobilization     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis

In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots.     

The Analysis of Correlative Growth in the Etiolated Oat Seedling in Relation to Carbon Dioxide and Nutrient Supply

To overcome the reduced extension growth of the coleoptile whichoccurs when oats are grown in air enriched with 5 per cent.CO¹, plants have been provided with nutrients via the roots.2 per cent, sucrose, glucose or mannitol so applied furtherpromoted the mesocotyl and further depressed the coleoptile.Root growth was also depressed. To induce promotion of coleoptile growth by externally appliedsucrose, seedlings were heated in darkness at 40° C. for3 hours so restricting selectively the growth of the mesocotyl.Promotion of the coleoptile, however, was not observed. Application of mixed Na and K nitrates occasioned an immediategrowth promotion of doleoptile and leaves in both the presenceand absence of CO², and also a.much less pronounced promotionof the mesocotyl in CO²; there was no effect in air. This enhancedgrowth of the coleoptile and leaves was coupled with a correspondinglygreater dry weight and also with an increased outflow of reservesfrom the endosperm into the plumule. Thus, while externally applied sugars seemed not to reach thecoleoptile, those made available from the endosperm as a resultof improved nitrogen supply were rapidly translocated to it.Simultaneous provision to the roots of nitrate and sucrose didnot improve the absorption and translocation of sugar. An analysis of covariance has been computed using the mesocotyland coleoptile length data together with the outflow from theendosperm and the conclusions so derived are discussed in relationto the problem of growth integration in etiolated oat seedlings.     

Invertase and maltase in the free space of the maize scutellum

When maize scutellum slices were incubated in solutions of sucrose or maltose, there was a release of glucose into the bathing solution. The pH optima for glucose release were 2.5 for sucrose and 3.5 for maltose. From measurement of rates of glucose uptake into slices in the presence or absence of sucrose, it is calculated that glucose uptake will introduce errors of 3–9%, depending on the sucrose concentration, in estimates of free-space sucrose-hydrolase activity at pH 2.5. At their respective pH optima, maltose was hydrolysed at a rate 2.5 times that of sucrose. When frozen-thawed slices were used the same pH optima were obtained, but rates of hydrolysis were increased. Raffinose and melezitose also were hydrolysed with pH optima of 2.5 and 3.5, respectively. α-Methyl glucose was not hydrolysed. A 60-min HCl treatment (pH 2) of scutellum slices destroyed 69% of the sucrose-hydrolase activity and 100% of the maltose-hydrolase activity. In contrast, sucrose uptake and sucrose synthesis from exogenous fructose were not affected by HCl treatment. It is concluded that there are two hydrolases, acid invertase and maltase; that they are either on or outside the plasmalemma (in the free space); and that they are not necessary to the disaccharide uptake processes either by supplying exogenous hexose or by acting as transporters.     

Differential Rates of Uptake of Glucose, Fructose and Sucrose by Pea Stem Protoplasts

Protoplasts from growing regions of etiolated pea stems takeup glucose more rapidly than fructose when supplied for briefperiods at low concentrations. The uptake of the two hexosesis differentially inhibited by galactoac and by reagents thatcurtail ATP synthesis, and uptake of one hexose is not preventedby the other, even at a 100-fold excess. Sucrose uptake is muchslower than that of either hexose and is correlated with theappearance of invertase activity in the medium. Label from [¹⁴C-glc]-sucroseis taken up more rapidly than from [¹⁴C-fru]-sucrose. It isconcluded that these cells take up supplied sucrose only afterhydrolysis to hexoses, which are then absorbed by differentcarrier-mediated processes. Key words: Glucose, fructose, invertase, pea, protoplast, sucrose     

Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize

Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP-dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP-linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi-linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Proline Metabolism and Transport in Maize Seedlings at Low Water Potential

The growing zone of maize seedling primary roots accumulatesproline at low water potential. Endosperm removal and excisionof root tips rapidly decreased the proline pool and greatlyreduced proline accumulation in root tips at low water potential.Proline accumulation was not restored by exogenous amino acids.Labelling root tips with [¹⁴C]glutamate and [¹⁴C]proline showedthat the rate of proline utilization (oxidation and proteinsynthesis) exceeded the rate of biosynthesis by five-fold athigh and low water potentials. This explains the reduction inthe proline pool following root and endosperm excision and theinability to accumulate proline at low water potential. Theendosperm is therefore the source of the proline that accumulatesin the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [¹⁴C]Prolinewas transported from the scutellum to other parts of the seedlingand reached the highest concentration in the root tip. Less[¹⁴C]proline was transported at low water potential but becauseof the lower rate of protein synthesis and oxidation, more accumulatedas proline in the root tip. Despite the low biosynthesis capacityof the roots, the extent of proline accumulation in relationto water potential is precisely controlled by transport andutilization rate.     

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

The proplastid fraction containing no cytosol and mitochondrionwas isolated from developing castor bean endosperm by stepwisesucrose density centrifugation. This fraction possesses thecapacity to synthesize LFAs from [u-14C]sucrose, [u-¹⁴C]-glucose,[u-¹⁴C]G-1-P, [u-¹⁴C]G-6-P, [2-¹⁴C]pyruvate and [1-¹⁴C]acetate.Little was incorporated from [1-¹⁴C]pyruvate into LFAs, butmuch into ¹⁴CO_a. Addition of cytosol to the proplastid fractiondid not enhance the LFA synthesis. From these data, the wholepath from sucrose to LFAs through glycolytic path and pyruvatedecarboxylation seems to be located within the proplastid indeveloping castor bean endosperm. The difference in utilizationof substrates indicates that the rate of LFA synthesis in castorbean proplastids is limited at a step between sucrose and hexosephosphate. In addition, experiments with CO₂ output and LFAsynthesis from [1-¹⁴C]glucose, [6-¹⁴C]glucose and [u-¹⁴C]G-6-Pstrongly suggest that the path flow branches actively throughG-6-P to the pentose phosphate path and little through acetylCoAto the TCA cycle. (Received May 12, 1975; )     

Impairment of sucrose utilization for cell wall formation in the roots of aluminum-damaged cotton seedlings

Shoots of normal cotton seedlings rapidly fixed a pulse of ¹⁴CO₂from the ambient atmosphere and translocated some of the resultinglabeled sucrose to the roots. Roots of these plants assimilatedmost of the radioactivity from a 10-min labeling pulse intoinsoluble cell wall materials and other stable metabolites within4 to 6 hr after the pulse. However, roots of cotton seedlingswhich had been exposed to 1 ppm of Al^3$ for 24 hr before labelingtended to accumulate the ¹⁴C-label as free sucrose. Histologicand microautoradiographic evidence suggested that Al^3$ impairedthe root's capacity to utilize sucrose in further metabolicproducts so that ¹⁴C-labeled sucrose was not polymerized intocell wall materials as it was in the roots of control plants. (Received July 7, 1971; )     

Invertase Activity and its Relation to Hexose Accumulation in Potato Tubers

Hexose accumulation was shown to occur in freshly harvestedmature potato tubers (Solanum tuberosum L.) both after storageat 10 ?C and when subsequently transferred to low temperature(3 ?C) storage. In general, changes in hexoses and sucrose werefound to be related to changes in acid invertase activity. Totalacid invertase activity (i.e. assayed after destroying the endogenousinvertase inhibitor present in the extracts) generally reflectedsugar changes more closely than did basal activity (i.e. assayedwith the inhibitor present). There was no evidence of a specificalkaline invertase. A comparison of the temperature responsesof cultivar Record with that of two SCRI² clones demonstrateddistinct genotypic variation in the extent of hexose accumulation.However, these differences were not always reflected by genotypicdifferences in total invertase activity. Key words: Invertase inhibitor, glucose, fructose, sucrose     

Metabolism of Radioactive Sugars by Tobacco leaf Disks

Destarched tobacco-leaf disks were floated on per cent. (w/v)solutions of sucrose uniformly labelled with ¹⁴C in either theglucose or fructose moiety, and on invert sugar in which onehexose only was so labelled. The experiments were carried outin an atmosphere of oxygen at 25° C. Seventy-five per cent,of the sugar lost from the external solutions was recoveredas starch, sucrose, fructose, glucose, and CO₂. With sucroseas the substrate, 30 per cent, of the material was recoveredas CO₂ and 17 per cent. each as starch, sucrose, fructose, andglucose. With invert sugar as the substrate, 30 per cent, wasagain recovered as CO₂ only 20 per cent. as the three sugarstogether, and 50 per cent. as starch. Whichever hexose was initiallylabelled and whether the sugar was supplied as sucrose or hexose,the relative specific activities of starch and sucrose in theleaf disks and of the CO₂ evolved were equal or nearly equalto that of the sugar supplied. With sucrose as the substratethe sucrose in the disks retained its asymmetry of label, andfree hexoses produced were similarly asymmetrically labelled.When invert sugar was the substrate the sucrose synthesizedwas strongly labelled in both moieties, as also were the freehexosea. It is concluded that fructose and glucose free or combinedin sucrose were equally available for starch synthesis and CO₂,formation, and that there can be no question of preferentialutilization of one or other hexose. Starch and CO₂ must arisefrom a common source in which readily formed derivatives ofthe hexoses are rapidly equilibrated. Free hexose cannot participatedirectly in either sucrose or starch synthesis. Accumulationof sugar not immediately metabolized and inversion of sucrosetake place at a site remote from the common pool. A scheme toaccommodate the results is discussed.