What Slows Down Phytoplasma Proliferation? Speculations on the Involvement of AtSEOR2 Protein in Plant Defence Signalling.

Considering the crude methods used to control phytoplasma diseases, a deeper knowledge on the defence mechanisms recruited by the plant to face phytoplasma invasion is required. Recently, we demonstrated that Arabidopsis mutants lacking AtSEOR1 gene showed a low phytoplasma titre. In wild type plants AtSEOR1 and AtSEOR2 are tied in filamentous proteins. Knockout of the AtSEOR1 gene may pave the way for an involvement of free AtSEOR2 proteins in defence mechanisms. Among the proteins conferring resistance against pathogenic bacteria, AtRPM1-interacting protein has been found to interact with AtSEOR2 in a high-quality, matrix-based yeast-two hybrid assay. For this reason, we investigated the expression levels of Arabidopsis AtRIN4, and the associated AtRPM1 and AtRPS2 genes in healthy and Chrysanthemum yellows-infected wild-type and Atseor1ko lines.

Reversible calcium-regulated stopcocks in legume sieve tubes.

Sieve tubes of legumes (Fabaceae) contain characteristic P-protein crystalloids with controversial function. We studied their behavior by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media containing either Ca(2)+ or chelators. Sr(2)+ and Ba(2)+, but not Mg(2)+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with which to control sieve tube conductivity.

Novel approach in plastid transformation.

Engineering the nuclear genome of plants is perceived to be associated with problems regarding biosafety and the stability of expression of the transgene. Alternative transformation strategies using the genomic outfit of the plastid promise to be more successful in this respect. Over the past few years progress has been made in screening procedures, and plastid transformation technology has allowed function to be assigned to open reading frames, massive expression of insecticidal agents and proteins involved in herbicide resistance, and the accumulation of biopolymers. Recently, the design of a novel femtoinjection technique that allows injection into chloroplasts has provided the opportunity to further manipulate and understand chloroplastic gene expression.

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.

Interaction between sieve element and companion cell and the consequences for photoassimilate distribution. Two structural hardware frames with associated physiological software packages in dicotyledons?

The unique properties of the sieve element/companion cell complex (SE/CC complex) are based on the concerted action of SE and CC. A prominent role in this interaction is played by the branched plasmodesmata (pore-plasmodesma units, PPUs) between SE and CC. The large molecular exclusion limit of the PPUs enables an intensive exchange of macromolecular materials and messengers. Further, the degree of symplasmic autonomy of the SE/CC complex and the structural/functional properties of the SE/CC modules may differ between the dicotyledonous families. The speculation is advanced that the difference in building blocks produces two distinct phloem frames ('hardware') with associated physiological programmes ('software'). The implications of two phloem types for photosynthate distribution and relative growth rate are discussed.

Quantification of symplastic continuity as visualised by plasmodesmograms: diagnostic value for phloem-loading pathways.

The use of plasmodesmatal frequency to correlate cell-cell symplastic transport capacity remains a contentious problem, as variation in cell shape, accurate determination of interface contact area between cell types, distribution (i.e. whether random or aggregated) and shape (i.e. whether single or branched), and state of permeability may confuse the issue. Additionally, variation in the methods used to determine the frequencies compounds the problem further. Data presented in this paper show that plasmodesmograms offer a means to visualise the potential transport pathway from mesophyll cells to sieve tubes. Furthermore, the results allow an instant appreciation of symplastic continuity or discontinuity and, accordingly, the potential symplastic and-or apoplastic stages involved in the overall loading process.

Dissimilar phloem loading in leaves with symplasmic or apoplasmic minor-vein configurations.

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to (14)CO2. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.

Symplastic isolation of the sieve element-companion cell complex in the phloem of Ricinus communis and Salix alba stems.

The anatomical and physiological isolation of the sieve element-companion cell complex (se-cc complex) was investigated in stems of Ricinus communis L. and Salix alba L. In Ricinus, the plasmodesmatal frequencies were in the proportions 8∶1∶2∶30, in the order given, at the interfaces between sieve tube-companion cell, sieve tube-phloem parenchyma cell, companion cellphloem parenchyma cell, and phloem parenchyma cellphloem parenchyma cell. The membrane potentials of the se-cc complex and the surrounding phloem-parenchyma cells sharply contrasted: the membrane potential of the se-cc complex was about twice as negative as that of the phloem parenchyma. Lucifer Yellow CH injected into the sieve element or into the companion cell remained within the se-cc complex. Dye introduced into phloem parenchyma only moved (mostly poorly) to other phloem-parenchyma cells. The distribution of the plasmodesmatal frequencies, the differential dye-coupling and the sharp discontinuities in membrane potentials indicate that the se-cc complexes constitute symplast domains in the stem phloem. Symplastic autonomy is discussed as a basic necessity for the functioning of the se-cc complex in the stem.

Mapping membrane potential differences and dye-coupling in internodal tissues of tomato (Solanum lycopersicum L.).

Symplastic continuity in internodal tissues of tomato was investigated by electrophysiological and micro-injection techniques. Recordings of potential differences (pd) were combined with iontophoretic injection of 5,6-carboxyfluorescein (CF) and Lucifer Yellow CH (LYCH). Mapping of pds and dye-coupling respectively labeled the potential sites of major solute uptake and the spatial differentiation in symplast permeability. The cells of the central pith had low negative pds (-23 mV) and limited dye-coupling. In the pith periphery, pds (-43 mV) and dye-coupling were more pronounced. The pith was symplastically isolated from the secondary xylem. Axial contact cells (pd -54 to -109 mV) showed a strong dye-coupling within the vessel-enclosing sheath. No dye transfer was observed from the vessel sheaths to other cells. Fibre-tracheids and libriform wood fibres were dye-coupled. Ray cells (pd -39 to -94 mV) transported dye to adjacent fibres (pd -18 to -41 mV). The reverse was not observed. Except for a few cases of limited radial cell-cell transport, no dye movement through rays was observed. Cambium cells were connected mainly axially (ray cell initials) or both axially and tangentially (fusiform initials). Radial dye-coupling of cambium cells was rare. The limitations of the present approach and the significance of the differential symplast permeability for xylem-to-phloem transfer are discussed.

Symplastic Transfer of Fluorescent Dyes from Mesophyll to Sieve Tube in Stripped Leaf Tissue and Partly Isolated Minor Veins of Commelina benghalensis.

We have stripped small (3 x 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves : Differences in ontogenic development, spatial arrangement and symplastic connections of the two sieve tubes in the minor vein.

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4-5 µm; wall thickness 0.12 µm) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 µm; wall thickness 0.2 µm) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.

Diurnal water balance of the cowpea fruit.

The vascular network of the cowpea (Vigna unguiculata [L.] Walp.) fruit exhibits the anatomical potential for reversible xylem flow between seeds, pod, and parent plant. Feeding of cut shoots with the apoplast marker acid fuchsin showed that fruits imported regularly via xylem at night, less frequently in early morning, and only rarely in the afternoon. The dye never entered seeds or inner dorsal pod strands connecting directly to seeds. Root feeding (early morning) of intact plants with (32)PO(4) or (3)H(2)O rapidly (20 min) labeled pod walls but not seeds, consistent with uptake through xylem. Weak subsequent (4 hours) labeling of seeds suggested slow secondary exchange of label with the phloem stream to the fruit. Vein flap feeding of subtending leaves with [(14)C]sucrose, (3)H(2)O, and (32)PO(4) labeled pod and seed intensely, indicating mass flow in phloem to the fruit. Over 90% of the (14)C and (3)H of fruit cryopuncture phloem sap was as sucrose and water, respectively. Specific (3)H activities of transpired water collected from fruits and peduncles were assayed over 4 days after feeding (3)H(2)O to roots, via leaf flaps, or directly to fruits. The data indicated that fruits transpired relatively less xylem-derived (apoplastic) water than did peduncles, that fruit and peduncle relied more heavily on phloem-derived (symplastic) water for transpiration in the day than at night, and that water diffusing back from the fruit was utilized in peduncle transpiration, especially during the day. The data collectively support the hypothesis of a diurnally reversing xylem flow between developing fruit and plant.

Phosphate uptake inLemna gibba G1: energetics and kinetics.

Phosphate uptake was studied by determining [(32)P]phosphate influx and by measurements of the electrical membrane potential in duckweed (Lemna gibba L.). Phosphate-induced membrane depolarization (ΔE m ) was controlled by the intracellular phosphate content, thus maximal ΔE m by 1 mM H2PO 4 (-) was up to 133 mV after 15d of phosphate starvation. The ΔE m was strongly dependent on the extracellular pH, with a sharp optimum at pH 5.7. It is suggested that phosphate uptake is energized by the electrochemical proton gradient, proceeding by a 2H(+)/H2PO 4 (-) contransport mechanism. This is supported also by the fusicoccin stimulation of phosphate influx. Kinetics of phosphate influx and of ΔE m , which represent mere plasmalemma transport, are best described by two Michaelis-Menten terms without any linear components.

Analysis of valine uptake by Commelina mesophyll cells in a biphasic active and a diffusional component.

Valine uptake by isolated Commelina benghalensis L. mesophyll cells was measured over a wide concentration range (10(-6)-4·10(-2) mol l(-1)). The uptake data were subjected to iterative fitting. Experiments with carbonyl cyanide mchlorophenyl hydrazone (CCCP), diethylstilbestrol (DES), and p-chloromercuriphenylsulphonic acid (PCMBS) provided evidence that the biphasic uptake kinetics of valine consists of a diffusional component and a biphasic active uptake. The data from the control experiments, were also best fitted to one diffusional component and two Michaelis-Menten systems. The presence of two carrier systems in the plasmalemma, however, was considered to be virtual for the following reasons: (1) Both phases of active uptake were equally decreased by high concentrations of K(+)-ions. (2) Fusicoccin stimulated the active uptake in both phases to the same extent. (3) Inhibitors of the proton-driven uptake (CCCP, DES, PCMBS) similarly inhibited the active uptake at all concentrations. (4) The active uptake equally responded in both phases to changes in the pH. (5) Light also promoted the active uptake over the whole concentration range. These results strongly indicate that, despite its biphasic character, the active uptake is due to one proton-driven carrier system.

Light-promoted diffusional amino acid efflux from Commelina leaf disks : Indirect control by proton pump activities.

The release (=the measured loss) of amino acids was studied in Commelina benghalensis leaf disks. The release is assumed to be the result of influx and efflux, therefore, both movements were investigated.The uptake of (14)C-labeled valine exhibited a biphasic isotherm. The uptake was pH-dependent, especially at low substrate concentrations (pH optimum 4.8). Signals for amino acid/proton co-transport were observed: stimulation of the uptake by fusicoccin (FC), inhibition by diethylstilbestrol (DES) or by high K(+) concentrations. In the light, the ATP level of the disks was maintained during the uptake period (2 h), in darkness the ATP content decreased from 87 to 24 nmol g(-1) fr. wt. However, light-promoted uptake, which is explained in the proton pump concept by an intensified proton extrusion as the result of high ATP production, was lacking.The release of amino acids was increased by washing with p-chloromercuriphenyl sulphonic acid (PCMBS), nystatin, 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or KCN. The release (Q10 about 1.5) was independent of the external pH and was linearly related to the intracellular amino acid concentration. Light enhanced the rate of release to the same extent at all intracellular concentrations. The present results suggest that the release is balanced by a, at least partially, proton-driven influx and a diffusional ligh-promoted efflux. A provisional model shows how the diffusional effulx can be indirectly controlled by a counter-flow fueled by the metabolism.

Kinetics of l-Alanine Escape from Xylem Vessels.

Labeled ((3)H or (14)C) l-alanine was perfused through the xylem vessels of isolated tomato internodes (Lycopersicon esculentum cv. Moneymaker) at various concentrations (10(-6) molar to 10(-2) molar). At each concentration the escape of l-alanine from the xylem vessels was apparently a first order process, which is in agreement with Horwitz' (1958, Plant Physiology 33:81-93) model for irreversible escape from the xylem vessels. The escape constant (K) decreased at higher concentrations of l-alanine, which implies that Horwitz' model is inappropriate to describe the kinetics of l-alanine escape, and that the escape at least partly is a saturable process. To obtain data that relate the concentration of l-alanine in the xylem vessels and the escape rate of the amino acid, average escape rates per internode were measured and the corresponding concentrations were calculated from the integrated form of the Michaelis-Menten equation.AS THE CONCENTRATION DEPENDENCE OF THE ESCAPE RATE WAS BIPHASIC, THREE POSSIBLE MECHANISMS WERE CONSIDERED, ESCAPE BEING CAUSED BY: (a) saturable amino acid uptake of cells around the xylem vessels and diffusion into the free space; (b) saturable uptake of the cells around the xylem vessels, but at higher amino acid concentrations in the xylem vessels the number of cells, that participate in the uptake, increases; (c) two, simultaneously operating, saturable uptake systems in the cells around the xylem vessels.

A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode disks.

Internode disks of tomato (Lycopersicon esculentum cv. Moneymaker) were shaken in glutamine and sucrose solutions. At low external pH (<±5.5), the uptake of these substances was accompanied with K(+) efflux, at high pH (>±5.5) with K(+) influx. Low concentrations of external K(+) (2 mmol l(-1)) stimulated the uptake of glutamine, which was strongly inhibited by the supply of high K(+) concentrations (20 mmol l(-1)). The effect of K(+) was particularly pronounced at high pH-values. Addition of CCCP in light reduced the uptake of glutamine to the same level as in the dark, and stopped the K(+) fluxes which coincided with the uptake. A model is presented wherein the movements of K(+) across the membrane are related to co-transport, depending on the membrane potential and the Nernst potential of K(+).

Different mass transfer rates of labeled sugars and tritiated water in xylem vessels and their dependency on metabolism.

Solutions of (14)C-sugars in tritiated water or solutions of (14)C- and (3)H-sugars were perfused by gravity through the xylem vessels of excised tomato internodes (Lycopersicon esculentum) mostly during 2 hours.Mass flow of a solution in plant vessels is found not to be in conflict with different mass transfer rates of sugar and water molecules. It is explained by individual lateral escape rates from the vessel for each single compound. The escape of tritiated water can be ascribed to diffusion, while the escape of sugars is apparently linked to the metabolism of the surrounding parenchyma cells (Q(10) sucrose uptake = 3.5). Lateral escape rates of sugars from the xylem vessels are in the proportion of sucrose-glucose-fructose (2.4:1.0:0.6). The accessibility of xylem parenchyma cells and their differential permeability to sugars control the longitudinal mass transfer of sugars in the xylem vessels.As sucrose and glucose do not compete for uptake from the vessels by the contiguous cells, separate uptake systems for both may be postulated.