Compartmentation of Assimilate Fluxes in Leaves

Abstract: Sugar levels in the apoplast of assimilate exporting leaves were studied in two groups of plant species with contrasting structures of companion cells in minor veins. These species are termed either "symplastic" (with intermediary cells) or "apoplastic" (with transfer or ordinary cells). Sugars were measured in intercellular washing fluid after extracting the apoplast by an infiltration-centrifugation technique. During the course of a day, sugar contents in the apoplast were, in general, lower in species with intermediary cells than in species with transfer or ordinary cells. In "symplastic" species, apoplastic sucrose concentrations were between 0.3 and 1 mM. In "apoplastic" species with transfer cells, they ranged between 2 and 6 mM. Apoplastic hexose contents were between 0.3 and 1 mM irrespective of presumed transport mode. "Symplastic" and "apoplastic" plants differed markedly in their response to a'translocation block. In "symplastic" plants, inhibition of assimilate export left apoplastic concentrations of sucrose and hexoses unchanged, whereas in "apoplastic" plants sugar levels increased, the maximal increase being observed with sucrose. In these plants, concentrations of sucrose were two to six times higher in the apoplast under export inhibition than in control leaves. The data suggest a different role of the leaf apoplast in the compartmentation and export of assimilates in the two plant groups under study.     

Impacts of ozone on Plantago major: apoplastic and symplastic antioxidant status

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O₃) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 70 nmol mol⁻¹ O₃ for 7 h d⁻¹ over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c . 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O₃ resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain 'unspecific' peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O₃ fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O₃ with plant age. By contrast, changes in 'unspecific' peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O₃ resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O₃.     

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.     

Guard cell apoplastic photosynthate accumulation corresponds to a phloem-loading mechanism

Apoplastic phloem loaders have an apoplastic step in the movement of the translocated sugar, prototypically sucrose, from the mesophyll to the companion cell-sieve tube element complex. In these plants, leaf apoplastic sucrose becomes concentrated in the guard cell wall to nominally 150 mM by transpiration during the photoperiod. This concentration of external sucrose is sufficient to diminish stomatal aperture size in an isolated system and to regulate expression of certain genes. In contrast to apoplastic phloem loaders and at the other extreme, strict symplastic phloem loaders lack an apoplastic step in phloem loading and mostly transport raffinose family oligosaccharides (RFOs), which are at low concentrations in the leaf apoplast. Here, the effects of the phloem-loading mechanism and associated phenomena on the immediate environment of guard cells are reported. As a first step, carbohydrate analyses of phloem exudates confirmed basil (Ocimum basilicum L. cv. Minimum) as a symplastic phloem-loading species. Then, aspects of stomatal physiology of basil were characterized to establish this plant as a symplastic phloem-loading model species for guard cell research. [(14)C]Mannitol fed via the cut petiole accumulated around guard cells, indicating a continuous leaf apoplast. The (RFO+sucrose+hexoses) concentrations in the leaf apoplast were low, <0.3 mM. Neither RFOs (<10 mM), sucrose, nor hexoses (all, P >0.2) were detectable in the guard cell wall. Thus, differences in phloem-loading mechanisms predict differences in the in planta regulatory environment of guard cells.     

Cadmium uptake by roots: Contribution of apoplast and of high- and low-affinity membrane transport systems

The aim was to measure the respective contributions of apoplast and symplast to the Cd root uptake and to explain the linear component of the symplastic absorption. Two plants were used, maize (Zea mays L.) and two ecotypes of alpine pennycress (Noccaea caerulescens (J. Presl & C. Presl) F.K. Mey.), with contrasted abilities to accumulate Cd. Their roots were exposed to labelled Cd solutions of increasing concentrations. Root Cd was physico-chemically fractioned to obtain the exchanged apoplastic, non-exchanged apoplastic and symplastic pools. For both species, the proportion of Cd retained by the cell walls increased with Cd concentration in the exposure solution (ranging from 0.05 to 50 μmol L^?1), from approximately 30% to 90% of the total root Cd. This was modeled using Freundlich isotherms. The non-exchanged apoplastic Cd was negligible at the highest exposure concentrations, but reached almost 30% of the total root uptake at the lowest ones. The symplastic influx in roots of both species fitted a Michaelis-Menten function associated with a linear one. The linear component of the symplastic influx could reflect absorption through a low-affinity transport system (LATS). The strong adsorption of Cd on root apoplast might act as a driving force to extract the metal from the soil, compete with the symplastic absorption and contribute to the amount of element taken up by the plant, at least in its roots.     

Apoplastic ascorbate metabolism and its role in the regulation of cell signalling

The apoplast has crucial functions in plant biology. It comprises all the compartments beyond the plasmalemma, including the cell wall. As the reservoir of information on the biotic and abiotic environment surrounding the cell and a major conduit of information between cells, the apoplast has functions in stress perception and the subsequent appropriate control of growth and defence. The oxidative burst phenomenon, caused by environmental challenges and pathogen attack in particular, oxidises the apoplast. Ascorbic acid (AA), the major and probably the only antioxidant buffer in the apoplast, becomes oxidised in these conditions. The apoplastic enzyme ascorbate oxidase (AO) also regulates the reduction/oxidation (redox) state of the apoplastic ascorbate pool. We propose that a key function of the oxidative burst and of AO is to modify the apoplastic redox state in such a way as to modify receptor activity and signal transduction to regulate defence and growth.     

Does high bicarbonate supply to roots change availability of iron in the leaf apoplast?

The role of the leaf apoplast in iron (Fe) uptake into the leaf symplast is insufficiently understood, particularly in relation to the supposed inactivation of Fe in leaves caused by elevated bicarbonate in calcareous soils. It has been supposed that high bicarbonate supply to roots increases the pH of the leaf apoplast which decreases the physiological availability of Fe in leaf tissues. The study reported here has been carried out with sunflower plants grown in nutrient solution and with grapevine plants grown on calcareous soil under field conditions. The data obtained clearly show that the pH of the leaf apoplastic fluid was not affected by high bicarbonate supply in the root medium (nutrient solution and field experiments). The concentrations of total, symplastic and apoplastic Fe were decreased in chlorotic leaves of both sunflower (nutrient solution experiment) and grapevine plants in which leaf expansion was slightly inhibited (field experiment). However, in grapevine showing severe inhibition of leaf growth, total Fe concentration in chlorotic leaves was the same or even higher than in green ones, indicative to the so-called `chlorosis paradox'. The findings do not support the hypothesis of Fe inactivation in the leaf apoplast as the cause of Fe deficiency chlorosis since no increase was found in the relative amount of apoplastic Fe (% of total leaf Fe) either in the leaves of sunflower or grapevine plants. It is concluded that high bicarbonate concentration in the soil solution does not decrease Fe availability in the leaf apoplast.     

OsTRXh1 regulates the redox state of the apoplast and influences stress responses in rice

The plant cell apoplast is the compartment beyond the cell plasmalemma, including the cell wall and intercellular space. Many environmental elements can trigger reactive oxygen species (ROS) burst at the plasma membrane which then alters the redox state of the apoplast. Recently, h-type thioredoxin (Trx), OsTRXh1, was identified to be involved in apoplastic redox state regulation in rice. OsTRXh1 is conserved redox-active Trx and can be secreted into the extracellular regions. Through transgenic rice plant, we found that OsTRXh1 regulated ROS accumulation in apoplast and influenced plant development and stress responses. This provides new insights into apoplastic redox state regulation pathway and expands our understanding of h-type Trxs function.     

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.     

Phloem loading. A reevaluation of the relationship between plasmodesmatal frequencies and loading strategies

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species. On this basis, two pathways of phloem loading have been proposed: symplastic where frequencies are high, and apoplastic where they are low. However, putative symplastic-loading species fall into at least two categories. In one, the plants translocate raffinose-family oligosaccharides (RFOs). In the other, the primary sugar in the phloem sap is sucrose (Suc). While a thermodynamically feasible mechanism of symplastic loading has been postulated for species that transport RFOs, no such mechanism is known for Suc transporters. We used p-chloromercuribenzenesulfonic acid inhibition of apoplastic loading to distinguish between the two pathways in three species that have abundant minor vein plasmodesmata and are therefore putative symplastic loaders. Clethra barbinervis and Liquidambar styraciflua transport Suc, while Catalpa speciosa transports RFOs. The results indicate that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, C. barbinervis and L. styraciflua load from the apoplast. C. speciosa, being an RFO transporter, loads from the symplast, as expected. Data from these three species, and from the literature, also indicate that plants with abundant plasmodesmata in the minor vein phloem have abundant plasmodesmata between mesophyll cells. Thus, plasmodesmatal frequencies in the minor veins may be a reflection of overall frequencies in the lamina and may have limited relevance to phloem loading. We suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.     

In comparison with nitrate nutrition,ammonium nutrition increases growth of the frostbite1 Arabidopsis mutant

Ammonium nutrition inhibits the growth of many plant species, including Arabidopsis thaliana. The toxicity of ammonium is associated with changes in the cellular redox state. The cellular oxidant/antioxidant balance is controlled by mitochondrial electron transport chain. In this study, we analysed the redox metabolism of frostbite1 (fro1) plants, which lack mitochondrial respiratory chain complex I. Surprisingly, the growth of fro1 plants increased under ammonium nutrition. Ammonium nutrition increased the reduction level of pyridine nucleotides in the leaves of wild‐type plants, but not in the leaves of fro1 mutant plants. The observed higher activities of type II NADH dehydrogenases and cytochrome c oxidase in the mitochondrial electron transport chain may improve the energy metabolism of fro1 plants grown on ammonium. Additionally, the observed changes in reactive oxygen species (ROS) metabolism in the apoplast may be important for determining the growth of fro1 under ammonium nutrition. Moreover, bioinformatic analyses showed that the gene expression changes in fro1 plants significantly overlap with the changes previously observed in plants with a modified apoplastic pH. Overall, the results suggest a pronounced connection between the mitochondrial redox system and the apoplastic pH and ROS levels, which may modify cell wall plasticity and influence growth.     

Ion distribution measured by electron probe X-ray microanalysis in apoplastic and symplastic pathways in root cells in sunflower plants grown in saline medium

Little is known about how salinity affects ions distribution in root apoplast and symplast. Using x-ray microanalysis, ions distribution and the relative contribution of apoplastic and symplastic pathways for delivery of ions to root xylem were studied in sunflower plants exposed to moderate salinity (EC=6). Cortical cells provided a considerably extended Na+ and Cl- storage facility. Their contents are greater in cytoplasm (root symplast) as compared to those in intercellular spaces (root apoplast). Hence, in this level of salinity, salt damage in sunflower is not dehydration due to extracellular accumulation of sodium and chloride ions, as suggested in the Oertli hypothesis. On the other hand, reduction in calcium content due to salinity in intercellular space is less than reduction in the cytoplasm of cortical cells. It seems that sodium inhibits the radial movement of calcium in symplastic pathway more than in the apoplastic pathway. The cell wall seems to have an important role in providing calcium for the apoplastic pathway. Redistribution of calcium from the cell wall to intercellular space is because of its tendency towards xylem through the apoplastic pathway. This might be a strategy to enhance loading of calcium to xylem elements and to reduce calcium deficiency in young leaves under salinity. This phenomenon may be able to increase salt tolerance in sunflower plants. Supplemental calcium has been found to be effective in reducing radial transport of Na+ across the root cells and their loading into the xylem, but not sodium absorption. Supplemental calcium enhanced Ca2+ uptake and influx into roots and transport to stele.     

Transport and action of ascorbate at the plant plasma membrane

The plasmalemma is both a bridge and a barrier between the cytoplasm and the outside world. It is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. In plants, ascorbate is an essential part of this dialogue. The concentration and ratio of reduced to oxidized ascorbate in the apoplast, for example, possibly modulates cell division and growth. The leaf apoplast contains millimolar amounts of ascorbate that protect the plasmalemma against oxidative damage. The apoplastic ascorbate-dehydroascorbate redox couple is linked to the cytoplasmic ascorbate-dehydroascorbate redox couple by specific transporters for either or both metabolites. Although evidence about the mechanisms driving ascorbate or dehydroascorbate transport remains inconclusive, these carrier proteins potentially regulate the level and redox status of ascorbate in the apoplast. The redox coupling between compartments facilitated by these transport systems allows coordinated control of key physiological responses to environmental cues.     

Rapid apoplastic pH measurement in Arabidopsis leaves using a fluorescent dye

In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant.     

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.     

Effect of nitrate infusion into the shoot apoplast on photosynthesis and assimilate transport in symplastic and apoplastic plants

The shoots of fireweed (Chamerion angustifolium (L.) Holub) and common flax (Linum usitatissimum L.) were infused with 50 mM KNO₃ solution to compare the influence of nitrate on photosynthesis and assimilate export from leaves in plants with the symplastic and apoplastic phloem loading, respectively. The infusion of nitrate in the shoots of both plant species lowered ¹⁴CO₂ fixation and enhanced the assimilate transport in the upward direction. Irrespective of the phloem loading type, the incorporation of ¹⁴C into sucrose decreased in nitrate-treated seedlings exposed to assimilation for short (3 min) periods. However, when shoots were sampled 3 h after ¹⁴CO₂ fixation, the content of ¹⁴C-labeled sucrose was higher in treated plants than in control seedlings infused with water. In fireweed, in contrast to flax, a similar temporal pattern was also characteristic for ¹⁴C incorporation into oligosaccharides. Within 3 h after nitrate infusion into the fireweed apoplast, the mitochondria and the cell vacuolar system underwent ultrastructural changes indicative of the increase in cytosolic osmotic pressure. At the same time, we observed accumulation of fibrillar inclusions in cell vacuoles of vascular bundles. It is concluded that the mechanisms of nitrate influence on photosynthesis and sugar export in leaves of symplastic and apoplastic plants are similar to a certain extent and involve the blocking of pores in phloem tubes, initiated by the NO-signaling system.     

Do pH changes in the leaf apoplast contribute to rapid inhibition of leaf elongation rate by water stress? Comparison of stress responses induced by polyethylene glycol and down‐regulation of root hydraulic conductivity

We have dissected the influences of apoplastic pH and cell turgor on short-term responses of leaf growth to plant water status, by using a combination of a double-barrelled pH-selective microelectrodes and a cell pressure probe. These techniques were used, together with continuous measurements of leaf elongation rate (LER), in the (hidden) elongating zone of the leaves of intact maize plants while exposing roots to various treatments. Polyethylene glycol (PEG) reduced water availability to roots, while acid load and anoxia decreased root hydraulic conductivity. During the first 30 min, acid load and anoxia induced moderate reductions in leaf growth and turgor, with no effect on leaf apoplastic pH. PEG stopped leaf growth, while turgor was only partially reduced. Rapid alkalinization of the apoplast, from pH 4.9 ± 0.3 to pH 5.8 ± 0.2 within 30 min, may have participated to this rapid growth reduction. After 60 min, leaf growth inhibition correlated well with turgor reduction across all treatments, supporting a growth limitation by hydraulics. We conclude that apoplastic alkalinization may transiently impair the control of leaf growth by cell turgor upon abrupt water stress, whereas direct hydraulic control of growth predominates under moderate conditions and after a 30-60 min delay following imposition of water stress.     

Predicting leaf-level fluxes of O3 and NO2: the relative roles of diffusion and biochemical processes

Pollutants like O₃ and NO₂ enter leaves through the stomata and cause damage during reactions with components of biological cell membranes. The steady-state flux rates of these gases into the leaf are determined by a series of physical and biochemical resistances including stomatal aperture, reactions occurring within the cell wall and the ability of the leaf to remove the products of apoplastic reactions. In the present study, multiple regression models incorporating stomatal conductance, apoplastic and symplastic ascorbate concentrations, and nitrate reductase (NR) activities were generated to explain the observed variations in leaf-level flux rates of O₃ and NO₂. These measurements were made on the plant Catharanthus roseus (Madagascar periwinkle). The best-fit model explaining NO₂ flux included stomatal conductance, apoplastic ascorbate and NR activity. This model explained 89% of the variation in observed leaf fluxes and suggested physical resistances, reaction between NO₂ and apoplastic ascorbate, and the removal rate of nitrate (generated by reactions of NO₂ and water) from the apoplast all play controlling roles in NO₂ flux to leaves. O₃ flux was best explained by stomatal conductance and symplastic ascorbate explaining 66% of the total variation in leaf flux. Both models demonstrate the importance of measuring processes other than stomatal conductance to explain steady-state leaf-level fluxes of pollutant gases.