Energized Uptake of Ascorbate and Dehydroascorbate From the Apoplast of Intact Leaves in Relation to Apoplastic Steady State Concentrations of Ascorbate

Abstract: Transport of ascorbate (AA) and dehydroascorbate (DHA) through the petiole into detached leaves of Lepidium sativum and other plant species via the transpiration stream, and energized uptake into leaf tissue, were measured indirectly by recording changes in membrane potential and apoplastic pH simultaneously with substrate‐stimulated respiration and transpiratory water loss. When 25 mM AA or DHA was fed to the leaves, steady state respiration at 25 °C was transiently increased by more than 50 % with AA and 70 % with DHA. Stimulation of respiration was accompanied by a transient breakdown of membrane potential followed by alkalinization of the leaf apoplast suggesting energized uptake at the expense of the transmembrane proton motive force. The average CO₂/AA ratio calculated from stimulated respiration during ascorbate uptake was 0.76 ± 0.26 (n = 17). The corresponding ratio for DHA was 1.38 ± 0.28 (n = 11). Far lower CO₂/substrate ratios were observed when NaCl or KCl were fed to leaves. The differences indicate either partial metabolism of AA and DHA in addition to energized transport, or less likely, higher energy requirement for transport of AA and DHA than for the inorganic salts. Maximum rates of energized AA transport into leaf tissue (deduced from maxima of extra respiration and calculated on the basis of CO₂/AA = 0.76) were close to 650 nmol m^‐2 leaf area s^‐1, i.e. far higher than most previously reported rates of transport. When the apoplastic concentration of AA was decreased below steady state levels during infiltration/centrifugation experiments, AA was released from leaf cells into the apoplast. This suggests that AA oxidation to DHA in the apoplast (as occurs during extracellular ozone detoxification) triggers energized transport of the DHA into the symplast and simultaneously AA release from the symplast into the apoplast, perhaps together with protons in a reversal of the energized uptake process.     

Energized Uptake of Sugars from the Apoplast of Leaves: A Study of Some Plants Possessing Different Minor Vein Anatomy

Solutions of sucrose, glucose, raffinose, and stachyose were fed via the petiole to detached leaves of plant species known to transfer sugars during photosynthesis into the phloem using either the apoplastic or the symplastic pathway of phloem loading. Symplastic phloem loaders, which translocate raffinose-type oligosaccharides and sucrose in the phloem, and apoplastic plants, translocating exclusively sucrose, were selected for this study. As the sugars arrived with the transpiration stream in the leaf blade within little more than a minute, dark respiration increased. Almost simultaneously, fluorescence of a potential-indicating dye, which had been infiltrated into the leaves, indicated membrane depolarization. Another fluorescent dye used to record the apoplastic pH revealed apoplastic alkalinization that occurred with a slight lag phase after respiration and membrane depolarization responses. Occasionally, alkalinization was preceded by transient apoplastic acidification. Whereas membrane depolarization and apoplastic acidification are interpreted as initial responses of the proton motive force across the plasma membrane to the advent of sugars in the leaf apoplast, the following apoplastic alkalinization showed that sugars were taken up from the apoplast into the symplast in cotransport with protons. This was true not only for glucose and sucrose, but also for raffinose and stachyose. Similar observations were made for sugar uptake not only in leaves of plants known to export sugars by symplastic phloem loading but also of plants using the apoplastic pathway. Increased respiration during sugar uptake revealed tight coupling between respiratory ATP production and ATP consumption by proton-translocating ATPase of the plasma membrane, which exports protons into the apoplast, thereby compensating for the proton loss in the apoplast when protons are transported together with sugars into the symplast. The extent of stimulation of respiration by sugars indicated that sugar uptake was not limited to phloem tissue. Ratios of the extra CO₂ released during sugar uptake to the amounts of sugars taken up were variable, but lowest values were lower than 0.2. When a ratio of 0.2 is taken as a basis to calculate rates of sugar uptake from observed maxima of sugar-dependent increases in respiration, rates of sugar uptake approached 350 nmol/(m² leaf surface s). Sugar uptake rates were half-saturated at sugar concentrations in the feeding solutions of about 10–25 mM indicating a low in vivo affinity of sugar uptake systems for sugars.     

Energy-dependent Solute Uptake Into the Symplast of Leaves: ATP/KCl, ATP/Sucrose, ATP/D-serine and H+/ATP Stoichiometries of Transmembrane Transport

Abstract: KCl, sucrose, D-serine and some other solutes were fed through the petiole to leaflets of Solanum tuberosum and uptake into the symplast was monitored. Solute transport was accompanied by changes in membrane potential, apoplastic pH and respiration. After termination of solute feeding, membrane potential, apoplastic pH and respiration returned to initial steady state values. From transpiration, solute uptake was calculated and compared to ATP production during stimulated respiration, assuming an ATP/CO₂ ratio of 5. On this basis, calculated ATP/KCl ratios of energized transport approached 0.5. Similar ATP/solute ratios were observed with sucrose, mannitol, methylglucose and D-serine. With glucose, many ratios were somewhat above 0.5, possibly because of some metabolization of imported glucose. We conclude that solute uptake is energized by the proton motive force across the plasma membrane. Low ATP/substrate ratios suggest that the H⁺/ATP ratio of proton export by the plasma membrane ATPase is not 1 as presently assumed but 2 in potato leaves, and that the contribution of the alternative cyanide-resistant oxidase to leaf respiration is small, if not negligible, in the dark.     

Apoplastic Transport of 14C-Photosynthates Measured under Drought and Nitrogen Supply

Using water infiltration of the plant and individual shoots with the subsequent intercellular liquid extraction by the pressure chamber, dynamics of the movement ¹⁴C-photosynthates from cell to apoplast, and ¹⁴C distribution among photosynthetic products in mesophyll cells and apoplast were studied. The relative quantity of ¹⁴C-photosynthetes in leaf apoplast depended on growing conditions; drought increased, and nitrate supply decreased it. When the middle leaves absorbed ¹⁴CO₂, photosynthates moving down in stem phloem appeared in intercellular space, where they were transported up by transpiration stream. ¹⁴C-photosynthates entering to the apex and young leaves were utilized a accumulated, and photosynthates transported to the mature leaves were reloaded into the phloem and reexported. Thus, photosynthates circulated through the plant and were redistributed to the plant organs according to their transpiration. In leaf apoplast photosynthetic sucrose was partly hydrolyzed to glucose and fructose. This increased under high nitrogen supply. The result indicate that apoplast sucrose hydrolysis is the basic cause of the reduction of photosynthate flux from leaves when the nitrate concentration in soil increases.     

Water Relations,CO2 Exchange,Water-use Efficiency and Growth of Welwitschia mirabilis Hook. fil. in three Contrasting Habitats of the Namib Desert1

CO₂ exchange, transpiration and leaf water potential of Welwitschia mirabilis were measured in three contrasting habitats of the Namib desert. From these measurements stomatal conductance, internal CO₂concentration and WUE were calculated. In two of the three habitats photosynthetic CO₂ uptake decreased and transpiration increased with increasing leaf age while in the third habitat CO₂ uptake increased and transpiration decreased with leaf age. Except for the stomata of young leaf sections in this habitat, stomata closed with increasing δw leading to a pronounced midday depression of CO₂ uptake. The high stomatal limitation of photosynthetic CO₂ uptake of glasshouse-grown plants was verified in the natural habitat. Photosynthetic CO₂ uptake saturated between 800 and 1300 μmol photons m^?2 s^?1depending on leaf age and habitat. CO₂ uptake had a broad temperature optimum declining significantly beyond 32 °C. Predawn leaf water potential reflected water availability and atmospheric conditions in the three habitats and ranged from ? 2.5 to ? 6.2 MPa. There was a pronounced diurnal course of leaf water potential in all habitats. During the day a gradient in water potential developed along the leaf axis with the lowest potential at the leaf's tip. With respect to whole plant balances of CO₂ exchange and transpiration, there were marked differences between Welwitschias in the three habitats. Despite a negative CO₂ balance over a period of five months, leaves in the driest habitat grew constantly at the expense of carbon reserves in the plant. Only at the wettest site did carbon gain exceed carbon demand for growth. The WUE of whole plants was insignificant in all habitats. The results were as contrasting as the habitats and plants and did not allow generalisations about adaptational features of Welwitschia mirabilis.     

Progressive Soil Drought Promotes the Export Function in the Birch (Betula platyphylla) Leaf

A greenhouse experiment, which imitated a short (4-day-long) and progressive (3-week-long) soil drought, was employed to assess, with an IR gas analyzer, leaf CO₂ exchange rate (CER) in intact one-year-old seedlings of Betula platyphylla as related to the flux of photosynthetically active radiation ranging from 0 to 1400 E/(m² s). The registered indices comprised leaf temperature, leaf transpiration conductivity, and the average daily increment of the leaf area. Within a week period following the transition from the short severe soil drought (20% H₂O per soil weight) to the conditions of sufficient water content (35–40%), the plants completely regained the initial leaf CER. Under the progressive soil drought, leaf CER was reduced by 30–35%, as compared to the conditions of sufficient water content, evidently due to a 3.7-fold drop in the transpiration conductivity as compared to the control plants. The apparent constant of Rubisco carboxylation and leaf respiration in the light were not affected by the drought period. The rate of leaf growth under the progressive drought was reduced by 64% as compared to the sufficient moisture conditions. Thus, under the progressive drought, the diminished stomatal conductivity reduced CO₂ concentration inside the leaf and lowered carbon photosynthetic assimilation. Meanwhile, the leaf source activity considerably increased in spite of diminished photosynthesis.     

Closure of Stomata in Water-Stressed Pine Needles Results from the Decreased Water Potential of the Mesophyll Apoplast in the Substomatal Cavity

Pine (Pinus sylvestris L.) seedlings grown under controlled conditions were subjected to water deficit (external water potentials ranging from–0.15 to–1.5 MPa) by adding polyethylene glycol 6000 (PEG) to the nutrient solution. Following this treatment, the dry weights of plant shoots and roots, as well as the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m), nonphotochemical quenching (NPQ) of chlorophyll excitations, photosynthetic CO₂/H₂O exchange, dark respiration of needles, and water potential of mesophyll apoplast in the substomatal cavity of pine needles, were measured. The imposed water deficit was followed by the inhibition of seedling growth, suppression of photosynthesis and transpiration, and by the decreased content of photosynthetic pigments. It is shown for the first time that the closure of stomata in the needles of water-stressed pine seedlings falls into the physiological reaction norm and is caused by the reduction of water potential in the mesophyll apoplast of the substomatal cavity.     

Involvement of respiratory processes in the transient knockout of net CO2 uptake in Mimosa pudica upon heat stimulation

Leaf photosynthesis of the sensitive plant Mimosa pudica displays a transient knockout in response to electrical signals induced by heat stimulation. This study aims at clarifying the underlying mechanisms, in particular, the involvement of respiration. To this end, leaf gas exchange and light reactions of photosynthesis were assessed under atmospheric conditions largely eliminating photorespiration by either elevated atmospheric CO₂ or lowered O₂ concentration (i.e. 2000 μmol mol^?1 or 1%, respectively). In addition, leaf gas exchange was studied in the absence of light. Under darkness, heat stimulation caused a transient increase of respiratory CO₂ release simultaneously with stomatal opening, hence reflecting direct involvement of respiratory stimulation in the drop of the net CO₂ uptake rate. However, persistence of the transient decline in net CO₂ uptake rate under illumination and elevated CO₂ or 1% O₂ makes it unlikely that photorespiration is the metabolic origin of the respiratory CO₂ release. In conclusion, the transient knockout of net CO₂ uptake is at least partially attributed to an increased CO₂ release through mitochondrial respiration as stimulated by electrical signals. Putative CO₂ limitation of Rubisco due to decreased activity of carbonic anhydrase was ruled out as the photosynthesis effect was not prevented by elevated CO₂.     

Evidence for Phloem loading from the apoplast: chemical modification of membrane sulfhydryl groups

The water-soluble, sulfhydryl-specific, chemical modifier p-chloromercuribenzenesulfonic acid reversibly inhibited the accumulation of exogenously supplied ¹⁴C-sucrose into leaf discs of Beta vulgaris. P-Chloromercuribenzenesulfonic acid treatment did not inhibit photosynthesis or respiration or induce membrane leakage to sucrose, indicating that the site of inhibition was the plasmalemma. The active loading of sucrose and ¹⁴CO₂-derived assimilates into the phloem and their translocation from the source leaf were inhibited by the nonpermeant modifier. Several nonpermeant sulfhydryl group modifiers also inhibited sucrose accumulation into leaf discs while two amino-reactive reagents had no effect. The results indicate that sugars are actively accumulated into the phloem from the apoplast and that membrane sulfhydryl groups may be involved.     

Atmospheric CO2 concentration does not directly affect leaf respiration in bean or poplar

It is a matter of debate if there is a direct (short‐term) effect of elevated atmospheric CO₂ concentration (C_a) on plant respiration in the dark. When C_a doubles, some authors found no (or only minor) changes in dark respiration, whereas most studies suggest a respiratory inhibition of 15–20%. The present study shows that the measurement artefacts – particularly leaks between leaf chamber gaskets and leaf surface, CO₂ memory and leakage effects of gas exchange systems as well as the water vapour (‘water dilution’) effect on DCO₂ measurement caused by transpiration – may result in larger errors than generally discussed. A gas exchange system that was used in three different ways – as a closed system in which C_a increased continuously from 200 to 4200 mmol (CO₂) mol^‐1 (air) due to respiration of the enclosed leaf; as an intermittently closed system that was repeatedly closed and opened during C_a periods of either 350 or 2000 mmol mol^‐1, and as an open system in which C_a varied between 350 and 2000 mmol mol^‐1– is described. In control experiments (with an empty leaf chamber), the respective system characteristics were evaluated carefully. When all relevant system parameters were taken into account, no effects of short‐term changes in CO₂ on dark CO₂ efflux of bean and poplar leaves were found, even when C_a increased to 4200 mmol mol^‐1. It is concluded that the leaf respiration of bean and poplar is not directly inhibited by elevated atmospheric CO₂.     

Direct and indirect effects of elevated CO2 on leaf respiration in a forest ecosystem

We measured the short‐term direct and long‐term indirect effects of elevated CO₂ on leaf dark respiration of loblolly pine (Pinus taeda) and sweetgum (Liquidambar styraciflua) in an intact forest ecosystem. Trees were exposed to ambient or ambient + 200 µmol mol^?1 atmospheric CO₂ using free‐air carbon dioxide enrichment (FACE) technology. After correcting for measurement artefacts, a short‐term 200 µmol mol^?1 increase in CO₂ reduced leaf respiration by 7–14% for sweetgum and had essentially no effect on loblolly pine. This direct suppression of respiration was independent of the CO₂ concentration under which the trees were grown. Growth under elevated CO₂ did not appear to have any long‐term indirect effects on leaf maintenance respiration rates or the response of respiration to changes in temperature (Q₁₀, R₀). Also, we found no relationship between mass‐based respiration rates and leaf total nitrogen concentrations. Leaf construction costs were unaffected by growth CO₂ concentration, although leaf construction respiration decreased at elevated CO₂ in both species for leaves at the top of the canopy. We conclude that elevated CO₂ has little effect on leaf tissue respiration, and that the influence of elevated CO₂ on plant respiratory carbon flux is primarily through increased biomass.     

Effects of long-term elevated atmospheric carbon dioxide onLolium perenne andTrifolium repens,using a simple photosynthesis model

Changes in gross canopy photosynthetic rate (PGc), produced by long-term exposure to an elevated atmospheric CO₂ level (626±50 µmol mol^-1), were modelled forLolium perenne L. cv. Vigor andTrifolium repens L. cv. Blanca, using a simple photosynthesis model, based on biochemical and physiological information (leaf gross CO₂ uptake in saturating light, P_max, and leaf quantum efficiency, ) and structural vegetation parameters (leaf area index, LAI, canopy extinction coefficient, k, leaf transmission, M). Correction of PGc for leaf respiration allowed comparison with previously measured canopy net CO₂ exchange rates, with the average divergence from model prediction amounting to about 6%. Sensitivity analysis showed that for a three-week old canopy, the PGc increase in high CO₂ could be attributed largely to changes in P_max and , while differences in canopy architecture were no longer important for the PGc-stimulation (which they were in the early growth stages). As a consequence of this increasing LAI with canopy age, the gain of daytime CO₂ uptake is progressively eroded by the increasing burden of canopy respiration in high-CO₂ grownLolium perenne. Modelling canopy photosynthesis in different regrowth stages after cutting (one week, two weeks,...), revealed that the difference in a 24-h CO₂ balance between the ambient and the high CO₂ treatment is reduced with regrowth time and completely disappears after 6 weeks.Abbreviations C350 ambient CO₂ treatment - C625 high CO₂ treatment - k canopy extinction coefficient - LAI leaf area index - LAI_max fitted LAI-maximum - M leaf transmission - NCER net CO₂ exchange rate - PGc gross canopy photosynthetic rate - Q photosynthetic photon flux density - Q₀ photosynthetic photon flux density at the top of the canopy - RDc canopy dark respiration rate - RDl leaf dark respiration rate - t regrowth time after cutting - T air temperature - leaf quantum efficiency - _LAI rate of initial LAI-increase with time     

Glucose-dependent respiration in suspensions of rabbit cortical tubules

Summary The effects of glucose on cellular respiration were examined in suspensions of rabbit cortical tubules. When glucose was removed from the bathing fluid, oxygen consumption (QO₂) decreased from 18.6±0.8 to 15.7±0.5 nmol O₂/mg protein·min (P<0.01). The transported but nonmetabolized analogue of glucose, -methyl-d-glucoside (MG), was found to support QO₂ to the same extent as glucose. These observations were also evident in the presence of butyrate, a readily oxidized substrate of the renal cortex. Additional studies with nystatin and ouabain indicated that glucose-related changes in QO₂ were the result of changes in Na, K-ATPase associated respiration. The effect of glucose was localized to the luminal membrane since phlorizin (10^–5 m), a specific inhibitor of liminalk glucose-sodium cotransport, also significantly reduced QO₂ by 10±1%. Phlorizin inhibition of QO₂ was also evident in the presence of MG but was abolished when glucose was removed from the bathing medium. Finally, measurement of NADH fluorescence showed that addition of glucose (5mm) to a tubule suspension causes an oxidation of NAD. These data are all consistent with glucose acting to increase respiration by stimulating sodium entry at the luminal membrane (via glucose-sodium cotransport) followed by increased sodium pump activity and its associated increase in mitochondrial respiration.     

Growth and maintenance components of leaf respiration of cotton grown in elevated carbon dioxide partial pressure

Elevated atmospheric carbon dioxide partial pressures have been shown to have variable direct and indirect effects on plant respiration rates. In this study, growth, leaf respiration, and leaf nitrogen and carbohydrate partitioning were measured in Gossypium hirsutum L. grown in 35 and 65 Pa CO₂ for 30d. Growth and maintenance coefficients of leaf respiration were estimated using gas exchange techniques both at night and during the day. Elevated CO₂ stimulated biomass production (107%) and net photo-synthetic rates (35–50%). Total day-time respiration (R_d) was not significantly affected by growth CO₂ partial pressure. However, night respiration (R_n) of leaves grown in 65 Pa CO₂ was significantly greater than that of plants grown in 35 Pa CO₂. Correlation of R_d and R_n with leaf expansion rates indicated that plants in both CO₂ treatments had equivalent growth respiration coefficients but maintenance respiration was significantly greater in elevated CO₂. Increased maintenance coefficients in elevated CO₂ appeared to be related to increased starch accumulation rather than to changes in leaf nitrogen.     

Effects of elevated atmospheric carbon dioxide on gas exchange and growth of white clover

Effects of rising atmospheric CO₂ concentrations on gas exchange, growth and productivity were investigated on an important grassland species, Trifolium repens L. cv. Blanca. Pure stands of this species were cultivated over an entire growing season in small acrylic greenhouses with an artificial atmosphere of ±367 or ±620 ppm CO₂, respectively. Effects on growth and development were examined in a functional growth analysis, while consequences for gas exchange were determined by photosynthesis and transpiration measurements on canopy level. The stands were regularly clipped for production assessment. Canopies grown at high CO₂ levels showed an average increase in productivity of almost 75%. Growth analysis indicated development of a larger foliage area as the major cause, particularly in the first days of regrowth after cutting. The growth advantage that began in this stage was maintained or bettered during the following weeks. The difference between gas exchange measurements expressed per unit leaf area and per unit ground area suggested that changes in net photosynthesis and respiration did not contribute to the increase in total yield. Transpiration declined under high CO₂ if expressed on a leaf area basis but total canopy transpiration was at least as large as in ambient CO₂ due to the larger leaf area. Water-use efficiency calculations on the summer data indicated a 35% improvement with a doubling of CO₂ concentration.     

Photosynthesis of Ivy Leaves (Hedera helix) after Heat Stress I. CO2-Gas Exchange and Diffusion Resistances

For an analysis of the inhibition of the photosynthetic CO₂-uptake after heat stress attached leaves of Hedera helix L. were heat-stressed for 30 min at various temperatures. Subsequently their photosynthetic CO₂-uptake, transpiration, respiration in light and darkness, and CO₂-compensation concentration were measured under optimal conditions. After heat stress the stomatal resistance increased only corresponding to the raised CO₂-concentration inside the leaves (due to the reduced CO₂-uptake). The physical resistance between the mesophyll cell walls and the chloroplasts remained unchanged after heat stress. A non-stomatal inhibition of the CO₂-uptake is indicated by a strong increase of the CO₂-compensation concentration after heat stress. This is hardly due to a stimulation of the respiration in light, as the CO₂-evolution into CO₂-free air in light was even reduced. Therefore, it must be concluded that the photosynthetic process itself is impaired after heat stress.     

Three‐dimensional microscale modelling of CO2 transport and light propagation in tomato leaves enlightens photosynthesis

We present a combined three‐dimensional (3‐D) model of light propagation, CO₂ diffusion and photosynthesis in tomato (Solanum lycopersicum L.) leaves. The model incorporates a geometrical representation of the actual leaf microstructure that we obtained with synchrotron radiation X‐ray laminography, and was evaluated using measurements of gas exchange and leaf optical properties. The combination of the 3‐D microstructure of leaf tissue and chloroplast movement induced by changes in light intensity affects the simulated CO₂ transport within the leaf. The model predicts extensive reassimilation of CO₂ produced by respiration and photorespiration. Simulations also suggest that carbonic anhydrase could enhance photosynthesis at low CO₂ levels but had little impact on photosynthesis at high CO₂ levels. The model confirms that scaling of photosynthetic capacity with absorbed light would improve efficiency of CO₂ fixation in the leaf, especially at low light intensity.     

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.