Relationship between polar basipetal auxin transport and acropetal Ca2+ transport into tomato fruits

The dependence of acropetal Ca²⁺ transport on polar basipetal indoleacetic acid (IAA) transport was investigated in excised tomato fruits ( Lycopersicon esculentum L. Mill.) using an in vitro fruit system. Auxin transport inhibitors like triiodobenzoic acid (TIBA), chlorofluorenolmethyl ester (CME) and naphthylphthalamic acid (NPA) were used in order to investigate the effect of restricted polar basipetal auxin transport on the acropetal transport of ⁴⁵Ca²⁺, ⁸⁶Rb⁺ and ⁹⁸Sr²⁺ into the same fruits. TIBA and CME inhibited basipetal transport of IAA. particularly in 10- to 12-day-old tomato fruits, and simultaneously restricted the acropetal transport of ⁴⁵Ca²⁺. The auxin transport inhibitors failed to significantly reduce the upward transport of ⁸⁶Rb⁺ and the transport of ⁹⁶Sr²⁺ was less inhibited than that of ⁴⁵Ca²⁺. TIBA and CME did not significantly affect the acropetal transport of labelled water into the fruit, nor the cation-exchange capacity or K⁺ and Mg²⁺ concentrations in the tomato fruit. These results support the view that a part of the Ca²⁺-specific acropetal transport into tomato fruits is associated with the polar basipetal IAA transport. This Ca²⁺ transport is independent of the transpiration stream into the fruit and the cation exchange capacity of the fruit tissue.     

Systemin transiently depolarizes the tomato mesophyll cell membrane and antagonizes fusicoccin-induced extracellular acidification of mesophyll tissue

The plant polypeptide signal systemin induces proteinase inhibitor synthesis in tomato leaves. We show here that systemin elicits a transient depolarization of the tomato mesophyll cell membrane. Furthermore it triggers a transient decrease in the external pH of the mesophyll tissue which is followed by a sustained pH increase. In the presence of fusicoccin (which has been shown to antagonize the synthesis of proteinase inhibitors) the depolarization and transient H⁺ efflux are attenuated whereas the slower phase of the sustained electroneutral H⁺ influx persists. These results suggest that systemin-induced changes in ion transport play a role in the early phases of systemin signal transduction.     

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall-bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 m M NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five m M CaCl₂ did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca²⁺ levels and ionic strength of the apoplast.     

Inhibition of brassinosteroid-induced epinasty in tomato plants by aminooxyacetic acid and Co2+

The inhibitory effects of aminooxyacetic acid (AOA) and cobalt chloride (CoCl₂) on brassinosteroid (BR)-induced epinasty in tomato plants ( Lycopersicon esculentum Mill. cv. Heinz 1350) are evaluated. CoCl₂ dramatically decreases petiole bending and ethylene production as the concentration increases from 50 to 200 μ M. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petiole, instead of accumulating, is reduced and does not change over the concentration range tested. Inhibition of BR-induced epinasty by AOA results from inhibition of ACC synthesis. There are dramatic reductions in petiole bending, ethylene and ACC production as the concentration of AOA is increased from 50 to 200 μ M. Maximum inhibition occurs when the plants are pretreated with the inhibitors. The degree of inhibition increases as the length of pretreatment increases from 1 to 4 h. The response of BR-treated plants to AOA and CoCl₂ is similar to the effect of auxin, indicating the integral relationship between BR and auxin.     

Fluxes of Na+, K+ and Cl-, and osmotic adjustment in Lycopersicon pimpinellifolium and L. esculentum during short- and long-term exposures to NaCl

NaCl (140 m M ) was applied to 14-day-old plants of salt-sensitive Lycopersicon esculentum Mill. cv. Volgogradskij and its wild relative L. pimpinellifolium Mill. accession PE-2. Changes in the relative growth rate of whole plant, and in the levels of inorganic and organic solutes in leaves, stems and roots were followed for 15 days after the application. Short-term salt exposure (4–6 days of salinization) resulted in enhanced relative growth rates for L. pimpinellifolium , but did not affect growth of L. esculentum , After 6 days of salinization, the relative growth rates of both species decreased significantly; leading to practically comparable growth rates for them by day 15. In all parts of both species, the contribution of organic solutes to the osmotic potential (Ψ_s) gradually decreased from 30% on day 0 to a value lower than 5% on day 4. In L. pimpinellifolium , compared to L. esculentum , short-term salt exposure resulted in (1) a higher percentage of adjustment of Ψ_s; and (2) increases in Na⁺ and K⁺ uptake rates, and in the levels of organic acids and proline (the level of which reached that of sugars, i.e., 10 μmol g^-1 dry weight. Conversely, in L. esculentum , drastic reductions of K⁺ uptake rates and organic acid levels occurred already on day 1. During long-term salt exposure, both species were able to adjust osmotically and both exhibited decreases in organic acid levels as well as in K⁺ uptake and accumulation rates in all parts. The results are discussed in an attempt to explain the adaptive responses during short-term salt exposure and the metabolic dysfunctions that lead to growth decrease after long-term exposure to salt.     

Ca2+ and peroxidase derived from cultured peanut cells

A 5% increase of Ca²⁺ content of the incubation medium for cultured peanut ( Arachis hypogaea L.) cells caused a rise of peroxidase (EC 1.11.1.7) activity in the medium, which could be abolished by the addition of the chelator EGTA [eth-yleneglycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. However, the determination of in vivo peroxidase synthesis in these cells showed that Ca²⁺ had a direct effect on the biosynthesis rather than on transport alone. This concept was re-enforced by the lack of effect by the ionophore A23187 on the transport. The Ca²⁺ content was 2 and 5 mol (mol protein⁻¹) for the cationic and anionic peanut peroxidase, respectively. The latter is different from the value reported for the anionic peroxidase from horseradish.     

Cation exchange in isolated xylem cell walls of tomato. I. Cd2+ and Rb+ exchange in adsorption experiments

Abstract Purified xylem cell walls were prepared from isolated xylem bundles of tomato (an inbred line of Lycopersicon esculentum Mill, cv. Tiny Tim). Adsorption and exchange experiments were carried out with ¹¹⁵Cd²⁺, ⁸²Rb⁺ and ⁸²Br^?. The application of γ-ray spectroscopy permitted the simultaneous measurement of several ions applied together. The cell-wall water volume was shown to be independent of the external pH and solution ionic strength, possibly due to the presence of lignin. The Donnan Free Space (DFS) volume could be determined as a constant 0.15 dm³ per kg cell-wall dry weight. Consequently, the total cell-wall cation exchange capacity (CEC) could be estimated based on the DFS volume, and amounted to approximately 1000 mol m^?3 negative charges. The results of Cd²⁺ -Rb⁺ exchange experiments indicated an apparent CEC value of about 350–450 mol m^?3 DFS, at external pH ～ 4. These data are in agreement with earlier reports on xylem wall CEC, and indicate the weak acid characteristics of the charge groups. The rational selectivity coefficient R^Cd_Rb, of the cell wall was shown to depend on external ion fractions and ionic strength, with a maximum R^Cd_Rb of 450 at ionic Cd²⁺ fraction near 0.3, based at the smallest experimental ionic strength of the external solution. The adsorption of Cd²⁺, applied at relatively high concentrations, was shown to be stimulated by simultaneous application of high Rb⁺ concentrations.     

Growth and gibberellin relations of the extreme dwarf dx tomato mutant

The extreme dwarf d ^x tomato ( Lycopersicon esculentum Mill.) mutant has very short internodes which were found to contain shorter and fewer epidermal cells. The leaves are highly abnormal. The mutant showed a substantial stem growth response to GA₃, without approaching normal stature or morphology. The active gibberellin GA₁ and its precursors GA₁₉ and GA₂₀ were identified by coupled gas chromatography-mass spectrometry (GC/MS) in d ^x shoots. Quantitative GC/MS revealed that GA₂₀ accumulated to far higher levels than normal in stems and leaves of the mutant.     

Phosphorylase a and Labile Metabolites During Anoxia: Correlation to Membrane Fluxes of K+ and Ca2+

The objective of the present study was to explore mechanisms responsible for activation of ion conductances in the initial phases of brain ischemia, particularly for the early release of K+ that precedes massive cell depolarization, and rapid downhill fluxes of K+, Na+, Cl-, and Ca2+. As it has been speculated that a K+ conductance can be activated either by an increase in the free cytosolic calcium concentration (Ca2+i) or by a fall in ATP concentration, the question arises whether the early increase in extracellular K+ concentration (K+e) is preceded by a rise in Ca2+i and/or a fall in ATP content. In the present experiments, ischemia was induced in rats by cardiac arrest, the time courses of the rise in K+e and cellular depolarization were determined by microelectrodes, and the tissue was frozen in situ through the exposed dura for measurements of levels of labile metabolites. including adenine nucleotides and cyclic AMP (cAMP), after ischemic periods of 15, 30, 60, and 120 s. Conversion of phosphorylase b to a was assessed, because it depends, among other things, on changes in Ca2+i. The K+e value rose within a few seconds following induction of ischemia, but massive depolarization (which is accompanied by influx of calcium) did not occur until after approximately 65 s. Activation of phosphorylase was observed already after 15 s and before glycogenolysis had begun. At that time, 3',5'-cAMP concentrations were unchanged, and total 5'-AMP concentrations were only moderately increased. The results demonstrate that a K+ conductance is activated at a time when the overall ATP concentration remains at 95% of control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.     

Galanin Reduces Carbachol Stimulation of Phosphoinositide Turnover in Rat Ventral Hippocampus by Lowering Ca2+ Influx Through Voltage-Sensitive Ca2+ Channels

The 29-amino-acid peptide galanin (GAL) caused concentration-dependent inhibition of the accumulation of 3H-inositol phosphates (3H-InsPs) induced by the muscarinic agonist carbachol (CARB; 10(-3)-10(-5) M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono-, bis-, tris-, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 microM) did not affect alpha-adrenergic or serotonergic type 2 receptor-mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H-InsPs nor affected muscarinic receptors in binding studies. Blockade of the T-, N-, and L-types of voltage-sensitive calcium channel (VSCC) with 200 microM Cd2+ reduced muscarinic receptor-mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 microM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 microM abolished the GAL inhibition of CARB-stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 microM Bay K 8644, a selective agonist of the L-type VSCC, prevented the inhibitory effect of GAL. Blockade of the L-type VSCC with nifedipine (1 microM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic inhibition of cortex microsomal Mg2+/Ca2+ ATPase-mediated Ca2+ uptake in the rat pilocarpine model following epileptogenesis

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.