STUDIES OF VEGETATIVE COMPATIBILITY-INCOMPATIBILITY IN HIGHER PLANTS. IV. THE DEVELOPMENT OF TENSILE STRENGTH IN A COMPATIBLE AND AN INCOMPATIBLE GRAFT

Three phases of adhesion between the stock and scion are observable during the formation of a compatible autograft in Sedum telephoides and an incompatible heterograft between Sedum telephoides and Solanum pennellii. The first phase of adhesion is similar in both systems in that it 1) lasts 2 to 3 days, and 2) is characterized by an average increase in tensile strength of 1 g breaking weight (BW)/mm² graft area (GA)/day. In the compatible Sedum autograft, the second phase of adhesion lasts from Days 3 to 11 after grafting and is correlated with a 28-fold increase in the tensile strength of the graft union to approximately 56 g BW/mm² GA by 11 days after grafting. The third phase of adhesion in the compatible autograft is characterized by a leveling off of the tensile strength of the graft union at approximately 56 g BW/mm² GA, roughly equal to that of an ungrafted internode. Graft formation is now complete. These results suggest that the ratio of the tensile strength of the graft union : tensile strength of a comparable ungrafted internode provides an estimate of the percent development of compatible autografts. In the incompatible heterograft between Sedum and Solanum, Phase II adhesion 1) lasts from Days 2 to 5 after grafting, and 2) peaks at 12 g BW/mm² GA at 5 days after grafting. Phase III adhesion in the incompatible heterograft occurs subsequent to Day 5 after grafting and is characterized by an average decrease in the tensile strength of the graft union of 0.3 g BW/mm² GA/day. The results of this study are discussed relative to the quantitative contributions of various structural events to the tensile strength of a graft union.     

Further Evidence For Cell Wall Deposition During Graft Formation

Cells of Sedum telephoides undergoing lethal cellular senescencein response to grafting with Solanum pennellii often possessplasmalemmal tubules (PT). The PT are unbranched, 27–31nm in diameter, and are bordered by a wall measuring 4.8–5.2nm in thickness. No regular substructure is discernible in thelumen of the PT. At 36 h after grafting PT extend into the cytoplasmof Sedum cells at the graft interface between Sedum and Solanum.By 10 days after grafting, however, PT are found embedded innewly deposited cell wall. These results indicate that cellwall deposition occurs during the early stages of graft development. grafting, plasmalemmal tubules, cell wall, Sedum telephoides, Solanum pennellii     

Wound healing and in vitro antiradical activity of five Sedum species grown within two sites of community importance in Emilia-Romagna (Italy)

Sedum genus includes more than 400 different species, many of which having ethnobotany interest. The skin healing is one of the most common therapeutic indication of Sedum spp. In this work, for the first time, we compared five different Sedum species grown in two sites of community importance in Emilia Romagna (Italy): Sedum acre L., Sedum album L., Sedum hispanicum L., Sedum rupestre L. and Sedum sexangulare L., analysing their total phenolic and flavonoid content, their antiradical capacity and the in vitro healing activity on human keratinocytes. Total phenolic content of the five species ranged from 35.41?±?1.18 to 90.22?±?1.03?µg gallic acid equivalent/mg of dry extract, being S. rupestre the richest one. Total flavonoid content ranged from 22.39?±?0.51 to 47.93?±?2.82?µg rutin equivalent/mg of extract and S. album resulted the species with the highest flavonoid content. Antiradical capacity was found to be related to the phenolic content of the extracts. All the extracts were active in wound healing assay and each one showed different kinetic of action and concentration-activity relationship. This study proposes few investigated Sedum species grown in Italy as promising agents for skin healing and suggests further phytochemical and biological investigations.     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonance The very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Sucrose phosphatase associated with vacuole preparations from red beet, sugar beet, and immature sugarcane stem

The specific phosphatase, sucrose phosphate phosphohydrolase (sucrose phosphatase, EC 3.1.3.24) was present in vacuole preparations from storage tissue of red beet (Beta vulgaris L.), sugar beet (Beta vulgaris L. cultivar Kawemono), and immature sugarcane (Saccharum spp. hybrid, cultivar NCO 310). In red beet vacuole preparations the specific activity of sucrose phosphatase, using the naturally occurring vacuole marker, betanin, as reference, was higher than the specific activity of cytoplasmic markers, phosphoenolpyruvate carboxylase and glucose 6-phosphate dehydrogenase, suggesting that sucrose phosphatase is associated with the vacuoles. High speed centrifugation of lysed vacuoles did not result in precipitation of the enzyme indicating that the enzyme is not tightly bound to the tonoplast. Sucrose phosphatase was more sensitive to inhibition by sodium vanadate and less sensitive to ammonium molybdate than was the nonspecific phosphatase which was also present in the extracts. Sucrose phosphatase might be part of the group translocator proposed recently to operate in the tonoplast of sugarcane and red beet.     

Immunological studies on phosphoenolpyruvate carboxylase in Sedum species with crassulacean acid metabolism or C3 photosynthesis

Abstract From Sedum morganianum, which is a plant species known to have constitutive crassulacean acid metabolism (CAM), phosphoenolpyruvate (PEP) carboxylase (E.C.4.1.1.31) has been extracted and purified by (NH₄)₂SC₄ precipitation, ion exchange chromatography and gel electrophoresis. A specific antibody to this purified enzyme was obtained by immunization of a rabbit. This antibody was used to compare the antigen–antibody reaction of PEP-carboxylases prepared from other Sedum species including constitutive, facultative and non-CAM plants. The experiments revealed partial immunological indentity of PEP-carboxylases obtained from the different sources.     

New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress

Background  

The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp.     

Alkaline phosphatase production during sporulation ofBacillus cereus

Cell-bound alkaline phosphatase ofBacillus cereus was produced during vegetative growth and sporulation in a complex medium. Addition of glucose repressed the sporulation process and the amount of enzyme synthesized increased. The time course of alkaline phosphatase production is very similar in both sporulating and non-sporulating cells. Irrespective of sporulation, alkaline phosphatase level shows a peak of activity in the exponential phase, and another in the stationary phase of growth. This preliminary data indicates differences betweenB. cereus, andB. subtilis in alkaline phosphatase characteristics.     

The presence and possible significance of cross-reactive antigens inRhizobium — legume associations

Soluble antigens of threeRhizobium species were compared by the agar-gel double-diffusion technique with those of eight legumes representing compatible and non-compatible hosts. Cross-reactive antigens were found between all the legume hosts and the three rhizobia. These common antigens among hosts and bacteria were not related to the specificity of compatibleRhizobium-legume associations. The cross-reactive antigens were absent between rhizobia and eight non-legume plants tested, but present between five out of eleven gramnegative phytopathogenic bacteria and legumes. The potential significance of cross-reactive antigens betweenRhizobium and legume hosts in nodule development is discussed.     

Metabolite profiling and assessment of metabolome compartmentation of soybean leaves using non-aqueous fractionation and GC-MS analysis

In the present study, non-aqueous fractionation (NAQF) and GC-MS were used to obtain a spatially resolved view of metabolism in mature leaves of soybean (Glycine max Merr.). NAQF of lyophilized soybean leaves was performed using CCl₄-n-heptane and ultracentrifugation that yielded a gradient comprised of six fractions. Chlorophyll content, and marker enzyme activities, phosphoenolpyruvate carboxylase (PEPC) and α-mannosidase, were utilized as stroma, cytosol and vacuole markers, respectively. GC-MS analyses of each fraction resulted in the identification of around 100 different metabolites. The distribution of these identified compounds showed a decreasing order from the vacuole to cytosol to chloroplast stroma. In other words, a greater number of identified compounds were found in the vacuole when compared to the cytosol or stroma. Levels of sugars, organic acids and fatty acids showed greater relative abundances in the vacuole with 50, 55, and 50% of the respective pools. A greater relative abundance of amino acids was observed in the cytosol where 45% of the total of amino acids content was recorded. The relatively large pool of sugars and phenolic acids in the vacuole compartment implies high levels of starch metabolism and phenylpropanoid biosynthesis. The low amino acids pool, on the other hand, suggests low nitrogen accumulation in the leaves of soybean. Hierarchical cluster analysis on the most abundant metabolites revealed three clusters containing 10, 20, and 2 of the 32 selected metabolites. The data were discussed in term of NAQF and GC-MS analysis of soybean mature leaves, and also in term of distribution and compartmentation of metabolites at subcellular levels.     

Subcellular localization of tryptophan decarboxylase,strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata

The subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus (L.) G. Don and Tabernaemontana divaricata (L.) R. Br. ex Roem. et Schult, was investigated. It was found that tryptophan decarboxylase is an extra-vacuolar enzyme, whereas strictosidine synthase is active inside the vacuole. Strong indications were obtained for the localization of strictosidine glucosidase on the outside of the tonoplast. The results suggest that tryptamine is transported into the vacuole where it is condensed with secologanin to form strictosidine, and that strictosidine passes the tonoplast and is subsequently hydrolysed outside the vacuole.Abbreviations AM -mannosidase - EDTA ethylenediaminetetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPLC highperformance liquid chromatography - MDH malate dehydrogenase - SG strictosidine glucosidase - SSS strictosidine synthase - TDC tryptophan decarboxylase     

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.     

Movement of Abscisic Acid Across the Plasmalemma and the Tonoplast of Guard Cells of Valerianella locusta

Uptake experiments and efflux compartmental analyses of abscisic acid (ABA) with acid treated epidermal peels of Valerianella locusta were performed to elucidate the mechanisms of transport of ABA across the plasmalemma and tonoplast of guard cells. ABA uptake across the plasmalemma is linearly correlated with external ABA concentration in the incubation medium. Under alkaline conditions ABA-uptake was not significantly above background, indicating that ABA uptake occurs mainly by diffusion of undissociated ABAH as the most permeable species, which is trapped afterwards in the alkaline cytosol as impermeable ABA^?. Efflux analysis of ABA revealed a saturable component of ABA transfer across the tonoplast. A Woolf-Augustinsson-Hofstee analysis suggested the existence of two transport systems for ABA at the tonoplast. The high affinity transport system had a K_M of 0.21 mol m^?3 and a V_max 85.8 amol ABA cell^?1 h^?1. Using the data of the uptake and efflux experiments we calculated the permeability coefficients of ABA for the plasmalemma and the tonoplast of guard cells, which are 2.46 10^?7 m s–¹ and 1.26 10^?8m s^?1, respectively. The distribution of the pH-probe (¹⁴C)-DMO between medium, cytosol and vacuole was investigated and used to calculate cytosolic and vacuolar pH. The vacuolar pH is too low to explain the high vacuolar ABA concentration by trapping of ABA^?, whereas the cytosol is sufficiently alkaline to act as an efficient anion trap. Therefore we conclude that ABA transport across the guard cell tonoplast is catalyzed by a saturable uptake component.     

Dormancy in bulbils of several herbaceous plants: Effects of photoperiod,light, temperature,oxygen and gibberellic acid

Effects of some environmental conditions (photoperiod, white and colored lights, temperature, partial oxygen pressure) and growth regulators (gibberellic acid, 2-chloroethyltrimethylammonium chloride) on induction and release of dormancy of the bulbils ofDioscorea batalas, Laportea bulbifera, Elatostema involucratum andSedum bulbiferum were investigated. Bulbils were formed under short-day conditions inLaportea andElatostema, under long-day conditions inSedum, and irrespective of photoperiods inDioscorea. In all species exceptSedum, immature bulbils required light, particularly blue or far red, for sprouting (photo-sprouting stage), and mature bulbils required a cold treatment (thermo-sprouting stage). The duration of photo-sprouting and thermo-sprouting stages and the degree of dependency on light or low temperature of sprouting differed from species to species. Sprouting of chilled mature bulbils of these species was promoted by light, especially by red or green light. Both immature and mature bulbils ofSedum sprouted under short-day conditions. Continuous irradiation with blue, far-red and green light markedly inhibited their sprouting. Oxygen at high concentration inhibited the sprouting of immature bulbils inDioscorea; in the other species it promoted sprouting regardless of the maturation of the bulbils. Applications of gibberellic acid caused the sprouting of bulbils the absence of light, chilling or photoperiodic treatment in all species exceptDioscorea, in which gibberellic acid inhibited sprouting. Polyphenol oxidase activity was very high in the homogenates ofDioscorea bulbils, and increased further when the bulbils had been treated with gibberellic acid. In the other species, little or no such activity was observed.     

Isolation and partial characterization of vacuoles from tobacco protoplasts

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.     

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.     

Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles

Vacuoles isolated from storage root tissue of red beet (Beta vulgaris L.) do not leak significant quantities of betanin, sucrose, Na⁺ or K⁺ during isolation. This indicates that analysis of vacuoles in vitro gives meanigful information about the compartmentation of solutes in vivo. Preparations of vacouoles were used to determine the distribution of glycinebetaine and proline between vacuole and cytoplasm in beet cells. Both compounds were detected in preparations of isolated beet vacuoles. In the case of glycinebetaine it was shown that this solute was associated with the vacuoles, not with the small number of other organelles which contaminated the preparations. The vacuolar pool accounted for 26 to 84% of the total tissue glycinebetaine and 17 to 57% of the proline. Concentrations of these compounds in vacuole and cytoplasm were calculated and were always higher in the cytoplasm than in the vacuole. The concentration gradient across the tonoplast varied considerably. The significance of these results is discussed in relation to the hypothesis that glycinebetaine and proline function as benign cytoplasmic osmotica.Abbreviations A₅₃₇ absorbance at 537 nm - MES 2-(N-morpholino)-ethanesulphonic acid - Na₂EDTA ethylenediaminetetraacetic acid, disodium salt - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl)methylamine     

Molecular Markers for Ion Compartmentation in Cells of Higher Plants: II. LIPID COMPOSITION OF THE TONOPLAST OF THE HALOPHYTE SUAEDA MARITIMA (L.) DUM.

Vacuoles of high purity were isolated from the leaves of thehalophyte Suaeda maritima (L.) Dum. The relative compositionsof phospholipids, phytosterols, and fatty acids in the tonoplastmembrane were determined and membrane fluidity was assessedby electron spin resonance. The characteristics of the tonoplastwere consistent with minimizing passive permeability to NaCI.The phospholipid: protein ratio (1.1: 1.0) was higher than thatrecorded in other membrane preparations, including vacuolesfrom beetroot storage material, commensurate with the low density(1.05 g cm^–3) of the S. maritima tonoplast. The tonoplastfatty acids were highly saturated and dominated by n-hexadecanoicacid and n-octadecanoic acid. Phytosterols identified by gaschromatography were cholesterol, campesterol, stigmasterol,and ß-sitosterol. Cholesterol was a trace percentagein protoplasts, but comprised 30% of the tonoplast sterols.Semi-quantitative analysis by chromatography on silica gel revealedan enrichment in the tonoplast of glycolipid which was not accountedfor as chloroplast contamination. The fluidity of the tonoplast,determined by electron spin resonance, was lower than the protoplasts,consistent with the high degree of saturation of the fatty acidchains. The relevance of the lipid composition of the tonoplastto its central role in ion compartmentation within the halophytecell is discussed. Key words: Ion compartmentation, membrane lipids, salinity, vacuole     

Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.