Movement and metabolism of oligogalacturonide elicitors in tomato shoots

We have studied the movement and metabolism of oligogalacturonides through shoots of tomato (Lycopersicon esculentum L. cv Rutgers). Oligomers of polygalacturonic acid were prepared by enzyme digestion and gel filtration. These were end-reduced with [³H]NaBH₄, using an improved reaction method, to yield oligoalditols. The radiolabelled oligomer of degree of polymerisation 6 was supplied to tomato shoots through their transpiration stream. Analysis of the distribution of radiolabel in the plant, and TLC of radiolabelled material recovered from the plant revealed the following: a) material recovered from the plant could be identified as an oligogalacturonide from its behaviour on TLC and susceptibility to digestion with polygalacturonase; b) end-reduced oligogalacturonides moved freely through the plant and were not complexed to high-molecularweight compounds and immobilised; c) during passage through the plant, modifications to the oligogalacturonide occurred, presumably as a consequence of metabolism in the apoplastic space. We found evidence of i) esterification of the molecule, and ii) shortening of the oligogalacturonide chain. The results show that in the assay for protease-inhibitor-inducing factor using cut shoots, oligogalacturonide elicitors can move into the leaves and act directly on the cells producing protease inhibitor.Abbreviations and Symbols d.p. degree of polymerisation - PIIF protease inhibitor inducing factor - R_f ratio of distance moved by solute to that of solvent front     

Separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography

A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.     

Galacturonic acid-induced changes in strawberry plant development <Emphasis Type="Italic">in vitro</Emphasis>

The oligogalacturonides derived from trifluoroacetic acid hydrolysis and their monomer galacturonic acid were tested for their effect on development of strawberry explants (Fragaria × ananassa) in vitro. The addition of oligogalacturonides or galacturonic acid in the plant tissue culture medium at concentrations of 0.1 or 1.0 mM increased shoot and leaf number in the presence of added benzyl adenine. The response to oligogalacturonides or galacturonic acid was maintained in the absence of benzyl adenine but was lessened. Conversely, root elongation was increased in the absence of benzyl adenine. A combined analysis of these experiments also indicated the increase in shoot or leaf number. High-performance anion exchange chromatography of the bioassayed oligogalacturonide samples using a pulsed amperometric detector revealed that the sample consisted of galactouronides with a degree of polymerization from 1 to 5 and the proportion of the monomer was very high (94%).     

A sensitive and rapid bioassay of homogalacturonan synthase using 2-aminobenzamide-labeled oligogalacturonides

Polygalacturonate 4-alpha-galacturonosyltransferase (GalA T) activity was detected in the microsomal fraction isolated from pumpkin (Cucurbia moschata Duchesne, cv. Tokyou-Kabocha) seedlings using UDP-GalA and 2-aminobenzamide (2AB)-labeled oligogalacturonides. A 2AB-labeled undecagalacturonide was elongated by the attachment of galacturonic acid (GalA) residues to give 2AB-labeled oligogalacturonides with a degree of polymerization (DP) between 12 and 17. Exogenous 2AB-labeled oligogalacturonide acceptors with a DP >3 are effective acceptor molecules for pumpkin GalA T.     

Characteristics of the phloem path: analysis and distribution of carbohydrates in the petiole of Cyclamen

Compartmentation fluxes of carbohydrates along the phloem path were analysed in the petiole of Cyclamen persicum (L.) Mill. Sucrose represented the dominant fraction (58-75% of soluble carbohydrates in the vascular symplast). Planteose (12-22%), glucose (3-8%) and fructose (3-13%) occurred in lower amounts (data from liquid chromatography, percentages of the total peak area). Starch was not detectable. Upon feeding leaves with ¹⁴CO2, 98% and 90% of radiolabel was recovered as sucrose in the vascular symplast after 3 h and 24 h, respectively. Thus, sucrose appeared to be the exclusive transport sugar in Cyclamen. Experiments with asymmetrically labelled sucrose revealed that there was no metabolism of translocated sucrose. Analysis of six consecutive petiole segments (each 2 cm in length) showed a homogeneous longitudinal distribution of these sugars differed markedly. On average, the sucrose concentration amounted to 4.7 and 0.4 mg g^-1 FM in the vascular apoplast and petiole parenchyma, respectively. Sucrose was unloaded with out hydrolysis and stored in the periphery of the phloem path. Planteose was identified as another storage saccharide. Sucrose synthesis by sucrose phosphate synthase occurred when isolated vascular bundles were incubated with [¹⁴C]glucose or [¹⁴C]fructose. These data suggest that the phloem path is characterized by both source and sink like activity.     

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Cytochemical localization of ATPase activity in phloem tissues ofRicinus communis

Summary Standard lead precipitation procedures have been used to examine the localization of ATPase activity in phloem tissues ofRicinus communis. Reaction product was localized on the plasma membrane of the companion cells associated with sieve elements and of parenchyma cells in phloem tissues from the leaf, petiole, stem and root. ATPase activity was also present on the plasma membrane and dispersed P-protein of sieve elements in petiole, stem and root tissue, but was absent from the plasma membrane of these cells in the leaf minor veins. Substitution of-glycerophosphate for ATP produced no change in the localization of reaction product in leaf tissue. These findings are discussed in relation to current theories on the mechanism of sugar transport and phloem loading.     

Host-Pathogen Interactions : XXXIII. A Plant Protein Converts a Fungal Pathogenesis Factor into an Elicitor of Plant Defense Responses

This paper describes the effect of a plant-derived polygalacturonase-inhibiting protein (PGIP) on the activity of endopolygalacturonases isolated from fungi. PGIP's effect on endopolygalacturonases is to enhance the production of oligogalacturonides that are active as elicitors of phytoalexin (antibiotic) accumulation and other defense reactions in plants. Only oligogalacturonides with a degree of polymerization higher than nine are able to elicit phytoalexin synthesis in soybean cotyledons. In the absence of PGIP, a 1-minute exposure of polygalacturonic acid to endopolygalacturonase resulted in the production of elicitor-active oligogalacturonides. However, the enzyme depolymerized essentially all of the polygalacturonic acid substrate to elicitor-inactive oligogalacturonides within 15 minutes. When the digestion of polygalacturonic acid was carried out with the same amount of enzyme but in the presence of excess PGIP, the rate of production of elicitor-active oligogalacturonides was dramatically altered. The amount of elicitor-active oligogalacturonide steadily increased for 24 hours. It was only after about 48 hours that the enzyme converted the polygalacturonic acid into short, elicitor-inactive oligomers. PGIP is a specific, reversible, saturable, high-affinity receptor for endopolygalacturonase. Formation of the PGIP-endopolygalacturonase complex results in increased concentrations of oligogalacturonides that activate plant defense responses. The interaction of the plant-derived PGIP with fungal endopolygalacturonases may be a mechanism by which plants convert endopolygalacturonase, a factor important for the virulence of pathogens, into a factor that elicits plant defense mechanisms.     

Untersuchungen zur funktionellen Anatomie des Leitgewebesystems im Blatt von Pelargonium zonale

Summary The petiole of Pelargonium zonale is traversed by 17 bundles, whose arrangement and form are typical for this plant. The bundles of the petiole are connected with the conducting system of the axis and with the main nerves by a system of phloem anastomoses in the leaf base and in the junction between the petiole and the leaf blade (Fig. 2). The anatomical findings were confirmed and extended by a study of the translocation of K-fluorescein and ¹⁴C. It could be shown that the metaphloem of the central petiole bundle is composed of phloem subunits, each of which is connected with the phloem of one certain main nerve only (Fig. 4). Accordingly, if fluorescein or ¹⁴CO₂ is applied to one main nerve, the dye or ¹⁴C-material is translocated exclusively in a small phloem area of the central bundle. Autoradiograms of the petioles indicate that the ¹⁴C-labelled assimilates (sucrose, glucose, fructose and amino acids) are translocated exclusively in the phloem. A lateral movement of the labelled material within the petiole was not observed. The metaphloem of the central petiole bundle of Pelargonium zonale revealed a functional organization of phloem subunits.

---

---

Teil einer Dissertation unter der wissenschaftlichen Leitung von Prof. Dr. J. Willenbrink.     

Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants

The movement of systemin, the 18-amino-acid polypeptide inducer of proteinase inhibitors in tomato (Lycopersicon esculentum L.) plants, was investigated in young tomato plants following the application of [¹⁴C]systemin to wounds on the surface of leaves. Wholeleaf autoradiographic analyses revealed that [¹⁴C]systemin was distributed throughout the wounded leaf within 30 min, and then during the next several hours was transported to the petiole, to the main stem, and to the upper leaves. The movement of [¹⁴C]systemin was similar to the movement of [¹⁴C]sucrose when applied to leaf wounds, except that sucrose was slightly more mobile than systemin. Analyses of the radioactivity in the petiole phloem exudates at intervals over a 5-h period following the application of [¹⁴C]systemin to a wound demonstrated that intact [¹⁴C]systemin was present in the phloem over the entire time, indicating that the polypeptide was either stable for long periods in the phloem or was being continually loaded into the phloem from the source leaf. The translocation pathway of systemin was also investigated at the cellular level, using light microscopy and autoradiography. Within 15 min after application of [³H]systemin to a wound on a terminal leaflet, it was found distributed throughout the wounded leaf and was primarily concentrated in the xylem and phloem tissues within the leaf veins. After 30 min, the radioactivity was found mainly associated with vascular strands of phloem tissue in the petiole and, at 90 min, label was found in the phloem of the main stem. Altogether, these and previous results support a role for systemin as a systemic wound signal in tomato plants.The authors acknowledge the Washington State University Electron Microscope Center and staff for their technical advice and collaboration. We also thank Greg Wichelns for growing our plants and Dr. Steven Doares for providing [³H]systemin. This research was supported in part by the Washington State College of Agriculture and Home Economics Project No. 1791 and National Science Foundation grants IBN 9117795 and IBN 9104542     

Mineral partitioning in the phloem during autumn senescence of beech leaves

Summary During the period of leaf senescence in fall, the minerals Mg, Ca, K, P, Cl, S, and Si were compared for occurrence and density in tissue compartments of leaf blade, petiole, and subtending stem of beech (Fagus sylvatica L.). Measurements were made by energy-dispersive X-ray microanalysis. The plant material was collected on 2,9, 16 and 23 October, and showed green, greenyellow, yellow, and red-brown autumn leaf coloration. Mg, K, and P were retrieved from the leaf blade prior to shedding, and deposited mainly in cortex and pith tissues of the stem. S and Ca remained in the leaf, and Si and Cl appeared to accumulate in the leaf prior to shedding. During the four stages of leaf senescence, the phloem compartments of the petiole showed considerable changes in mineral content. In addition, leaf senescence in several cases was accompanied by ion shifting from symplastic to apoplastic compartments and vice versa.     

Cation and Chloride Partitioning through Xylem and Phloem within the Whole Plant of Ricinus communis L. under Conditions of Salt Stress

Uptake and partitioning through the xylem and phloem of K⁺,Na⁺, Mg²⁺ , Ca²⁺ and Cl^– were studied over a 9 d intervalduring late vegetative growth of castor bean (Ricinus communisL.) plants exposed to a mean salinity stress of 128 mol m^–3NaCl. Empirically based models of flow and utilization of eachion within the whole plant were constructed using informationon ion increments of plant parts, molar ratios of ions to carbonin phloem sap sampled from petioles and stem internodes andpreviously derived information on carbon flow between plantsparts in xylem and phloem in identical plant material. Salientfeatures of the plant budget for K⁺ were prominent depositionin leaves, high mobility of K⁺ in phloem, high rates of cyclingthrough leaves and downward translocation of K⁺ providing theroot with a large excess of K⁺ . Corresponding data for Na⁺showed marked retention in the root, lateral uptake from xylemby hypocotyl, stem internodes and petioles leading to low intakeby young leaf laminae and substantial cycling from older leavesback to the root. The partitioning of the anionic componentof NaCl salinity, Cl^–, contrasted to that of Na⁺ in thatit was not substantially retained in the root, but depositedmore or less uniformly in stem, petiole and leaf lamina tissues.The flow pattern for Mg²⁺ showed relatively even depositionthrough the plant but some preferential uptake by young leaves,generally lesser export than import by leaf laminae, and a returnflow of Mg²⁺ from shoot to root considerably less than the recordedincrement of the root. Ca²⁺ partitioning contrasted with thatof the other ions in showing extremely poor phloem mobility,leading to progressive preferential accumulation in leaf laminaeand negligible cycling of the element through leaves or root.Features of the response of Ricinus to salinity shown in thepresent study were discussed with data from similar modellingstudies on white lupin (Lupinus albus L.) and barley (Hordeumvulgare L.) Key words: Ricinus communis L, potassium, sodium, chloride, calcium, magnesium, phloem, xylem, transport, partitioning, salinity     

INTERNAL PHLOEM IN THE PULVINUS OF SOYBEAN PLANTS

Glycine max, like many species of Fabaceae, has pulvini at the base of the petiole. In this structure, the vascular cylinder is constricted and consists of a ring of phloem surrounding a ring of xylem. A combination of light and transmission electron microscopy and histochemistry showed that, in addition, there are groups of internal phloem strands in the pulvinar pith. This was confirmed by direct observation of sieve plates and crystalline P-protein inclusions typical of leguminous sievetube members. Serial sections through the stem–pulvinus–petiole revealed that a spatial reorientation of the vascular tissue in the pulvinus resulted in the formation of internal phloem strands, which are continuous with the external phloem bundles above and below the pulvinus. Using 6(5)carboxyfluorescein (6CF) as a fluorescent tracer of phloem transport, we have shown that the internal phloem was active. In most of the experiments, when 6CF was applied to a source leaf, the internal phloem was not stained when the stem was girdled between the source leaf and the roots. Thus, we suggest that the internal phloem of the pulvinus of soybean is specialized for transport toward the root.     

Imaging the Vascular Continuity of Prunus avium Petioles During Leaf Senescence Using Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) micro-imaging was used to studythe distribution of mobile protons in the petiole and stem ofPrunus avium throughout the period of senescence of an attachedleaf. The base of the NMR micro-imaging probe was modified toallow access to the coil from below, enabling a branch of theintact tree to be inserted into the probe which, in turn, wasinserted into the base of the NMR cryomagnet. A multi-sliceimage was used to determine the final orientation of the petiolein the probe. NMR spin echo imaging parameters were investigatedby using variable relaxation delays (D1) ranging from 0·1to 4·0 s. Transverse slices of the petiole with a D1ranging between 1·0 to 4·0 s produced bright imagescorresponding to the cortex and leaving the xylem vessels asa dark horseshoe-shaped area. Reducing D1 to 0·25 s or0·1 s produced images with more detail, differentiatingthe phloem and xylem vessels from the surrounding cortex. A longitudinal slice, 0·5 mm thick, through the stemand subtending petiole was imaged repeatedly over 6 d, duringwhich the leaf senesced and abscinded. The images showed detailsof the vascular strands in the stem with a section continuouswith the petiole. During leaf senescence the development ofthe abscission zone in the petiole was observed. The petioleremained hydrated throughout leaf senescence, demonstratingthe usefulness of NMR imaging to probe the vascular integrityof the intact plant. Key words: Spin echo pulsed gradient, cherry, petiole, vascular integrity     

The Substrate Preference and Histochemical Localization Argue against the Direct Role of Cucumber Stress-Related Anionic Peroxidase in Lignification

The substrate preference and the localization of cucumber (Cucumis sativus L.) stress-related anionic peroxidase (srPRX) were investigated in order to assess whether this activity correlates with the lignification. The results showed that none of the purified srPRX isoenzymes (PRX 1 –3) could oxidize the lignin monomer analog syringaldazine. The srPRX immunospecific signal was found to be highly abundant in both the extrafascicular and fascicular phloem regions in cucumber stem and leaf petiole. In Nicotiana, Petunia and Dahlia, the srPRX homologs were specifically deposited in both outer and inner phloem elements of stem and in both abaxial and adaxial phloem of leaf stems. The srPRX mRNA expression analysis showed similar pattern as for immunolocalization. The subcellular localization of immunospecific srPRX demonstrated that at least part of the peroxidase could be ionically-bound to phloem cell wall.     

Dynamic changes in petiole specific conductivity in red maple (Acer rubrum L.), tulip tree (Liriodendron tulipifera L.) and northern fox grape (Vitis labrusca L.)

Diurnal variation in petiole specific hydraulic conductivity and simultaneous measurements of leaf water potential were recorded in red maple, tulip tree and fox grape. Petiole specific conductivity was determined from in situ measurements of water flow into the distal (leaf‐bearing) end of an attached petiole as a function of applied hydrostatic pressure and petiole dimensions. The hydraulic properties of the petiole dominated the measurements, indicating that this technique can be used for rapid estimates of petiole hydraulic conductivity. There was a significant decrease in petiole specific conductivity associated with increasingly more negative leaf water potentials in maple and tulip tree, but not in grape. Petiole specific conductivity increased during the afternoon while the plant was actively transpiring and the xylem sap was under tension. The recovery of petiole conductivity during the afternoon suggests that hydraulic conductivity reflects a dynamic balance between a loss of hydraulic conductivity with increasing water stress, and its restoration as tension within the xylem decreases. Three experimental manipulations were applied to red maple and tulip tree to examine the sensitivity of diurnal changes in petiole conductivity to various physiological perturbations. Both phloem girdling and application of HgCl₂ to the transpiration stream resulted in a marked decrease in the degree to which petiole specific conductivity recovered as xylem tension relaxed during the afternoon. Delivery of a surfactant to the xylem, however, did not significantly alter the relation between leaf water potential and petiole hydraulic conductivity.     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

Oligogalacturonides are able to induce flowers to form on tobacco explants

Oligogalacturonides (α-1,4-D-galactosyluronic acid oligomers) are fragments of the homogalacturonan component of the primary cell walls of higher plants. Treatment of cell walls with endopolygalacturonase (EPG) releases the polysaccharides rhamnogalacturonan-1 (RG-I) and rhamnogalacturonan-II (RG-II), and variously sized oligogalacturonide fragments of homogalacturonan. The EPG-released sycamore cell wall components are able to regulate several morphogenetic processes of tobacco thin-cell layer (TCL) explants. We followed one of these morphogenesis-regulating activities, namely, the induction of flower formation on TCLs, to purify the biologically active component from EPG-released material. Saponification of the methyl and acetyl esters of the EPG-released material did not reduce the flower-inducing activity. However, EPG treatment of the deesterified EPG-released material destroyed the flower-inducing activity, establishing that the active substance contained several, consecutive α-1,4-linked galactosyluronic acid residues that are required for the flower-inducing activity. The flower-inducing activity was purified and shown to be α-1,4-linked Oligogalacturonides with a degree of polymerization (DP) of 12-14, which exhibited half-maximum activity at approximately 0.4 μM. Smaller oligogalacturonides, RG-I and RG-II, did not, even at higher concentrations, induce flowers to form. The ability of oligogalacturonides to stimulate the formation of flowers on tobacco explants provides further evidence of the pleiotropic nature of this oligosaccharin.