Regulation of Fructan Metabolism in Leaves of Barley (Hordeum vulgare L. cv Gerbel)

Excised primary leaf blades of barley (Hordeum vulgare L. cv Gerbel) rapidly synthesized large quantities of fructan in the light and, upon transfer to the dark, they rapidly degraded it again. In the course of such a light/dark cycle the activities of sucrose-sucrose-fructosyltransferase (SST), fructan hydrolase, and invertase were measured in cell-free extracts of the blades. SST activity increased 20-fold within 24 hours in the light and disappeared again upon transfer to the dark during a similar period of time. Cycloheximide inhibited the increase of SST activity in the light indicating de novo synthesis. The loss of SST activity in the dark, however, was unaffected by cycloheximide. No SST activity appeared in the light if photosynthesis was inhibited by lowering the CO₂ concentration in the atmosphere. However, SST activity and fructan synthesis were induced even in the dark and at a low CO₂ concentration when the leaf blades were immersed in a solution of sucrose. Several other sugars, maltose and fructose in particular, had the same effect. Trehalose induced SST activity but no fructan synthesis occurred. The activities of fructan hydrolase and invertase changed little during the light/dark cycle. It is suggested that the control of SST activity in conjunction with the supply of photosynthates plays a key role in the regulation of fructan metabolism.     

Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

低pH对水稻黄化叶片硝酸还原酶活性暗诱导的调节

在低pH条件下,水稻离体黄化叶片的硝酸还原酶(NR)活性能在暗中诱导产生,其诱导过程约有2h的滞后期,亚胺环已酮(CHI,5ppm)和Na_2WO_4(25 mmol/L)能完全抑制这种诱导作用。在最适pH 3.0时,H~3标记氨基酸掺入NR的量比pH 7.0时约高2倍,表明酶活性的产生与酶蛋白的重新合成有关。 当低pH暗诱导时,BA(5ppm)和ABA(15ppm)能使酶活性分别提高约30％和80％,但它们都不能取代低pH在NR活性暗诱导中的作用。当存在1ppm CHI的时候,BA仍促进NR活性,而ABA则加强CHI对酶活性的抑制作用,这提示BA与ABA在低pH暗诱导条件下促进NR活性的机制是不同的。在pH 7.0的光诱导条件下,ABA对NR活性起抑制作用。     

Control of delta-Aminolevulinic Acid and Chlorophyll Accumulation in Greening Maize Leaves upon Light-Dark Transitions

The accumulation of δ-aminolevulinic acid (ALA) was studied in greening maize (Zea mays) leaves which were transferred to darkness and reilluminated after various periods of time. The system synthesizing ALA decays in the dark with a half-life of about 80 minutes. The onset of enzyme decay after transfer to darkness shows a 40-minute lag. The accumulation of ALA in the presence of levulinic acid in leaves transferred to darkness corresponds to that expected from the estimated half-life of the enzyme synthesizing ALA. On the other hand, the accumulation of protochlorophyll upon transfer to darkness in the absence of levulinic acid stops much earlier. It is suggested that a control point exists in the pathway between ALA and protochlorophyll, preventing utilization of the accumulated ALA upon transfer of greening leaves to darkness. This is supported by the observed effects of low intensities of monochromatic light (648 nm) on ALA and chlorophyll accumulation.     

Changes of Sucrose-Phosphate Synthase Activity in Barley Primary Leaves during Light/Dark Transitions

The activity of sucrose-phosphate synthase (SPS) in 9-day-old barley (Hordeum vulgare L.) primary leaves was measured over a 24-hour period. Extractable enzyme activity was constant in the light, decreased 50 to 60% during the first one-half hour of darkness, and then returned to full activity before the start of the normal light period. Decreases of SPS activity in the dark were fully reversed by less than 10 minutes of illumination. In contrast to results with barley, the measurable activity of SPS in soybean, spinach, and pea leaves was unchanged during the first hour of darkness. Changes of SPS activity in barley primary leaves were stable upon gel filtration. The exact biochemical mechanism responsible for the enzyme activity changes in barley leaf extracts is unknown. The above findings support the suggestion by de Fekete (1973 Eur J Biochem, 10: 73-80) that SPS is controlled by posttranslational protein modification. These results are discussed in relation to the regulation of photosynthetic sucrose metabolism.     

Trehalose Toxicity in Cuscuta reflexa: SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING

Trehalose, an α,α-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [¹⁴C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding.     

Relationship between light conditions and behavior of the freshwater snail Biomphalaria glabrata (Say)

The influence of light upon behavior of Biomphalaria glabrata was investigated in snails submitted for 48 h to one of the following regimes: normal light cycle, continuous darkness, continuous light. Time-lapse cinematography was used to provide data about snail locomotor activity in response to (a) continuous light or darkness; (b) light or dark phases; (c) light transitions. Snails were significantly less active under continuous light than under continuous or intermittent darkness. Under the normal light cycle, the activity rate was significantly higher in the dark than in the light. Changes from light to dark corresponded to increases in the activity rate which persisted long afterwards. No significant variation in activity occurred upon changes from dark to light.     

Ethylene Release from Leaves of Xanthium strumarium L. and Zea mays L.

The release of ethylene into sealed Erlenmeyer flasks by intactleaves and leaf discs of Xanthium strumarium L. a C₃ plant andZea mays L. a C₄ plant were compared both in white light andin darkness. The effects of the presence or absence of addedCO₂ (in the form of sodium bicarbonate) the photosynthetic inhibitor3-[3,4-dichlorophenyl]-l, l-dimethyl urea (DCMU) and 1-aminocyclopropane-1-carboxylicacid (ACC), the precursor of ethylene in higher plants, werealso investigated. The rate of ethylene release from leaf tissue of Xanthium inthe absence of added CO₂ was markedly reduced in the light (i.e.at the CO₂ compensation point). Treatments that would enhancethe CO₂ availability to the tissue (i.e. added bicarbonate,darkness, treatment with DCMU) allowed higher levels of ethylenerelease. Incubation of the tissue with ACC considerably enhancedthe release of ethylene compared to that from the correspondingcontrol tissue without ACC. However, the pattern of ethylenerelease induced by the various treatments was similar with orwithout added ACC. When tissue, in the absence of added CO₂, was transferred fromlight to darkness, and back to light for 90 min periods, theethylene release rates Increased during the interposed darkperiod but resumed the lower rate during the final light period.The addition of CO₂ in the light resulted in a similar rateof ethylene release to that found in the dark. The overall pattern of ethylene release from Zea leaf tissuesubjected to light and dark in the presence or absence of addedCO₂ was similar to that of Xanthium. However, two or three timesmore ethylene was released from maize leaves in the light whenCO² was added compared to that generated in the dark. This isin marked contrast to Xanthium, where, under the light conditionsused, the ethylene release rate in the dark equalled or exceededthat occurring in the light, even in the presence of high levelsof CO₂. A very low rate of ethylene release was observed atthe CO₂ compensation point of maize. A speculative model is presented to explain how photosyntheticactivity might act as a key factor in regulating ethylene evolutionfrom leaf tissue in these experiments. It invokes the conceptof an inhibition by CO₂ of ethylene retention or breakdown thuspermitting more ethylene to be released from the leaves.     

THE EFFECT OF LIGHT INTENSITY AND SUCROSE FEEDING ON THE FINE STRUCTURE IN CHLOROPLASTS AND ON THE CHLOROPHYLL CONTENT OF ETIOLATED LEAVES

Changes in the fine structure of proplastids of etiolated leaves exposed to various conditions of light and darkness for 24 and 48 hours were investigated, and the chlorophyll content of the leaves so treated was determined in vivo. The light treatments were given while the leaves were floated on tap water or on a 0.2 M sucrose solution. Leaves floated on water under low light intensity (2 foot-candles) were low in chlorophyll and contained plastids with concentric rows of vesicles. Transferring the leaves back to darkness resulted in the disappearance of the concentric rigs and re-formation of vesicular centers together with straight rows of vesicles and tubules, evenly spaced throughout the stroma. Chloroplasts of leaves floated on a sucrose solution under low light showed large vesicular centers together with stacks of rows of elongated tubules. The same chloroplast structure was found in leaves floated on a sucrose solution in the dark, after having been exposed to weak light for 24 hours. Chlorophyll content in these leaves was the same as in leaves floated on water under high light intensity, where the chloroplasts had normal grana and lamellae. The effect of the investigated factors on plastid development is discussed.     

NADH-Nitrate Reductase Inhibitor from Soybean Leaves

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.

The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO₃. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V_max.

The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.

Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.

     

Ryegrass leaf fructan synthesis is oxygen dependent and abolished by endomembrane inhibitors

Valid models are the foundation of systems biology. However, even well-established models may warrant reassessment. A testable feature of the currently accepted vacuolar model for fructan biosynthesis is its independence from metabolic energy at substrate level. The effects of limiting energy provision on fructan biosynthesis in grass leaves were determined. It was found that, in darkness in air, the rate of fructan accumulation was reduced to half relative to a light control. In darkness under anoxia the process was immediately abolished. In the light, the leaf sucrose concentration remained high, but in darkness +/- O(2), 40% of this sucrose was rapidly degraded. The constant rate of dark-aerobic fructan accumulation was independent of the decrease in sucrose concentration. Constant rates of aerobic fructan synthesis were independent of marked changes in extractable polymerase rates. In the dark under anoxia, fructan accumulation was abolished but leaves maintained > or = 80% of the extractable polymerase. Extractable polymerase rates cannot explain the rates of fructan accumulation observed in vivo, if the process is vacuolar. It was shown that the results were inconsistent with a vacuolar site for fructan synthesis. Six inhibitors of endomembrane function were shown to abolish fructan synthesis in vivo.     

Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., `Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.     

Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation.

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.     

Light-induced Development of Polyribosomes and the Induction of Nitrate Reductase in Corn Leaves

Nitrate reductase activity was induced by nitrate in green corn (Zea mays) leaves in either light or darkness. The induction process required oxygen in darkness but not in light. A light treatment was required before the enzyme could be induced in etiolated leaves.     

Dependency of Nitrate Reduction on Soluble Carbohydrates in Primary Leaves of Barley under Aerobic Conditions

Nitrate reduction was studied as a function of carbohydrate concentration in detached primary leaves of barley (Hordeum vulgare L. cv Numar) seedlings under aerobic conditions in light and darkness. Seedlings were grown either in continuous light for 8 days or under a regimen of 16-hour light and 8-hour dark for 8 to 15 days. Leaves of 8-day-old seedlings grown in continuous light accumulated 4 times more carbohydrates than leaves of plants grown under a light and dark regimen. When detached leaves from these seedlings were supplied with NO₃⁻ in darkness, those with the higher levels of carbohydrates reduced a greater proportion of the NO₃⁻ that was taken up. In darkness, added glucose increased the percentage of NO₃⁻ reduced up to 2.6-fold depending on the endogenous carbohydrate status of the leaves. Both NO₃⁻ reduction and carbohydrate content of the leaves increased with age. Fructose and sucrose also increased NO₃⁻ reduction in darkness to the same extent as glucose. Krebs cycle intermediates, citrate and succinate, did not increase NO₃⁻ reduction, whereas malate slightly stimulated it in darkness.

In light, 73 to 90% of the NO₃⁻ taken up was reduced by the detached leaves; therefore, an exogenous supply of glucose had little additional effect on NO₃⁻ reduction. The results indicate that in darkness the rate of NO₃⁻ reduction in primary leaves of barley depends upon the availability of carbohydrates.

     

Light Scattering as an Indicator of the Energy State in Leaves of the Crassulacean Acid Metabolism Plant Kalanchoë pinnata

Both transmittance changes in a weak beam of green light (light scattering) and the slow decay of chlorophyll a fluorescence were used as indicators of the energy state of leaves of a Crassulacean acid metabolism plant, Kalanchoë pinnata, at frequent intervals during 12-hour light/12-hour dark cycles. To induce light scattering and fluorescence changes, leaves were exposed to red light for 6 minutes. When measurements were made during the light period, the leaves were kept in darkness for 6 minutes before illumination. In the middle of the light period, when malic acid decarboxylation was very active and stomatal conductance was low, light scattering changes were small and indicated that the energy state of leaves was low. This result was supported by determination of adenylate levels. Light scattering and ATP/ADP ratios increased during the late light period when the tissue was deacidified. Illumination produced maximum light scattering changes between the 2nd and 5th hour of the dark period, when rates of dark CO₂ fixation were highest. Light scattering and fluorescence measurements taken from leaves, which were illuminated with red or far-red light in the presence or absence of O₂ showed that, in addition to linear electron transport, K. pinnata has the potential for both cyclic and pseudocyclic electron transport. The results are relevant with regard to the high ATP demand during Crassulacean acid metabolism.     

Induction of a Carbon-Starvation-Related Proteolysis in Whole Maize Plants Submitted to Light/Dark Cycles and to Extended Darkness

Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283–292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.     

Light/Dark profiles of sucrose phosphate synthase, sucrose synthase, and Acid invertase in leaves of sugar beets

The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves.     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.