Apoplastic domains and sub-domains in the shoots of etiolated corn seedlings

Light Green, an apoplastic probe, was applied to the cut mesocotyl base or to the cut coleoptile apex of etiolated seedlings of Zea mays L. cv. Silver Queen. Probe transport was measured and its tissue distribution determined. In the mesocotyl, there is an apoplastic barrier between cortex and stele. This barrier creates two apoplastic domains which are non-communicating. A kinetic barrier exists between the apoplast of the mesocotyl stele and that of the coleoptile. This kinetic barrier is not absolute and there is limited communication between the apoplasts of the two regions. This kinetic barrier effectively creates two sub-domains. In the coleoptile, there is communication between the apoplast of the vascular strands and that of the surrounding cortical tissue. No apoplastic communication was observed between the coleoptile cortex and the mesocotyl cortex. Thus, the apoplastic space of the coleoptile cortex is a sub-domain of the integrated coleoptile domain and is separate from that of the apoplastic domain of the mesocotyl cortex.     

Movement of Lucifer Yellow CH in potato tuber storage tissues: A comparison of symplastic and apoplastic transport

The fluorescent dye Lucifer Yellow CH (LYCH) was introduced directly into the symplast of potato (Solanum tuberosum L.) tuber storage parenchyma by microinjection and also into the apoplast through cuts made in the stolon cortex. Microinjected LYCH moved away rapidly from a single storage cell and spread radially via the symplast. When the microinjected tissue was subsequently fixed in glutaraldehyde and sectioned the dye was seen clearly to be localised in the cytoplasm but not in the vacuole. In comparison, when LYCH was introduced into cuts made in the stolon cortex the dye entered the tuber by the xylem and subsequently spread apoplastically. No movement of dye was observed in the phloem. In glutaraldehyde-fixed tissues, in which LYCH was introduced to the apoplast, the dye was found within xylem vessels, in the cell walls and in intercellular spaces. Wall regions, possibly associated with plasmodesmata, became stained by the dye as it moved through the apoplast. Three hours after introduction of the dye to the stolon, intense deposits of LYCH were found in the vacuoles of all cells in the tuber, many aligned along the tonoplast. Differentiating vascular parenchyma elements contained large amounts of dye within enlarging vacuoles. However, with the exception of plasmolysed and-or damaged cells, LYCH was absent from the cytoplasm following its introduction to the plasmalemma it is suggested that the most likely pathway from the cell wall to the vacuole was by endocytosis, the dye being transported across the cytoplasm in membrane-bound vesicles. Clathrin-coated vesicles were abundant in the storage cells, providing a possible endocytotic pathway for dye movement. The significance of these observations is discussed in relation to the movement of LYCH in plant tissues and to the movement of solutes within and between storage cells of the tuber.Abbreviation LYCH Lucifer Yellow CH     

Cell walls as reservoirs of potassium ions for reversible volume changes of pulvinar motor cells during rhythmic leaf movements

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K⁺ content, the apoplastic K⁺ content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K⁺ content, shrunken tissue a low total but high apoplastic K⁺ content. Thus, part of the K⁺ transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K⁺ fraction shuttled between protoplasts and walls was found to be 30–40% of the total transported K⁺ fraction. Furthermore, 15–20% of the total K⁺ content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5–10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K⁺-storing capacity on pH and [K⁺] of the equilibration solution. The high apoplastic K⁺ contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K⁺ ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K⁺] in the water free space of the apoplast.Abbreviations and symbols DFS Donnan free space - DW dry weight - pK negative logarithm of the equilibrium constant K of the acidic group - WFS water free space - water potential; Indices - cw cell wall - t tissue     

The integration of whole-root and cellular hydraulic conductivities in cereal roots

The hydraulic conductivities of excised whole root systems of wheat (Triticum aestivum L. cv. Atou) and of single excised roots of wheat and maize (Zea mays L. cv. Passat) were measured using an osmotically induced back-flow technique. Ninety minutes after excision the values for single excised roots ranged from 1.6·10^-8 to 5.5·10^-8 m·s^-1·MPa^-1 in wheat and from 0.9·10^-8 to 4.8·10^-8 m·s^-1·MPa^-1 in maize. The main source of variation was a decrease in the value as root length increased. The hydraulic conductivities of whole root systems, but not of single excised roots, were smaller 15 h after excision. This was not caused by occlusion of the xylem at the cut end of the coleoptile. The hydraulic conductivities of epidermal, cortical and endodermal cells were measured using a pressure probe. Epidermal and cortical cells of both wheat and maize roots gave mean values of 1.2·10^-7 m·s^-1·MPa^-1 but in endodermal cells (measured only in wheat) the mean value was 0.5·10^-7 m·s^-1·MPa^-1. The cellular hydraulic conductivities were used to calculate the root hydraulic conductivities expected if water flow across the root was via transcellular (vacuole-to-vacuole), apoplasmic or symplasmic pathways. The results indicate that, in freshly excised roots, the bulk of water flow is unlikely to be via the transcellular pathway. This is in contrast to our previous conclusion (H. Jones, A.D. Tomos, R.A. Leigh and R.G. Wyn Jones 1983, Planta 158, 230–236) which was based on results obtained with whole root systems of wheat measured 14–15 h after excision and which probably gave artefactually low values for root hydraulic conductivity. It is now concluded that, near the root tip, water flow could be through a symplasmic pathway in which the only substantial resistances to water flow are provided by the outer epidermal and the inner endodermal plasma membranes. Further from the tip, the measured hydraulic conductivities of the roots are consistent with flow either through the symplasmic or apoplasmic pathways.Symbols L _{p, cell} cell hydraulic conductivity - L _{p, root} root hydraulic conductivity - L _{p, root} calculated root hydraulic conductivity - root reflection coefficient     

Electrical resistance and ion movement through excised discs of sugar beet root tissue

Electrical and tracer techniques were used to investigate the movement of Na⁺, K⁺ and Cl^? through discs of a range of thicknesses cut from the root tissue of sugar beet, Beta vulgaris L. cv. Amono. At low external concentration the electrical resistance across a discs is less than that of an equivalent volume of solution. This does not appear to be due to a low resistance symplastic pathway but rather, to an enhanced concentration of cation in the apoplast. The resistance is proportional to the thickness of tissue. Although measurement of diffusion potential gives about 25 mV difference across the disc for a ten-fold change in cation concentration, there is little discrimination between K⁺ and Na⁺. The observed tracer kinetics of ⁸⁶Rb through the disc are consistent with those of diffusion, with a coefficient of diffusion, D, of 0.19 × 10^?9 m² s^?1 and a tissue partition coefficient, k, of 0.27 (or of 2.7 if referred to the cell wall phase only). ³⁶Cl gives a similar value for D, but has a k reduced by a factor of 3.3, a result that is consistent with the diffusion potential observation. However, a much larger discrimination would have been expected from the chemically measured cation exchange capacity.     

Phospholipids are present in extracellular fluids of imbibing sunflower seeds and are modulated by hormonal treatments

Phospholipids are well known messengers involved in developmental and stress responses mediating intracellular signalling. It has been hypothesized that phospholipids exist which could participate in intercellular communication events through the apoplast of sunflower (Helianthus annuus) seeds. Here it is shown that extracellular washing fluids (EWFs) obtained from seeds imbibed for 2 h contain diverse phospholipids. Lipid profiling by electrospray ionization tandem mass spectrometry revealed that the EWFs have a particular composition, with phosphatidic acid (PA) and phosphatidylinositol (PI) being the major phospholipids. These profiles are clearly distinct from those of seed extract (SE), and comparative SDS-PAGE of EWF and SE, followed by intracellular and plasma membrane marker analyses, allowed a significant contamination of the EWF to be discarded. Treatment of the seeds with 100 microM jasmonic acid (JA) induces changes in the profile of EWF phospholipids, leading to a decrease in PI content, while the accumulation of phosphatidylinositol 4-phosphate (PI4P) and specific PA species is observed. On the other hand, the EWF from seeds subjected to 50 microM abscisic acid (ABA) treatment exhibit an increase in PA and phosphatidylglycerol levels. To our knowledge, this is the first report on the existence of phospholipids as extracellular components of seeds. Moreover, the modulation of PA, PI, and PI4P levels by hormonal treatments further suggests their contribution to intercellular communication in planta.     

Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development

Fluorescein diacetate (FDA) was used as a vital stain to assaymembrane integrity (cell viability) in mesocarp tissue of thedeveloping grape (Vitis vinifera L.) berry in order to testthe hypothesis that there is a substantial loss of compartmentationin these cells during ripening. This technique was also usedto determine whether loss of viability was associated with symptomsof a ripening disorder known as berry shrivel. FDA fluorescenceof berry cells was rapid, bright, and stable for over 1 h atroom temperature. Confocal microscopy detected FDA stainingthrough two to three intact surface cell layers (300–400µm) of bisected berries, and showed that the fluorescencewas confined to the cytoplasm, indicating the maintenance ofintegrity in both cytoplasmic as well as vacuolar membranes,and the presence of active cytoplasmic esterases. FDA clearlydiscriminated between living cells and freeze-killed cells,and exhibited little, if any, non-specific staining. Propidiumiodide and DAPI, both widely used to assess cell viability,were unable to discriminate between living and freeze-killedcells, and did not specifically stain the nuclei of dead cells.For normally developing berries under field conditions therewas no evidence of viability loss until about 40 d after veraison,and the majority (80%) of mesocarp cells remained viable pastcommercial harvest (26 °Brix). These results are inconsistentwith current models of grape berry development which hypothesizethat veraison is associated with a general loss of compartmentationin mesocarp cells. The observed viability loss was primarilyin the locule area around the seeds, suggesting that a localizedloss of viability and compartmentation may occur as part ofnormal fruit development. The cell viability of berry shrivel-affectedberries was similar to that of normally developing berries untilthe onset of visible symptoms (i.e. shrivelling), at which timeviability declined in visibly shrivelled berries. Berries withextensive shrivelling exhibited very low cell viability (15%). Key words: Apoplast, berry shrivel, compartmentation, DAPI, FDA, fluorescence, fruit ripening, locule, propidium iodide Received 19 September 2007; Revised 16 December 2007 Accepted 26 December 2007     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane