Hydrogen Peroxide-Dependent Oxidation of 3,4-Dihydroxyphenylalanine in Vacuoles of Mesophyll Cells of Vicia faba L. Participation of Peroxidase in the Oxidation

Peroxidase activity and 3,4-dihydroxyphenylalanine (DOPA) werefound in vacuoles isolated from mesophyll protoplasts of Viciafaba L. A peroxidase isozyme localized in vacuoles migratedto the cathode during electrophoresis at pH 8.7, indicatingthat the vacuole peroxidase was a basic isozyme. When isolatedvacuoles were treated with 2 mM H₂O₂, dopachrome, a productof oxidation of DOPA, was formed in a reaction that was inhibitedby KCN and NaN₃. These results suggest that DOPA can serve asa donor of electrons to the peroxidase in vacuoles. (Received December 25, 1989; Accepted March 22, 1990)     

Cytochemical localization of peroxidase in soybean suspension culture cells and protoplasts: Intracellular vacuole differentiation and presence of peroxidase in coated vesicles and multivesicular bodies

Summary The ultrastructural localization of peroxidase in soybean (Glycine max L.) suspension culture cells and protoplasts is reported. In cells peroxidase is found primarily in the cell wall and at the tonoplast. Protoplasts and cells contain a vacuolar system which is differentiated with respect to peroxidase content since some vacuoles are found which do not contain peroxidase reaction product. The Golgi dictyosomes, coated and smooth vesicles contain peroxidase. Some of the multivesicular bodies have the reaction product as well. The results are discussed in terms of the pathways of sorting of peroxidase between the cell wall and vacuoles of cultured cells.     

ATP Sulphurylase and O-Acetylserine Sulphydrylase in Isolated Mesophyll Protoplasts and Bundle Sheath Strands of S-deprived Maize Leaves

In Zea mays L. (cv. XL 72 A) leaves sulphur deficiency causedreduction of soluble protein and chlorophyll contents, whereasATP sulphurylase (EC 2.7.7.4 [EC] ) and O-acetylserine sulphydrylase(EC 4.2.95.9 [EC] ) activities increased with the increasing of S-deprivationtime. The two enzymes exhibited the maximum activity after 5d (ATP sulphurylase) and 3 d (O-acetylserine sulphydrylase)from the beginning of deprivation period. The activities weredifferently distributed between mesophyll protoplasts and bundlesheath strands. The results suggest that the activity of thetwo enzymes may be induced sequentially and differently regulatedin the two types of cells. Key words: ATP sulphurylase, Bundle sheath strands, Mesophyll protoplasts, O-acetylserine sulphydrylase, Sulphur deprivation, Zea     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

Stimulation of the oxidative decarboxylation of indole-3-acetic acid in citrus tissues by ethylene

Ethylene has been shown to stimulate the degradation of indole-3-acetic acid (IAA) in citrus leaf tissues via the oxidative decarboxylation pathway, resulting in the accumulation of indole-3-carboxylic acid (ICA). Preliminary data indicated that ethylene stimulates only the first step of this pathway, i.e. the decarboxylation of IAA which leads to the formation of indole-3-methanol. The effect of ethylene seems to be a specific one since 2,5-norbornadiene, an ethylene action inhibitor, significantly inhibited the stimulation of IAA decarboxylation by ethylene. It has long been suggested that peroxidase or a specific form of the peroxidase complex (`IAA oxidase') catalyse this step. However, we did not observe a clear effect of ethylene on the peroxidase system. An alternative possibility, that the stimulatory effect of ethylene on IAA catabolism results from increased formation of hydrogen peroxide (H₂O₂), a co-factor for peroxidase activity, was verified by direct measurements of H₂O₂ in the tissues or by assaying the activity of gluthathione reductase, which has been shown to be induced by oxygen species. This possibility is further supported by the observations showing that IAA decarboxylation in control tissues was enhanced to the level detected in ethylene-treated tissues by application of H₂O₂.     

Glucose Transport in Vitis vinifera L. Protoplasts

Transport of glucose and its analogues was studied in grapevine(Viris vinfera L. cv. Soultanina) leaf protoplasts. The transportsystem was hexose specific and the stereospecificity was closelyrelated to carbon-1 of the glucose molecule. Glucose structuralanalogues were not metabolized beyond the stage of phosphorylationand differences between these compounds and glucose were observedin their transport rates and in their specificity for the carrier.Concentration-dependent uptake of labelled glucose by grapevineprotoplasts was linear for concentrations higher than 1?5 molm^–3 at lower concentrations a saturating pattern was observed.The carrier was driven by the proton motive force and the substrateentered the cell probably in an unchanged form. Efflux studieswere not useful as an indication of the rate of metabolism orassimilation of transported compounds in grapevine protoplasts. Key words: Sugar transport, protoplasts, grapevine     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

Conversion of Indole-3-butyric Acid to Indole-3-acetic Acid by Cuttings of Grapevine (Vitis vinifera) and Olive (Olea europea)

The metabolism of indolebutyric acid (IBA) in hardwood cuttingsof grapevine (Vitis vinifera cv. Perlette) and green cuttingsof olive (Olea europea cvs. Manzanillo, Kalamata and Koroneiki)was investigated. Radioactive IBA which was synthesized in ourlaboratory was used in these studies. Cuttings of both oliveand grapevine converted IBA to IAA. The identity of IAA wasconfirmed by high performance liquid chromatography and gas-liquidchromatography. The stability of IBA, its slow transport from the site of applicationat the base of the cutting and its conversion to IAA in thecutting are probably the factors which make this compound agood root promoter. ¹Contribution from the Agricultural Research Organization, theVolcani Center, Bet Dagan, Israel. No. 619-E, 1982 series. (Received April 28, 1983; Accepted April 12, 1984)     

Stimulation of the Uptake of Sorbitol into Vacuoles from Apple Fruit Flesh by Abscisic Add and into Protoplasts by Indoleacetic Acid

The uptake of sorbitol into vacuoles from immature flesh ofapple fruit (Maluspumila Mill, var domestica Schneid.) was facilitatedby 10^–6 M ABA, while such uptake into protoplasts wasnot stimulated. By contrast, the application of 10^–5 MIAA facilitated uptake of sorbitol into protoplasts but didnot significantly into vacuoles. (Received July 17, 1990; Accepted December 25, 1990)     

The Subcellular Localization of Isoperoxidases in Capsicum annuum Leaves and their Different Expression in Vegetative and Flowered Plants

The subcellular localization of leaf peroxidases (EC 1.11.1.7)and their expression in vegetative and flowered plants has beenstudied in Capsicum annuum (var. annuum) in order to assesswhether the expression of new peroxidase isoenzymes can characterizethe floral state which determines the beginning of reproductivedevelopment. The results showed that floral development is accompaniedby a significant increase in the level of soluble (non-sedimentable)leaf peroxidase, independently of leaf position along the internodes,and therefore independently of the leaf age. An analysis ofthe leaf peroxidase isoenzyme patterns along the internodesfor vegetative and flowered plants shows that the increase inperoxidase activity is due to a general increase in the activityof all the pre-existing peroxidase isoenzymes, although isoenzymeB₂ and, especially, isoenzyme A₁ showed a distinctive and majorincrease in activity. These two isoenzymes are mainly ionically-boundto cell walls, probably in equilibrium with the same isoenzymesmoving freely in the cell-wall free spaces. The differs fromother peroxidase isoenzymes, such as isoperoxidase B₆, whichis mainly located in the covalently-bound cell-wall fractionand in mesophyll vacuoles. These results are discussed in thelight of a possible role of cell wall peroxidases as markersof the floral state in Capsicum annuum morphogenesis.Copyright1993, 1999 Academic Press Capsicum, floral state, leaf peroxidases, subcellular localization, vegetative state     

Cloning and characterisation of a basic IAA oxidase associated with root induction in Vitis vinifera.

Changes in apoplastic peroxidases during auxin-induced in vitro rooting of cultured grapevine (Vitis vinifera L. cv. Touriga) stems have been studied. The largest increase in peroxidase activity (EC 1.11.1.7) was associated with the early stages of root initiation and could be attributed to an increase in activity of an apoplastic 36 kDa cationic peroxidase (PxB2). Relative to other peroxidases, PxB2 demonstrated high indole-3-acetic acid (IAA) oxidase activity and apparently contributed the majority of potential IAA oxidase activity in rooting tissues. The distribution of this peroxidase in developing roots additionally associates it with early phases of growth restriction. PxB2 was purified from cell wall extracts prepared from the basal 1 cm of rooting stems. Microsequencing and subsequent cloning of its corresponding 3' truncated cDNA (encoding 255 amino acids of the mature protein) revealed it to have a typical class III peroxidase structure. The results suggest that this class III peroxidase with IAA oxidase activity is important for the control of IAA levels during root initiation and development.     

Ferricyanide Reduction by Guard Cell Protoplasts

Guard cell protoplasts of Commelina communis L. reduced exogenousferricyanide at pH values lower than 5?0; upon addition of NADH,reduction of ferricyanide by guard cell protoplasts was stimulatedover the pH range 4?0 to 9?0 with two peaks of activity at pH5?0 and between pH 8?0 and pH 9?0. Calcium chloride (1?0 molm^–3) and MgCl2 (1?0 mol m^–3) increased the NADH-stimulatedreduction of ferricyanide. Superoxide dismutase and cyanidehad little effect on the NADH-stimulated reduction of ferricyanideby guard cell protoplasts, but, salicylhydroxamic acid completelyinhibited this activity. The NADH-stimulated reduction of ferricyanidealso occurred in the cell-free supernatant. Horseradish peroxidasedid not reduce ferricyanide in the absence of NADH over a broadrange of pH (4?0 to 9?0). However, in the presence of NADH,horseradish peroxidase reduced ferricyanide over the pH range5?0 to 9?0 with maximal activity at pH 8?0. The NADH-stimulatedreduction of ferricyanide by horseradish peroxidase showed similarproperties to those observed with guard cell protoplasts. Mannitol,superoxide dismutase, and cyanide did not inhibit the NADH-stimulatedreduction of ferricyanide by horseradish peroxidase; SHAM, however,completely inhibited the reduction of ferricyanide by horseradishperoxidase. Catalase inhibited the NADH-stimulated reductionof ferricyanide by horseradish peroxidase by 20%, while absenceof oxygen in the assay medium stimulated this activity over60%. We propose that the reduction of ferricyanide in the presenceof NADH by guard cell protoplasts, can be explained in termsof peroxidase activity associated with the plasma membrane andsecreted to the extracellular medium. However, the capacityof guard cell protoplasts to reduce ferricyanide at acid pHvalues where little peroxidase activity occurs may indicatethe presence of a plasma membrane redox system in guard cellsof C. communis. Key words: Commelina, guard cell protoplasts, ferricyanide reduction, peroxidase, redox system     

Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation

Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI-TOF-MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation.     

Increased Production of IAA by Rhizoctonia solani is Induced by Culture Filtrate from Rice Suspension Cultures

To investigate the role of the plant hormones produced by fungi,we tried to construct a system to examine the interaction betweenRhizoctonia solani Kühn MAFF305219 and rice cells in suspensionculture (Oc). R. solani was previously found to produce IAA,with the main biosynthetic pathway via the indole-3-pyruvatepathway. The amount of IAA in the medium produced by R. solaniwas increased by cocultivation with rice cells (Oc) and by culturefiltrate (CF) of Oc. Further analysis revealed that the factor(s)that induced the enhanced accumulation of IAA was sensitiveto heat, to freezing and thawing and lyophilization, and themolecular weight was estimated to more than 10,000. These resultssuggest that the active agent(s) in the medium was (a) proteinor a proteinous substance. Among suspension cultures of variousplants, Oc and another line of rice cells (Ok) had the abilityto induce the accumulation of IAA in the fungal medium 4 h afterinoculation but other cultures of plant cells were ineffective.The promotive effect of rice CF on the accumulation of IAA wasalso observed with some strains of R. solani that belong toa different anastmosis group from MAFF305219. Thus, the accumulationof IAA was not related to the host specificity. (Received July 28, 1997; Accepted October 27, 1997)     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)     

Isolation of Rhizobium leguminosarum biovar viciae Variants with Indole-3-acetamide Hydrolase Activity

The IAA biosynthetic pathway from tryptophan to IAA via IAM(IAM pathway) was investigated in Rhizobium spp. (fast-growingrhizobium). Southern hybridization with the bam gene, a structuralgene for IAM hydrolase (the enzyme that converts IAM to IAA)cloned from Bradyrhizobium japonicum J1063, indicated that homologoussequences exist among wild-type Rhizobium spp. However the IAMpathway has not been detected biochemically in free-living bacteria.When 5-methyltryptophan-resistant strains were screened forRhizobium leguminosarum biovar viciae K5 which has DNA sequenceswith high homology to the bam gene, spontaneous mutants showingIAM hydrolase activity were isolated. The results suggest thepossibility that the activity of IAM hydrolase is suppressedin free-living state in Rhizobium leguminosarum biovar viciaeK5. In addition we detected the peak at the same t_R of IAM byHPLC analysis using two columns when a large amount of L-tryptophanwas added to the suspension of 5-methyltryptophan-resistantvariants. Whether or not tryptophan-2-monooxygenase activity,however, actually works in Rhizobium cells remained to be solved. (Received September 20, 1989; Accepted March 6, 1990)     

Efficient Conversion of L-Tryptophan to Indole-3-Acetic Acid and/or Tryptophol by Some Species of Rhizoctonia

In a study of various phytopathogenic fungi, we found that fungithat belong to the genus Rhizoctonia produce IAA efficientlyfrom tryptophan. R. solani Kühn MAFF-305219, in particular,produced large amounts of tryptophol (Tol), which was assumedto be a specific by-product of the indole-3-pyruvate (IPy) pathway,in addition to IAA. Therefore, this fungus seemed suitable foranalysis of the function and the regulation of the biosynthesisof auxin by a fungal pathogen. Under normal aerobic conditions,the ratio of IAA to Tol synthesized by this strain was higherthan that under less aerobic conditions. In metabolic studieswith various indole derivatives, R. solani converted L-tryptophanand indole-3-acetaldehyde to IAA and Tol, but other indole derivativeswere scarcely metabolized. These results suggest that both IAAand Tol are synthesized from tryptophan through the IPy pathwayin Rhizoctonia. (Received May 27, 1996; Accepted July 8, 1996)     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

The Permeability of the Guard Cell Plasma Membrane and Tonoplast

Uptake experiments and efflux compartmental analysis of planthormones, osmotica and toxins using ‘isolated’ guardcells of Valerianella locusta and guard cell protoplasts (GCP)of Vicia faba were performed in order to study the permeabilityproperties of guard cell plasma membrane and tonoplast. Theplasma membrane of guard cells exhibits a higher permeabilitythan plasma membranes of mesophyll cells for most solutes investigated.The permeability coefficients (P_s calculated for the guard cellplasma membranes are also significantly higher than the P_s valuesfor the guard cell tonoplast. This applies also for protonatedABA. We suppose that the high permeability for ABAH could bepart of the target cell properties. A Collander analysis demonstratesa linear correlation between P_s, values and the ratio K_r/M_r^1,5for both plasma membrane (r = 0.87) and for the tonoplast (r=0.93). Because of deviations from the observed correlations,the permeation of some solutes (ABA, GA, IAA through the tonoplast;methylamine through the plasma membrane) seems to be facilitatedby an additional transport mechanism. The Collander analysisof the plasma membrane of GCP shows very similar results tothe analysis of the plasma membrane of ‘isolated’guard cells, indicating that isolation of protoplasts does notalter the permeability of the guard cell plasma membrane. Key words: Permeability coefficient, guard cells, plasma membrane, tonoplast     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)