Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole

When illuminated leaf discs and detached leaves of spinach ( Spinacia oleracea L. cv. Estivato) were exposed to 0.4 and 0.25 μl 1^-1 H₂S, respectively, pool sizes of cysteine and glutathione increased. In the dark, apart from these compounds, the level of γ-glutamyl-cysteine also increased. Incubation of leaf discs with 1.0 m M buthionine sulfoximine (BSO) resulted in the accumulation of cysteine only, both in the light and in darkness. When glycine was supplied to the petioles of detached leaves exposed to H₂S in the dark, the accumulation of glutathione was stimulated, while γ-glutamyl-cysteine accumulation was prevented completely. Glycolate and glyoxylate, precursors of glycine in the glycolate pathway, had nearly the same effect as glycine. Although other amino acids were apparently taken up equally well as glycine when supplied to the petiole, they were much less effective, or not effective at all, in restoring glutathione synthesis in the dark. These results provide evidence, that H₂S-induced glutathione accumulation in spinach leaves in the dark is limited by the availability of glycine, giving rise to the accumulation of the metabolic precursor γ-glutamyl-cysteine.     

Sulphate induced accumulation of glutathione and frost-tolerance of spinach leaf tissue

Water-extractable sulfhydryl content of spinach leaf discs increased up to four-fold when they were incubated with sulphate (10–100 m M ) for 20 h in light or darkness. The accumulated sulfhydryl compound was reduced glutathione. An increased glutathione content did not result in a higher frost-tolerance of the spinach leaf discs. Both freezing temperature and time of exposure to freezing, determined as the point at which 50% of the cells were killed, remained unchanged after incubation with sulphate. These observations suggest that a sulfhydryl compound as glutathione does not play a direct role in protection of plants against freezing injury.     

Stimulation of ACC-dependent ethylene production in citrus leaf discs by light

The influence of light and darkness incubation on in vivo ethylene forming enzyme (EFE) activity in citrus ( Citrus sinensis L. Osbeck cv. Salustiana) mature leaf discs was studied. Leaf discs incubated in light produced higher amounts of ethylene than in darkness. Transfer of discs from light to the dark resulted in a marked inhibition of EFE activity, whereas transfer of discs from the dark to light enhanced ethylene forming activity considerably. Light did not affect 1-aminocyclopropane-l-carboxylie acid (ACC) uptake. Incubation in a CO₂-eniiched atmosphere enhanced EFE activity both in light and in darkness, but light stimulation of EFE activity was apparently not affected by CO₂. Effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, inhibitor of photosynthetic electron flow) and KCN (inhibitor of cytochrome oxidase) were studied. DCMU at 0.2 m M inhibited EFE activity in light, whereas no effect was detected in the dark. On the other hand 1 m M KCN stimulated EFE activity in the light, and no significant effect was observed in the dark. CoCl₂ at 1 m M inhibited ACC-dependent ethylene production, suggesting that ethylene production from ACC is mediated by EFE in citrus leaf discs both in light and in the dark. Cycloheximide also inhibited EFE activity in the light and no effects were detected in the dark. Therefore protein synthesis in light (perhaps EFE synthesis) could be required for the light stimulation of the in vivo EFE activity.     

Plant responses to H2S and SO2 fumigation. II. Differences in metabolism of H2S and SO2 in spinach

Fumigation of spinach (Spinacia oleracea L. cvs Estivato and Monosa) with H₂S or SO, for 1 to 6 days resulted in accumulation of sulfhydryl (SH) compounds in the shoots of both H₂S- and SO₂-exposed plants. The sulfate concentration in shoots of SO₂-exposed plants increased linearly with time. SH accumulation showed saturation kinetics as a function of time as well as H₂S concentration, ascribed to the internal H₂S concentration in the plant and the availability of substrates for glutathione synthesis, respectively. SH compounds accumulated more at lower exposure temperatures, whereas sulfate accumulation was more pronounced at higher temperatures. These results are discussed in relation to the possible foliar uptake of H₂S and SO₂, the temperature dependence of uptake and the water solubility of these gases. The possibility of SO₂-induced H₂S emission rather than sulfate accumulation as a source for SH accumulation is also discussed. Cessation of fumigation resulted in a decrease in SH compounds and sulfate content that could be accounted for by sulfur metabolism and growth, respectively.     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.     

Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C4 plants

When leaf discs of a C₄ species, Alternanthera pungens (L.) H.B. and K. or Amaranthus hypochondriacus L., were preincubated in 7.5 m M NH₄Cl, the pH of the cell sap increased by nearly 0.3 unit, while the activity of phosphoenolpyruvate carboxylase (PEPC) about doubled compared to the cell sap from control leaf discs (preincubated in 5 m M Tricine‐KOH, pH 8.5). The sensitivity of PEPC to L ‐malate (a feedback inhibitor) decreased marginally as a result of cytosolic alkalization. The pH of the cell sap and PEPC activity decreased by nearly 0.4 unit and 50%, respectively, when leaf discs were incubated in weak organic acids such as propionic, butyric or salicylic acid. Thus, our results demonstrate a marked modulation in vivo of cell sap pH and PEPC activity in leaf discs from C₄ plants by external alkalizing or acidifying reagents. The rise in PEPC activity due to alkalization of leaf discs was not sensitive to cycloheximide, implying that cytosolic protein synthesis was not involved in the activation of PEPC. Despite the marked increase in the PEPC activity due to the base‐loading of leaf discs, the change in malate sensitivity of the enzyme was only marginal, indicating that there was no significant increase in the extent of PEPC‐phosphorylation. Besides the physiological significance, the technique of acid/ base‐loading may be an important tool for studying the regulation of PEPC in leaf discs of C₄ species, since the activity of PEPC could be enhanced apparently without phosphorylation of the enzyme.     

Stimulation of h(2)s emission from pumpkin leaves by inhibition of glutathione synthesis

The effect of inhibitors of glutathione (GSH) synthesis, namely gamma-methyl glutamic acid, d-glutamic acid, cystamine, methionine-S-sulfoximine (MSX), buthionine-S-sulfoximine, and GSH itself, on the emission of H(2)S was investigated. All these compounds stimulated H(2)S emission from pumpkin (Cucurbita pepo L. cv Small Sugar Pumpkin) leaf discs in response to sulfate. MSX and GSH were the most effective compounds, stimulating H(2)S emission from leaf discs of mature pumpkin leaves by about 80% in response to sulfate. Both inhibitors did not appreciably enhance H(2)S emission in response to l-cysteine and inhibited H(2)S emission in response to sulfite.Treatment with MSX or GSH enhanced the uptake of sulfate by pumpkin leaf discs, but did not affect the incorporation of sulfate into reduced sulfur compounds. Inhibition of GSH synthesis by MSX or GSH caused an increase in the pool size of cysteine, and, simultaneously, reduced the incorporation of labeled sulfate into cysteine. The incorporation of labeled sulfate into the sulfite and sulfide pools of the cells are stimulated under these conditions.These observations are consistent with the idea that inhibition of GSH synthesis leads to an elevated cysteine pool that inhibits further cysteine synthesis. The H(2)S emitted under these conditions appears to arise from diversion of a precursor of the sulfur moiety of l-cysteine. Therefore, stimulation of H(2)S emission in response to sulfate upon inhibition of GSH synthesis may reflect a role of H(2)S emission in keeping the cysteine concentration below a critical level.     

Development of pH sensitivity of sucrose uptake during ageing of Vicia faba leaf discs

Uptake of [U-¹⁴C]-sucrose (40 m M ) by fresh and aged peeled leaf discs of broad bean ( Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m -chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10^−H M cycloheximide or 5.7 × 10⁻⁵ M actinomycin D. Similarly, when the tissues were treated with 10⁻³ M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca²⁺ (10⁻² M ) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10⁻³ M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins.
The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism.     

Freezing injury in spinach leaf tissue: Effects on water-soluble proteins, protein-sulfhydryl and water-soluble non-protein-sulfhydryl groups

Freezing of spinach leaf discs ( Spinacia aleracea L. cv. Estivato) resulted in an irreversible and parallel loss of protein-sulfhydryl (SH) and water-soluble protein. This decrease was inversely related to the increase in freezing injury as determined by the loss of electrolytes from the tissue after thawing. Loss of proteins and protein-SH occurred during freezing of the tissue and was not enhanced by thawing. The parallel decreases in content of soluble proteins and SH groups make it impossible to determine whether oxidation of protein-SH groups is the primary step in decline of protein content. During freezing the content of non-protein-SH compounds, mainly glutathione (GSH), was decreased to a lesser extent than that of protein-SH. Contrary to protein-SH, the levels of non-protein-SH declined substantially after thawing. The data indicate that GSH is not directly involved in protection of soluble proteins against freezing-induced denaturation.     

The effect of short-term H2S fumigation on water-soluble sulphydryl and glutathione levels in spinach

Abstract. Short-term fumigation of Spinacia oleracea with 380 μg m⁻³ H₂S (250 ppb) resulted in a rapid accumulation of water-soluble SH-compounds in the shoots. After 1 h exposure a substantial increase in the SH-content was already detectable and maximal accumulation, three- to four-fold that in control plants, was observed after 24 h of exposure. Irradiation during H₂S exposure only slightly affected the rate and level of SH-accumulation. H₂S fumigation did not affect the water-soluble SH-content of the roots. Glutathione was the sole water-soluble SH-compound accumulating upon exposure to H₂S. It was calculated that during the first hour of exposure to 380 μg m⁻³ H₂S 39% of the possible absorbed H₂S was converted into glutathione. The SH-content of the water-soluble proteins of the shoots was not affected by H₂S exposure. When fumigation was stopped, a rapid decrease in glutathione content was observed and after 48 h the content was comparable to that of the control plants. Contrary to H₂S, SO₂ fumigation did not result in a rapid accumulation of glutathione in spinach shoots. The possible role of glutathione accumulation during H₂S fumigation is discussed.     

Calcium Requirement for Fast Axonal Transport in Frog Motoneurons

Abstract: Calcium is required to sustain fast axonal transport in sensory neurons of frog and cat. We studied the Ca²⁺ dependence of fast axonal transport in the motoneurons of the lower spinal cord from frog. The accumulation of acetylcholinesterase at a crush on the ventral roots was used to follow axonal transport. Two types of experiments were performed: modification of the medium bathing the ventral roots, alone, and modification of the medium bathing the spinal cord and ventral roots. Incubation (17-18 h) of the ventral roots in Ca²⁺-free medium markedly inhibited acetylcholinesterase transport, a finding that demonstrates a Ca²⁺ requirement for fast axonal transport in motoneurons; when 4 m M MgCl₂ was added to the Ca²⁺-free medium, transport was also greatly reduced. During incubation of the ventral roots in normal medium supplemented with 0.18 m M CoCl₂ transport proceeded normally; but when the Co²⁺ concentration was raised to 1.8 m M , transport was diminished as drastically as in the Ca²⁺-free medium. Incubation of the spinal cord and ventral roots in medium containing 0.18 m M CoCl₂ did not reduce the accumulation of acetylcholinesterase at the crush. Similarly, accumulation of acetylcholinesterase at a crush on the dorsal root was not significantly reduced by exposure of the dorsal root ganglion and root to 0.18 m M Co²⁺. Exposure of sensory cell bodies to 0.18 m M Co₂₊ thus produces differential effects on transport of acetylcholinesterase and on transport of newly synthesized radiolabeled protein.     

Effect of high and low sulfate concentrations on adenosine 5'-phosphosulfate sulfotransferase activity from Lemna minor

The effect of Na₂SO₄ concentrations from 0 to 17.6 m M in the nutrient solution of Lemna minor L. strain 6580 on adenosine 5'-phosphosulfate sulfotransferase activity was examined. Routinely, the plants were cultivated on 0.88 mA SO₄²⁻. The enzyme activity was increased by 50 to 100% after transfer to 0 or 0.0088 m M SO₄²⁻. Transfer back to 0.88 m M rapidly decreased the enzyme activity to the initial level. Cultivation on 17.6 mM Na₂SO₄ redueed extractable adenosine 5'-phosphosulfate sulfotransferase by 50%. The original level was rapidly re-established on 0,88 m M . In control experiments, a decrease in adenosine 5'-phosphosulfate sulfotransferase activity was also induced by K₂ SO₄, whereas NaCl caused a small increase. This indicates that the observed effects are dependent on the sulfate ion. ATP-sulfurylase activity measured for comparison was only significantly affected by the omission of sulfate, which induced a 20% increase, indicating that this enzyme activity from Lemna minor is less suseeptible to changes in medium sulfate than adenosine 5'-phosphosulfate sulfotransferase. A close relationship between adenosine 5'-phosphosulfate sulfotransferase activity and the content of asparagine, glutamine, non-protein thiols and sulfate in the tissue was detected, indicating a positive control mechanism induced by amides and a negative mechanism induced by thiols and sulfate.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

Interaction between exogenous glycine betaine and the photorespiratory pathway in canola leaf discs

The uptake and accumulation of exogenously supplied glycine betaine (GB) by canola (which never accumulates GB in response to stress) leaf discs has been found to induce damage to some of their structural and functional components. As a consequence some free amino acids were accumulated, particularly glutamine and glycine. Similar results were obtained with leaf discs of Arabidopsis thaliana i.e. another cruciferous plant that does not naturally produce significant amounts of GB. In contrast no changes in glutamine and glycine contents were observed in response to the GB treatment in leaf discs of spinach, a natural producer of GB. The change in glutamine content might be related to the senescing effects caused by the GB treatment. Glycine accumulation in response to GB has been more thoroughly studied with canola leaf discs. It only occurred under light conditions and was suppressed under non-photorespiratory conditions. The accumulation of glycine in canola leaf discs in response to GB was either restricted when GB was added in the presence of aminooxyacetate (an inhibitor of transaminases) or enhanced when added in the presence of aminoacetonitrile (an inhibitor of glycine decarboxylation by mitochondria). Both compounds are known to block the glycolate pathway. Glycine accumulation was not found in leaf discs of Zea mays treated in the light in the presence of GB. These results suggest that the absorbed GB could exert destabilizing effects on the photorespiration of the C₃ cruciferous plants canola and Arabidopsis via competitive effects between GB and glycine at the mitochondrial step of the glycolate pathway. The mechanism of the GB effect remains to be elucidated as well as that of its apparent compatibility in spinach, the well known natural producer of GB.     

Characteristics of assimilatory sulfate transport in Rhodobacter sulfidophilus

Abstract Sulfate uptake was investigated with four species of phototrophic sulfur bacteria. Rhodobacter sulfidophilus and Chromatium vinosum took up ³⁵S-labeled sulfate added in micromolar concentrations. Sulfate uptake by C. vinosum was expressed only under sulfate starvation. R. sulfidophilus took up 10 μM sulfate almost completely and accumulated it up to 5300-fold, also when grown with excess sulfate. Sulfite (1 mM) as an intermediate of sulfate assimilation inhibited sulfate uptake completely within 1 min. Moderate inhibition was observed with cysteine (1 mM) and none with sulfide (1 mM). Transport was not dependent on the cations K⁺, Na⁺, Li⁺ or protons, but was sensitive to uncouplers and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). The accumulation of sulfate correlated with the ATP concentration in the cells, indicating an ATP-dependent uptake mechanism.     

Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris)

The antagonistic effects of ethylene and Ag⁺ on the metabolism of [1-¹⁴C]indole-3-acetic acid (IAA) and on the rates of ethylene production were studied in tobacco leaf discs ( Nicotiana rustica var. Brasilia ). During the first 10 h of incubation, Ag⁺-pretreated leaf discs contained more free [¹⁴C]IAA than untreated ones due to decreased oxidative decarboxylation, and the discs also produced more ethylene. Exogenously supplied ethylene nullified these effects of Ag⁺. However, the most pronounced effect of Ag⁺ in increasing ethylene production, as well as the strongest antagonistic effect of exogenous ethylene, were found between 24 and 48 h of incubation. During this time span no effect on the level of free IAA and on its decarboxylation could be observed. It is suggested that ethylene exerted its autoinhibitory effect by a feedback control on the IAA-induced ethylene biosynthesis. Possible mechanisms for the autoinhibitory effect of ethylene are discussed.     

Involvement of abscisic acid and ethylene in the responses of citrus seedlings to salt shock

The responses of salt‐sensitive citrus rootstocks to 200 m M NaCl were periodically determined on seedlings of citrange Carrizo ( Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf) during 30 days. The stressed seedlings adjusted osmotically, reduced stomatal conductance, increased proline content and ethylene production, and showed massive leaf abscission (92%). The salt shock also increased abscisic acid (ABA) and aminocyclopropane‐1‐carboxylic acid (ACC) in roots, xylem fluid and leaves, and in addition promoted Cl⁻ accumulation. The pattern of change of ABA, ACC and proline followed a two‐phase response: an initial transient increase (10‐12 days) overlapping with a gradual and continuous accumulation. This biphasic response appears to be compatible with the proposal that the transitory hormonal rises are induced by the osmotic component of salinity, whereas the Cl⁻ increase determines the subsequent accumulations. During the second phase, Cl⁻ levels correlated with abscission in leaves. Production of leaf ethylene was also concomitant with the increase in the abscission rate. Salt‐induced abscission was either reduced with CoCl₂ (52%) or inhibited with silver thiosulphate (14%). The results suggest that in salt‐stressed citrus, leaf abscission is induced by the chloride build‐up through a mechanism that stimulates leaf ACC synthesis and further conversion to ethylene.     

Interactive effects of Ca2+ and NaCl salinity on the ionic relations and proline accumulation in the primary root tip of Sorghum bicolor

The effects of supplemental Ca²⁺ supply and NaCl salinity on the ionic relations and levels of proline and other amino acids in the primary root of Sorghum bicolor (cv. Hegari) seedlings were investigated. Two days of exposure to 150 m M NaCl resulted in a 50-fold increase in the proline level in the 0–10 mm root tips of seedlings supplied with 5.0 m M Ca²⁺, but only a 4-fold increase in seedlings with 0.5 m M Ca²⁺. In contrast to the high levels of proline in the root tip, proline accumulation was only modest in the expanded tissues of the root. The enhancement of proline accumulation in the root tip of salinized seedlings with the Ca²⁺ supplement may be related to their more favorable tissue K to Na ratio. Thus, elevated Ca²⁺ may mitigate the NaCl-induced inhibition of S. bicolor root growth via the maintenance of net K to Na selectivity and the enhancement of proline accumulation in the root tip.     

Salt tolerance of the reed plant Phragmites communis

Reed plants ( Phragmites communis Trinius) were grown at NaCl concentrations up to 500 m M and their growth, mineral contents and leaf blade osmotic potential were determined. Addition of NaCl up to 300 m M did not affect growth significantly. Sucrose, Cl^-and Na⁺ concentrations in the shoots increased with the salinity of the medium and the shoot water content decreased. K⁺ always contributed most to the leaf osmotic potential. Even in the presence of 250 m M NaCl in the rooting medium, the leaf blade contained only 50 mM Na⁺, suggesting that the plants have an efficient mechanism for Na⁺ exclusion. ²²Na⁺ uptake experiments suggested that the retranslo-cation of absorbed Na⁺ from shoots to the rooting medium lowered the uptake of Na⁺.