Sink anatomy in relation to solute movement in rice (Oryza sativa L.): A summary of findings

The results of studies on assimilate and water transport in the developing caryopsis of rice are summarised. Evidence is presented for a symplastic movement of solutes as far as the aleurone layer. However, transport into the apoplast at the nucellus/aleurone interface appears to be a necessary step due to the absence of plasmodesmata at this site. It is suggested that water leaves the caryopsis during grain filling by the isolated cell walls of the pigment strand, the suberised walls of these cells functioning to isolate the apoplast from the symplast and thereby allowing opposing fluxes of water and assimilates to occur in the dorsal region of the grain.     

The cellular pathway of photosynthate transfer in the developing wheat grain. I. Delineation of a potential transfer pathway using fluorescent dyes

A potential cellular pathway for photosynthate transfer between the crease phloem and the starchy endosperm of the developing wheat grain has been delineated using fluorescent dyes. Membrane permeable and impermeable dyes have been introduced into the grain through the crease phloem, the endosperm cavity or the dorsal surface of the starchy endosperm. The movement of the symplastic tracer 5-(6)-6-carboxyfluorescein (CF) derived from 5-(6)-6-carboxyfluorescein diacetate (CFDA), from either direction between the crease phloem and the endosperm cavity, indicated that the symplastic pathway was operative from the crease phloem to the nucellar projection. Furthermore, the inward movement of apoplastic tracer trisodium, 3-hydroxy-5,8,10-pyrentrisulphonate (PTS) from the endosperm cavity and that of CF following plasmolysis showed that there was a high resistance to solute transfer within the apoplast of the pigment strand. All dyes entered the modified aleurone and adjacent sub-aleurone bordering the endosperm cavity. Subsequent movement of the symplastic tracers CF and sulphorhodamine G (SRG) into and through the endosperm was rapid. However, the movement of apoplastic tracers PTS and Calcofluor White (CFW) was relatively slow and with tissue plasmolysis, CF was confined to the cytoplasm of the modified aleurone and subaleurone cells. Together, these results demonstrate that there is a high resistance to solute movement within the apoplast of the cells bordering the endosperm cavity. We propose that photosynthate transfer is via the symplast to the nucellar projection where membrane exchange to the endosperm cavity occurs. Uptake from the cavity is by the modified aleurone and small endosperm cells prior to transfer through the symplast to and through the starchy endosperm.     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.     

A possible stress physiological role of abscisic acid conjugates in root-to-shoot signalling

Abscisic acid (ABA) conjugates, predominantly their glucose esters, have recently been shown to occur in the xylem sap of different plants. Under stress conditions, their concentration can rise substantially to levels that are higher than the concentration of free ABA. External ABA conjugates cannot penetrate apoplastic barriers in the root. They have to be hydrolysed by apoplastic enzymes in the root cortex. Liberated free ABA can then be redistributed to the root symplast and dragged directly across the endodermis to the stele. Endogenous ABA conjugates are formed in the cytosol of root cells, transported symplastically to the xylem parenchyma cells and released to the xylem vessels. The mechanism of release is unknown; it may include the action of ABC-transporters. Because of its extremely hydrophilic properties, ABA-GE is translocated in the xylem of the stem without any loss to the surrounding parenchyma. After arrival in the leaf apoplast, transporters for ABA-GE in the plasmalemma have to be postulated to redistribute the conjugates to the mesophyll cells. Additionally, apoplastic esterases can cleave the conjugate and release free ABA to the target cells and tissues. The activity of these esterases is increased when barley plants are subjected to salt stress.     

Abscisic acid in the xylem: where does it come from, where does it go to?

Abscisic acid is a hormonal stress signal that moves in the xylem from the root to the different parts of the shoot where it regulates transpirational water loss and leaf growth. The factors that modify the intensity of the ABA signal in the xylem are of particular interest because target cells recognize concentrations. ABA(xyl), will be decreased as radial water flow through the roots is increased, assuming that radial ABA transport occurs in the symplast only. Such dilutions of the plant hormone concentration can be compensated in different ways, which help to keep the ABA-concentrations in the xylem constant: (i) apoplastic bypass flows of ABA, (ii) ABA flows between the stem parenchyma and the xylem during transport and (iii) the action of beta-D-glucosidases that release free ABA from its conjugates to the root cortex and the leaf apoplast. The significance of reflection coefficients (sigma(ABA)), permeability coefficients of membranes (P(S)(ABA)) and apoplastic barriers for ABA is discussed.     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy

Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved.     

Accumulation of sugars in the xylem apoplast observed under water stress conditions is controlled by xylem pH

Severe water stress constrains, or even stops, water transport in the xylem due to embolism formation. Previously, the xylem of poplar trees was shown to respond to embolism formation by accumulating carbohydrates in the xylem apoplast and dropping xylem sap pH. We hypothesize that these two processes may be functionally linked as lower pH activates acidic invertases degrading sucrose and inducing accumulation of monosaccharides in xylem apoplast. Using a novel in vivo method to measure xylem apoplast pH, we show that pH drops from ~6.2 to ~5.6 in stems of severely stressed plants and rises following recovery of stem water status. We also show that in a lower pH environment, sugars are continuously accumulating in the xylem apoplast. Apoplastic carbohydrate accumulation was reduced significantly in the presence of a proton pump blocker (orthovanadate). These observations suggest that a balance in sugar concentrations exists between the xylem apoplast and symplast that can be controlled by xylem pH and sugar concentration. We conclude that lower pH is related to loss of xylem transport function, eventually resulting in accumulation of sugars that primes stems for recovery from embolism when water stress is relieved.     

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.     

cGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.     

Apoplastic synthesis of nitric oxide by plant tissues

Nitric oxide (NO) is an important signaling molecule in animals and plants. In mammals, NO is produced from Arg by the enzyme NO synthase. In plants, NO synthesis from Arg using an NO synthase-type enzyme and from nitrite using nitrate reductase has been demonstrated previously. The data presented in this report strongly support the hypothesis that plant tissues also synthesize NO via the nonenzymatic reduction of apoplastic nitrite. As measured by mass spectrometry or an NO-reactive fluorescent probe, Hordeum vulgare (barley) aleurone layers produce NO rapidly when nitrite is added to the medium in which they are incubated. NO production requires an acid apoplast and is accompanied by a loss of nitrite from the medium. Phenolic compounds in the medium can increase the rate of NO production. The possible significance of apoplastic NO production for germinating grain and for plant roots is discussed.     

Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L

The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the `apoplastic' ABA, increased before `bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.

Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period.

     

PH as a stress signal

The pH of the xylem sap of plants experiencing a range of environmental conditions can increase by over a whole pH unit. This results in an increased ABA concentration in the apoplast adjacent to the stomatal guard cells in the leaf epidermis, by reducing the ability of the mesophyll and epidermal symplast to sequester ABA away from this compartment. As a result the guard cell ABA receptors become activated and the stomata close, enabling the plant to retain water. Were it not for the low concentration of ABA ubiquitous to all land plants, the increase in the pH of the apoplast adjacent to the guard cell would induce stomatal widening, and cause excessive water loss. Not only does ABA prevent this potentially harmful phenomenon, but it also converts the pH increase to a signal which can bring about plant protection.     

What Is Phloem Unloading?

Several studies of phloem unloading have failed to distinguish between transport events occurring at the sieve element/companion cell boundary and subsequent short-distance transport through parenchyma cells. Indirect evidence has been obtained for symplastic unloading in storage and utilization sinks. In other sinks transfer to the apoplast may occur, but not necessarily at the sieve element/companion cell complex, and the evidence for apoplastic phloem unloading is equivocal, as is the role of apoplastic acid invertase in this process. The ability of several types of sink cells to accumulate sugars from the apoplast is discussed in the conflicting light of functional symplastic continuity between sink cells. Attention is drawn to the complexity of the postunloading pathway in many sinks and the difficulty of determining the exact sites of symplast/apoplast solute exchange. Potential future areas for study in the field are highlighted.     

The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity

In the developing wheat grain, photosynthate is transferred longitudinally along the crease phloem and then laterally into the endosperm cavity through the crease vascular parenchyma, pigment strand and nucellar projection. In order to clarify this cellular pathway of photosynthate unloading, and hence the controlling mechanism of grain filling, the potential for symplastic and apoplastic transfer was examined through structural and histochemical studies on these tissue types. It was found that cells in the crease region from the phloem to the nucellar projection are interconnected by numerous plasmodesmata and have dense cytoplasm with abundant mitochondria. Histochemical studies confirmed that, at the stage of grain development studied, an apoplastic barrier exists in the cell walls of the pigment strand. This barrier is composed of lignin, phenolics and suberin. The potential capacity for symplastic transfer, determined by measuring plasmodesmatal frequencies and computing potential sucrose fluxes through these plasmodesmata, indicated that there is sufficient plasmodesmatal cross-sectional area to support symplastic unloading of photosynthate at the rate required for normal grain growth. The potential capacity for membrane transport of sucrose to the apoplast was assessed by measuring plasma membrane surface areas of the various cell types and computing potential plasma membrane fluxes of sucrose. These fluxes indicated that the combined plasma membrane surface areas of the sieve element–companion cell (se–cc) complexes, vascular parenchyma and pigment strand are not sufficient to allow sucrose transfer to the apoplast at the observed rates. In contrast, the wall ingrowths of the transfer cells in the nucellar projection amplify the membrane surface area up to 22-fold, supporting the observed rates of sucrose transfer into the endosperm cavity. We conclude that photosynthate moves via the symplast from the se–cc complexes to the nucellar projection transfer cells, from where it is transferred across the plasma membrane into the endosperm cavity. The apoplastic barrier in the pigment strand is considered to restrict solute movement to the symplast and block apoplastic solute exchange between maternal and embryonic tissues. The implications of this cellular pathway in relation to the control of photosynthate transfer in the developing grain are discussed.     

Programmed cell death in cereal aleurone

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.     

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.     

Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure

Petioles of water‐sufficient intact Vicia faba L. plants were infused with 1 µm abscisic acid (ABA) to simulate the import of root‐source ABA. This protocol permitted quantitative ABA delivery, up to 300 pmol ABA over 60 min, to the leaf without ambiguities associated with perturbations in plant–water status. The ABA concentrations in whole‐leaf samples and in apoplastic sap increased with the amount infused; ABA degradation was not detected. The ABA concentration in apoplastic sap was consistent with uptake of imported ABA into the leaf symplast, but this interpretation is qualified. Our focus was quantitative cellular compartmentation of imported ABA in guard cells. Unlike when leaves are stressed, the guard‐cell symplast ABA content did not increase because of ABA infusion (P = 0·48; 3·0 ± 0·5 versus 4·0 ± 1·2 fg guard‐cell‐pair^?1). However, the guard‐cell apoplast ABA content increased linearly (R² = 0·98) from ?0·2 ± 0·5 to 3·1 ± 1·3 fg guard‐cell‐pair^?1 (≈ 3·1 µm ) and was inversely related to leaf conductance (R² = 0·82). Apparently, xylem ABA accumulates in the guard‐cell wall as a result of evaporation of the apoplast solution. This mechanism provides for integrating transpiration rate and ABA concentration in the xylem solution.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.