Vacuolar localization of wound-induced carboxypeptidase inhibitor in potato leaves

The wound-induced carboxypeptidase inhibitor in potato leaves was shown to be localized in the central vacuoles of the cells. The inhibitor was quantified by immunological assays (ELISA) in protoplasts and vacuoles isolated from upper unwounded leaves of 5- to 6-week old potato plants that had been wounded on their lower leaves 48 hours earlier to induce the accumulation of the carboxypeptidase inhibitor. The regulation of the synthesis and compartmentation of the inhibitor is similar to that of wound-induced serine proteinase Inhibitors I and II in potato and tomato leaves and appears to be part of an induced defense response against attacking pests.     

Preparation and purification of glucanase and chitinase from bean leaves

Glucanase (endo-β-1, 3-glucan 3-glucanohydrolase, EC 3.2.1.6, laminarinase, callase) and chitinase (poly-β-1, 4-[2-acetamido-2-deoxy] -d-glucoside glycanohydrolase, EC 3.2.1.14) were extracted from ethylene-treated bean (Phaseolus vulgaris L. cv. Red Kidney) leaves and purified on hydroxyapatite and carboxymethyl Sephadex columns. The glucanase prepared was homogeneous as judged by analytical centrifugation data, electrophoresis, and antibody-antigen reactions. On the basis of gel filtration, antibody-antigen reactions, and amino acid analysis, the molecular weight was estimated to be between 11,500 and 12,500. However, ultracentrifugation gave a higher estimate of 34,000. The glucanase had an isoelectric point near pH 11 and was specific for β-1, 3-linkages. The chitinase was only partially purified as judged by electrophoretic behavior.     

Identification of chitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene-induced abscission

Abstract. Total RNA was extracted from bean leaf abscission zones at different times after the induction of abscission by ethylene. The RNA was translated in the wheat germ system and the products analysed by SDS-PAGE. Products of molecular weight (raw) 42, 32 and 17 kD were seen to accumulate substantially during the induction. An attempt was made to establish that the mRNA species which produced the 32 kD product, which was coded for the ethylene-regulated enzyme chitinase. Mature chitinase (30 kD) was purifed from ethylene-treated abscission zones and used to raise monospecific antibodies in rabbits. These antibodies recognized the 32 kD product and mature chitinase. The 2 kD difference in molecular weight was due to the presence of the signal sequence which could be removed by microsomal membranes. Chitinase was also detected by enzymatic assay and immunoblotting of crude homogenates from ethylene-treated abscission zones. Chitinase appears to be ubiquitous in bean plants and probably does not have a direct role in abscission.     

Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves

Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.     

Vacuolar solutes in the upper epidermis of barley leaves

The concentrations of vacuolar solutes in different cells of the upper epidermis of the third leaf of barley (Hordeum vulgare L.) were studied in leaves of different ages grown under different irradiances (120 or 400 mol photons·m^–2·s^–1). Vacuolar saps were extracted from individual cells located at various positions between adjacent veins and were analysed for their osmolality and the concentrations of K⁺, Ca²⁺, Cl^–, NO ₃ ^– and malate. Each ion showed a cell-specific distribution within the epidermis that was both quantitatively and qualitatively dependent on the leaf developmental stage and on the light level. During leaf ageing, Ca²⁺ accumulated preferentially in interstomatal cells (i.e. those located between longitudinally adjacent stomata) at concentrations up to 180 mM. Under low light conditions, this was accompanied by a more or less equal decrease in K⁺ concentration. Epidermal malate was found only in plants grown continuously or transiently under the high irradiance and reached highest concentrations in trough and interstomatal cells (60 to 150mM). Chloride concentration was highest in cells overlying the veins (designated as ridge cells) and lowest in cells located between the veins (trough cells), while NO ₃ ^– exhibited the reverse distribution, although the precise patterns were age-dependent. Epidermal osmolality increased with age, but the intercellular differences in the osmolalities were small compared to differences in vacuolar solute composition. A cell-to-cell analysis of the region surrounding the stomata showed that the steepest changes in the vacuolar solute composition of epidermal cells occurred at the boundary between ridge or trough cells and the adjacent near-stomatal cells.Abbreviations EDX analysis energy dispersive X-ray analysis We wish to thank Andrew Davies and Alison Bell (Bangor) for their technical advice. This work was financed as an Agricultural and Food Research Council Linked Research Group project between Bangor and Rothamsted (grants LR5/187 and 521).     

Vacuolar localization of the enzymatic synthesis of hydroxycinnamic acid esters of malic acid in protoplasts from Raphanus sativus leaves

Protoplasts from leaves of radish ( Raphanus sativus L. var. sativus ) were examined for the subcellular localization of p -coumaric, caffeic, ferulic and sinapic acid esters of malic acid and the enzyme(s) involved in their syntheses. Vacuoles isolated from leaf protoplasts contained all the hydroxycinnamic acid esters as well as all the dependent enzyme activities. Protein from leaf vacuoles was shown to form the hydroxycinnamoylmalic acids, using the corresponding hydroxycinnamic acid glucose esters (1-O-acyl glucosides) as acyl donors. It is proposed that the vacuole is the cell compartment for synthesis and deposition of the hydroxycinnamoylmalic acids.     

Carboanhydrase from bean leaves

A highly purified preparation of carboanhydrase from the leaves of the bean Vicia faba var. major (Harz) F. Janthina was obtained. The enzyme was homogeneous during analytical disc-electrophoresis in polyacrylamide gel and analytical ultracentrifugation. The molecular weight of the enzyme is 270,000. The enzyme molecule contains six Zn atoms, has a quaternary structure and is made up of six subunits with molecular weights of 45,000. Some properties of the enzymes from bean leaves and pea leaves were compared. The enzymes differ in molecular weights and behaviour during DEAE-cellulose chromatography and gel filtration through Sephadex G-200.     

Protochlorophyllide resynthesis in dark-grown bean leaves

The protochlorophyllide content of dark-grown bean leaves was determined at various ages. It was detectable the second day after germination and reached a maximum on about the tenth day.     

A chitinase with antifungal activity from the mung bean

A chitinase with antifungal activity was isolated from mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, (NH₄)₂SO₄ precipitation, ion-exchange chromatography on CM-Sepharose, high-performance liquid chromatography (HPLC) on Poros HS-20, and gel filtration on Sephadex G-75. The protein exhibited a molecular mass of 30.8 kDa in SDS–polyacrylamide gel electrophoresis. Its pI was 6.3 as determined by isoelectric focusing. The specific activity of the chitinase was estimated to be 3.81 U/mg. The enzyme expressed its optimum activity at pH 5.4 and was stable from 40 to 50 °C. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum, and Sclerotium rolfsii.     

The development of plastocyanin in greening bean leaves

The plastocyanin content of etiolated bean leaves (Phaseolus vulgaris L.) was measured, and the development of the protein in response to light was followed. Measurements were made by quantitative extraction of plastocyanin and a sensitive assay with an O₂ electrode. The electron-paramagnetic-resonance (e.p.r.) signal of oxidized plastocyanin was used as an independent check on the validity of the assay method, and on the thoroughness of extraction. After an initial lag period, the amount of plastocyanin in greening bean leaves increased to reach a maximum after 50h illumination. The chlorophyll/plastocyanin ratio reached a maximum value of 200 irrespective of the light intensity at which greening was carried out, suggesting that the synthesis of the two components is co-ordinated. Experiments involving treatment of etiolated seedlings with brief periods of light of different spectral composition indicated that phytochrome is involved in plastocyanin synthesis. The lack of inhibition of plastocyanin synthesis by specific inhibitors of chloroplast protein synthesis suggests that the protein is synthesized on cytoplasmic ribosomes. The data are discussed in relation to the development of ferredoxin in greening bean leaves.     

Metabolism of cytidine and uridine in bean leaves

The metabolism of cytidine-2-¹⁴C and uridine-2-¹⁴C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.     

Heat sensitivity of mycelium and haustoria in bean leaves infected with bean rust

Bean leaves experimentally inoculated with bean rust were subjected to heat treatment at 50° for 20 s, at different times after inoculation, and the modifications induced by heating in the pathogen and host cells were studied by electron microscopy, at different times after the treatment. This induced no alterations either in healthy or infected leaf cells. On the contrary, it caused severe ultrastructural alterations in the pathogen. These alterations were already visible in the haustoria and hyphae at the end of the treatment, became progressively more severe with time, and led to the death of the pathogen two days after the treatment. From this time onwards the haustoria appeared shrunken, extremely electron-dense, and encased in a mass of granular material, seemingly secreted by the host cell. No encasement was ever found around haustoria apparently dead by senescence in untreated leaves.     

Superoxide dismutases from kidney bean leaves

Three isozymes of superoxide dismutase were found in the solubleextract of kidney bean leaves. Two of them, purified to nearhomogeneous states, were inhibited by cyanide and by the antibodyto spinach Cu, Zn-superoxide dismutase. Thus, both isozymeswere the Cu and Zn containing enzyme that has a molecular weightof 32,000 and is composed of two subunits of equal size. Anotherisozyme was insensitive to cyanide and to the antibody, butwas inactivated by acetone or heating. The cyanideinsensitiveisozyme was not inactivated by H₂O₂ showing that this isozymeis Mn-superoxide dismutase. (Received June 13, 1979; )     

Intracellular localization of a class IV chitinase from yam

Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci. Biotechnol. Biochem., 68, 1508-1517 (2004)). But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants. In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated. The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles. This is the first report presenting the existence of vacuolar type class IV chitinase.     

Quantitative studies on ferredoxin in greening bean leaves

Two methods of measuring small amounts of the iron-sulphur protein ferredoxin are described. One involves measurements of the signal at g=1.96 produced by reduced ferredoxin in an e.p.r. (electron-paramagnetic-resonance) spectrometer; the other depends on the rate of ferredoxin-dependent electron transport in a chloroplast bioassay measured in an O(2) electrode. These methods of measurement were used to examine the development of ferredoxin during the greening of etiolated bean leaves. Ferredoxin is present in low concentrations in the leaves and cotyledons of 14-day-old etiolated beans (Phaseolus vulgaris L. var. Canadian Wonder), and develops in a linear manner with time when the leaves are illuminated. This synthesis appears to be independent of chlorophyll synthesis during the early stages of greening. However, the chlorophyll/ferredoxin ratio reaches a final value of approx. 360 irrespective of the light intensity, indicating the existence of a control mechanism operative in deciding the stoicheiometry of these components in the mature chloroplast. The ferredoxin synthesis appears to be light-dependent, and red light is the most effective in its promotion. The effect of red illumination is not reversed by far-red light, indicating the absence of a phytochrome control of ferredoxin synthesis. From experiments using specific inhibitors of chloroplast protein synthesis, it is concluded that ferredoxin is synthesized on cytoplasmic ribosomes.