Arabidopsis PLDs with C2-domain function distinctively in hypoxia.

Hypoxia (oxygen deprivation) causes metabolic disturbances at physiological, biochemical and genetic levels and results in decreased plant growth and development. Phospholipase D (PLD)-mediated signaling was reported for abiotic and biotic stress signaling events in plants. To investigate the participatory role of PLDs also in hypoxia signaling, we used wild type of Arabidopsis thaliana and 10 pld isoform mutants containing C2-domain. Hypoxia-induced changes in three major signaling players, namely, cytosolic free calcium (Ca2+ cyt ), reactive oxygen species (ROS) and phosphatidic acid (PA), were determined in mesophyll protoplasts. The Ca2+ cyt and ROS levels were monitored by fluorescence microscopy and confocal imaging, while PA levels were quantified by an enzymatic method. Our findings reveal that the elevations of cytosolic calcium and PA are reduced in all the 10 mutants dysfunctional in PLD isoforms. The hypoxia-related changes in both calcium and ROS show different kinetic patterns depending on the type of PLD studied. Pharmacological experiments confirm that both external and internal sources contribute to calcium and ROS accumulation under hypoxia. PLDα1-3, PLDß1 and PLDγ1-3 are likely involved in calcium signaling under hypoxia as well as in PA production, while all investigated PLDs, except for PLDγ3, take part in ROS elevation.

Phospholipases AtPLDζ1 and AtPLDζ2 function differently in hypoxia.

Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane-cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLDζ1 and PLDζ2 uniquely possess Ca2+ -independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pldζ1 and pldζ2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia-induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca2+cyt. ], hydrogen peroxide (H2 O2 ) and PA. The concentrations of cytosolic Ca2+ and H2 O2 were determined by fluorescence microscopy and the fluorescent dyes Fura 2-AM and CM-H2 DCFDA, specific for calcium and H2 O2 , respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLDζ1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLDζ2 is involved in cytosolic Ca2+ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pldζ1, while the PA level is unchanged in pldζ2. Thus, it is likely that AtPLDζ2 is involved in PA production by a calcium signaling pathway, while AtPLDζ1 is more important in ROS signaling.

Cadmium spiked soil modulates root organic acids exudation and ionic contents of two differentially Cd tolerant maize (Zea mays L.) cultivars.

Our earlier work described that the roots of two maize cultivars, grown hydroponically, differentially responded to cadmium (Cd) stress by initiating changes in medium pH depending on their Cd tolerance. The current study investigated the root exudation, elemental contents and antioxidant behavior of the same maize cultivars [cv. 3062 (Cd-tolerant) and cv. 31P41 (Cd-sensitive)] under Cd stress. Plants were maintained in a rhizobox-like system carrying soil spiked with Cd concentrations of 0, 10, 20, 30, 40 and 50 µmol/kg soil. The root and shoot Cd contents increased, while Mg, Ca and Fe contents mainly decreased at higher Cd levels, and preferentially in the sensitive cultivar. Interestingly, the K contents increased in roots of cv. 3062 at low Cd treatments. The Cd stress caused acidosis of the maize root exudates predominantly in cv. 3062. The concentration of various organic acids was significantly increased in the root exudates of cv. 3062 with applied Cd levels. This effect was diminished in cv. 31P41 at higher Cd levels. Cd exposure increased the relative membrane permeability, anthocyanin (only in cv. 3062), proline contents and the activities of peroxidases (POD) and superoxide dismutase (SOD). The only exception was the catalase activity, which was diminished in both cultivars. Root Cd contents were positively correlated with the secretion of acetic acid, oxalic acid, glutamic acid, citric acid, and succinic acid. The antioxidants like POD and SOD exhibited a positive correlation with the organic acids under Cd stress. It is likly that a high exudation of dicarboxylic organic acids improves nutrient uptake and activities of antioxidants, which enables the tolerant cultivar to acclimatize in Cd polluted environment.

Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification.

MAIN CONCLUSION: The heterologous expression of AtPCS1 in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

Calcium supply effects on wheat cultivars differing in salt resistance with special reference to leaf cytosol ion homeostasis.

Salinity causes changes in cytosolic Ca(2+), [Ca(2+)]cyt, Na(+), [Na(+)]cyt and pH, pH cyt , which induce specific reactions and signals. Reactions causing a rebalancing of the physiological homeostasis of the cytosol could result in plant resistance and growth. Two wheat cultivars, Triticum aestivum, Seds1 and Vinjett, were grown in nutrient solution for 7 days under moderate salinity (0 and 50 mM NaCl) with and without extra addition of 5 mM CaSO4 to investigate the seedling-ion homeostasis under salinity. In the leaf protoplasts [Ca(2+) ]cyt, [Na(+)]cyt and pH cyt were detected using acetoxymethyl esters of the ion-specific dyes, Fura 2, SBFI and BCECF, respectively, and fluorescence microscopy. In addition, both cultivars were grown for 3 weeks at 0, 50 and 125 mM NaCl with, or without, extra addition of 5 mM CaSO4 to detect overall Na(+) and Ca(2+) concentrations in leaves and salinity effects on dry weights. In both cultivars, salinity decreased [Ca(2+)]cyt, while at extra Ca(2+) supplied, [Ca(2+)]cyt increased. The [Ca(2+) ]cyt increase was accompanied by increase in the overall Ca(2+) concentrations in leaves and decrease in the overall Na(+) concentration. Moreover, irrespective of Ca(2+) treatment under salinity, the cultivars reacted in different ways; [Na(+) ]cyt significantly increased only in cv. Vinjett, while pH cyt increased only in cv. Seds1. Even at rather high total Na(+) concentrations, the cytosolic concentrations were kept low in both cultivars. It is discussed whether the increase of [Ca(2+)]cyt and pH cyt can contribute to salt tolerance and if the cytosolic changes are due to changes in overall Ca(2+) and Na(+) concentrations.

Ion Changes and Signaling under Salt Stress in Wheat and Other Important Crops.

High concentrations of sodium (Na+), chloride (Cl-), calcium (Ca2+), and sulphate (SO42-) are frequently found in saline soils. Crop plants cannot successfully develop and produce because salt stress impairs the uptake of Ca2+, potassium (K+), and water into plant cells. Different intracellular and extracellular ionic concentrations change with salinity, including those of Ca2+, K+, and protons. These cations serve as stress signaling molecules in addition to being essential for ionic homeostasis and nutrition. Maintaining an appropriate K+:Na+ ratio is one crucial plant mechanism for salt tolerance, which is a complicated trait. Another important mechanism is the ability for fast extrusion of Na+ from the cytosol. Ca2+ is established as a ubiquitous secondary messenger, which transmits various stress signals into metabolic alterations that cause adaptive responses. When plants are under stress, the cytosolic-free Ca2+ concentration can rise to 10 times or more from its resting level of 50-100 nanomolar. Reactive oxygen species (ROS) are linked to the Ca2+ alterations and are produced by stress. Depending on the type, frequency, and intensity of the stress, the cytosolic Ca2+ signals oscillate, are transient, or persist for a longer period and exhibit specific "signatures". Both the influx and efflux of Ca2+ affect the length and amplitude of the signal. According to several reports, under stress Ca2+ alterations can occur not only in the cytoplasm of the cell but also in the cell walls, nucleus, and other cell organelles and the Ca2+ waves propagate through the whole plant. Here, we will focus on how wheat and other important crops absorb Na+, K+, and Cl- when plants are under salt stress, as well as how Ca2+, K+, and pH cause intracellular signaling and homeostasis. Similar mechanisms in the model plant Arabidopsis will also be considered. Knowledge of these processes is important for understanding how plants react to salinity stress and for the development of tolerant crops.

Cytosolic Sodium Influx in Mesophyll Protoplasts of Arabidopsis thaliana, wt, sos1:1 and nhx1 Differs and Induces Different Calcium Changes.

The sodium influx into the cytosol of mesophyll protoplasts from Arabidopsis thaliana cv. Columbia, wild type, was compared with the influx into sos1-1 and nhx1 genotypes, which lack the Na+/H+ antiporter in the plasma membrane and tonoplast, respectively. Changes in cytosolic sodium and calcium concentrations upon a 100 mM NaCl addition were detected by use of epifluorescence microscopy and the sodium-specific fluorescent dye SBFI, AM, and calcium sensitive Fura 2, AM, respectively. There was a smaller and mainly transient influx of Na+ in the cytosol of the wild type compared with the sos1-1 and nhx1 genotypes, in which the influx lasted for a longer time. Sodium chloride addition to the protoplasts' medium induced a significant increase in cytosolic calcium concentration in the wild type at 1.0 mM external calcium, and to a lesser extent in nhx1, however, it was negligible in the sos1-1 genotype. LiCl inhibited the cytosolic calcium elevation in the wild type. The results suggest that the salt-induced calcium elevation in the cytosol of mesophyll cells depends on an influx from both internal and external stores and occurs in the presence of an intact Na+/H+ antiporter at the plasma membrane. The Arabidopsis SOS1 more effectively regulates sodium homeostasis than NHX1.

Silicate Inhibits the Cytosolic Influx of Chloride in Protoplasts of Wheat and Affects the Chloride Transporters, TaCLC1 and TaNPF2.4/2.5.

Chloride is an essential nutrient for plants, but high concentrations can be harmful. Silicon ameliorates both abiotic and biotic stresses in plants, but it is unknown if it can prevent cellular increase of chloride. Therefore, we investigated the influx of Cl- ions in two wheat cultivars different in salt sensitivity, by epifluorescence microscopy and a highly Cl--sensitive dye, MQAE, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide, in absence and presence of potassium silicate, K2SiO3. The Cl--influx was higher in the salt-sensitive cv. Vinjett, than in the salt-tolerant cv. S-24, and silicate pre-treatment of protoplasts inhibited the Cl--influx in both cultivars, but more in the sensitive cv. Vinjett. To investigate if the Cl--transporters TaCLC1 and TaNPF2.4/2.5 are affected by silicate, expression analyses by RT-qPCR were undertaken of TaCLC1 and TaNPF 2.4/2.5 transcripts in the absence and presence of 100 mM NaCl, with and without the presence of K2SiO3. The results show that both transporter genes were expressed in roots and shoots of wheat seedlings, but their expressions were differently affected by silicate. The TaNPF2.4/2.5 expression in leaves was markedly depressed by silicate. These findings demonstrate that less chloride accumulates in the cytosol of leaf mesophyll by Si treatment and increases salt tolerance.

Anoxia-induced elevation of cytosolic Ca2+ concentration depends on different Ca2+ sources in rice and wheat protoplasts.

The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1 mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.

Potassium Efflux and Cytosol Acidification as Primary Anoxia-Induced Events in Wheat and Rice Seedlings.

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K+ concentration, [K+]cyt, and cytosolic pH, pHcyt, in rice and wheat, plants with different tolerances to hypoxia. The [K+]cyt and pHcyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K+ from protoplasts of both plants after a lag-period of 300-450 s. The [K+]cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K+ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K+]cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pHcyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K+ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.

Indoleacetic Acid Levels in Wheat and Rice Seedlings under Oxygen Deficiency and Subsequent Reoxygenation.

The lack of oxygen and post-anoxic reactions cause significant alterations of plant growth and metabolism. Plant hormones are active participants in these alterations. This study focuses on auxin-a phytohormone with a wide spectrum of effects on plant growth and stress tolerance. The indoleacetic acid (IAA) content in plants was measured by ELISA. The obtained data revealed anoxia-induced accumulation of IAA in wheat and rice seedlings related to their tolerance of oxygen deprivation. The highest IAA accumulation was detected in rice roots. Subsequent reoxygenation was accompanied with a fast auxin reduction to the control level. A major difference was reported for shoots: wheat seedlings contained less than one-third of normoxic level of auxin during post-anoxia, while IAA level in rice seedlings rapidly recovered to normoxic level. It is likely that the mechanisms of auxin dynamics resulted from oxygen-induced shift in auxin degradation and transport. Exogenous IAA treatment enhanced plant survival under anoxia by decreased electrolyte leakage, production of hydrogen peroxide and lipid peroxidation. The positive effect of external IAA application coincided with improvement of tolerance to oxygen deprivation in the 35S:iaaM × 35S:iaaH lines of transgene tobacco due to its IAA overproduction.

Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.

A shift in sensitivity to auxin within development of maize seedlings.

The auxin-induced changes in cytosolic concentrations of Ca(2+) and H(+) ions were investigated in protoplasts from maize coleoptile cells at 3rd, 4th and 5th day of development of etiolated seedlings. The shifts in [Ca(2+)](cyt) and [H(+)](cyt) were detected by use of fluorescence microscopy in single protoplasts loaded with the tetra[acetoxymethyl]esters of the fluorescent calcium binding Fura 2, or pH-sensitive carboxyfluorescein, BCECF, respectively. Both the auxin-induced shifts in the ion concentrations were specific to the physiologically active synthetic auxin, naphthalene-1-acetic acid (1-NAA), and not to the non-active naphthalene-2-acetic acid (2-NAA). Regardless of the age of the seedlings, the rise in [Ca(2+)](cyt) was prior to the acidification in all investigated cases. The maximal acidification coincided with the highest amplitude of [Ca(2+)](cyt) change, but not directly depended on the concentration of 1-NAA. Within aging of the seedlings the amplitude of auxin-induced [Ca(2+)](cyt) elevation decreased. The shift in auxin-induced acidification was almost equal at 3rd and 4th day, but largely dropped at 5th day of development. The acidification was related to changes in the plasma membrane H(+)-ATPase activity, detected as phosphate release. The decrement in amplitude of both the tested auxin-triggered reactions well coincided with the end of the physiological function of the coleoptile. Hence the primary auxin-induced increase in [Ca(2+)](cyt), which is supposed to be an important element of hormone signal perception and transduction, can be used as a test for elucidation of plant cell sensitivity to auxin.

Calcium improves apoplastic-cytosolic ion homeostasis in salt-stressed Vicia faba leaves.

Salinity disturbs both apoplastic and cytosolic Ca2+ and pH ([Ca2+]apo, [Ca2+]cyt, pHapo and pHcyt) homeostasis, and decreases plant growth. Seedlings of Vicia faba L. cv. Fuego were cultivated in hydroponics for 7 days under control, salinity (S), extra Ca (Ca) or salinity with extra Ca (S+Ca) conditions. The [Ca2+]apo, and pHapo in the leaves were then recorded in parallel by a pseudoratiometric method, described here for the first time. Lower [Ca2+]apo and higher pHapo were obtained under salinity, whereas extra Ca supply increased the [Ca2+]apo and acidified the pHapo. Moreover, the ratiometric imaging recorded that [Ca2+]cyt and pHcyt were highest in S+Ca plants and lowest in control plants. After all pretreatments, direct addition of NaC6H11O7 to leaves induced a decrease in [Ca2+]apo in control and S+Ca plants, but not in S and Ca plants, and only slightly affected pHapo. Addition of NaCl increased [Ca2+]cyt in protoplasts from all plants but only transiently in protoplasts from S+Ca plants. Addition of NaCl decreased pHcyt in protoplasts from Ca-pretreated plants. We conclude that Ca supply improves both apoplastic and cytosolic ion homeostasis. In addition, NaC6H11O7 probably causes transport of Ca from the apoplast into the cytosol, thereby leading to a higher resting [Ca2+]cyt.

Silicate reduces cadmium uptake into cells of wheat.

Cadmium (Cd) is a health threat all over the world and high Cd content in wheat causes high Cd intake. Silicon (Si) decreases cadmium content in wheat grains and shoot. This work investigates whether and how silicate (Si) influences cadmium (Cd) uptake at the cellular level in wheat. Wheat seedlings were grown in the presence or absence of Si with or without Cd. Cadmium, Si, and iron (Fe) accumulation in roots and shoots was analysed. Leaf protoplasts from plants grown without Cd were investigated for Cd uptake in the presence or absence of Si using the fluorescent dye, Leadmium Green AM. Roots and shoots of plants subjected to all four treatments were investigated regarding the expression of genes involved in the Cd uptake across the plasma membrane (i.e. LCT1) and efflux of Cd into apoplasm or vacuole from the cytosol (i.e. HMA2). In addition, phytochelatin (PC) content and PC gene (PCS1) expression were analysed. Expression of iron and metal transporter genes (IRT1 and NRAMP1) were also analysed. Results indicated that Si reduced Cd accumulation in plants, especially in shoot. Si reduced Cd transport into the cytoplasm when Si was added both directly during the uptake measurements and to the growth medium. Silicate downregulated LCT1 and HMA2 and upregulated PCS1. In addition, Si enhanced PC formation when Cd was present. The IRT1 gene, which was downregulated by Cd was upregulated by Si in root and shoot facilitating Fe transport in wheat. NRAMP1 was similarly expressed, though the effect was limited to roots. This work is the first to show how Si influences Cd uptake on the cellular level.

Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots.

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were cultivated for 3 weeks at different root and shoot temperatures and the plasma membranes (PM) from roots were purified by aqueous two-phase partitioning and analyzed for lipid composition and ATPase activities. Lipid analyses, undertaken immediately after PM purification from the roots, showed that a low root zone temperature (10 degrees C) decreased the ratio between the major lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A low temperature in the root environment increased the mol% of PE and decreased the mol% of phosphatidic acid (PA), independent on the shoot growth temperature. A low temperature also decreased the mol% of linoleic acid (18:2) and increased mol% of linolenic acid (18:3) in the analyzed lipid classes, especially in PC and PE. The ratio between acyl chain lipids and protein generally increased in PM from roots grown at 10 degrees C, compared with higher temperature. The changes in lipid composition correlated with changes in ATPase activities, detected as hydrolyses of MgATP. The kinetic parameters, K(m) and V of the PM H(+)ATPase in roots increased at a low cultivation temperature, independent on shoot temperature. Moreover, Arrhenius analyses showed that the transition temperature was independent of both root or shoot growth temperature at 10-24 degrees C, whereas the activation energy of the ATPase was dependent on the growth temperature of the root, and independent on shoot temperature. Thus, acclimation processes can take place in roots, irrespective of the shoot temperature.

Uptake of sodium in quince, sugar beet, and wheat protoplasts determined by the fluorescent sodium-binding dye benzofuran isophthalate.

The uptake of sodium into protoplasts of quince (Cydonia oblonga Mill, clone BA29), sugar beet (Beta vulgaris L. cv. Monohill), and wheat (Triticum aestivum L. cv. Kadett) was determined by use of the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate (SBFI-AM). In the presence of 1 mM CaCl2, little sodium was taken up in the cytosol of quince mesophyll cells compared to cytosols of sugar beet and wheat. Upon addition of 40 mM NaCl, approximately the same amount of sodium was taken up in leaf and root protoplasts of wheat, but no sodium was taken up in quince. However, in calcium-free medium, obtained by addition of ethylene glycol tetra acetic acid (EGTA), quince protoplasts transiently took up sodium in the cytosol when 200-400 mM NaCl was added to the protoplast medium. Moreover, after cultivation of quince in the presence of 200 mM sodium for 4 weeks, the cytosol of isolated protoplasts did not take up any sodium at all from a calcium-free medium. The results show that protoplasts from salt tolerant quince only temporarily take up sodium in the cytosol and that they have a mechanism for fast extrusion of sodium from that compartment. These mechanisms are probably important for the high salt tolerance of quince. Calcium blocks the sodium uptake into the cytosol of both quince and wheat protoplasts.

Cadmium-induced rhizospheric pH dynamics modulated nutrient acquisition and physiological attributes of maize (Zea mays L.).

Cadmium (Cd) is a highly mobile toxic element in soil-plant systems that interferes with plant growth and nutrient acquisition by modulations in the rhizospheric environment. The current study investigated the influence of maize roots on the medium pH, alterations in nutrient uptake, and impact on the plant's physiological attributes under Cd stress. Among the nine maize cultivars, subjected to Cd stress (9.15 mg/kg of sand), one was identified as Cd tolerant (3062-Pioneer) and the second as Cd sensitive (31P41-Pioneer). The selected maize cultivars were grown in nutrient solutions supplemented with 0, 10, 20, 30, 40, or 50 µM CdCl2 under controlled conditions and a starting pH of 6.0. The rhizospheric pH dynamics were monitored each day up to 3 days. Both cultivars caused medium basification; however, the response was different at low (10 and 20 µM) Cd treatments (sensitive cultivar caused medium basification) and at higher (50 µM) Cd treatment (tolerant cultivar caused medium basification). Furthermore, higher Cd was accumulated by the sensitive cultivar which was predominantly found in the roots. Higher Cd levels in the medium resulted in increased uptake and translocation of both Cd and K (in the tolerant cultivar) or only Cd (in the sensitive cultivar). Uptake of other nutrients (Ca, Zn, and Fe) was antagonistically affected by Cd stress in both cultivars. Moreover, Cd stress significantly impaired chlorophyll content, catalase activity, and total protein content; irrespective of the genotype. The malondialdehyde (MDA) content was found to increase, in both cultivars, together with Cd level. However, the extent to which Cd interfered with the studied attributes was more pronounced in the sensitive cultivar as compared to the tolerant one. It is concluded that the maize roots responded to Cd stress by initiating modulations of medium pH which might be dependent on Cd tolerance levels. The study results may help to develop strategies to reduce Cd accumulation in maize and decontamination of metal-polluted soil sediments.

Auxin induces an increase of Ca2+ concentration in the cytosol of wheat leaf protoplasts.

Auxin addition to protoplasts isolated from leaves of 6-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) induced a rapid increase in the cytosolic calcium concentration [Ca2+]cyt. The shifts in [Ca2+]cyt were detected by use of fluorescence microscopy in single protoplasts loaded with the calcium binding tetra[acetoxymethyl]ester of the fluorescent dye, Fura 2. Addition of the synthetic auxin naphthyl acetic acid, 1-NAA, induced an increase in [Ca2+]cyt within 5-10s, while the physiologically non-active analogue, 2-NAA, did not. The amplitude of calcium increase depended on the concentration of 1-NAA. Since the process was affected by different concentrations of Ca2+ in the external medium, and since the calcium channel blockers (nifedipine and verapamil) postponed and inhibited the reaction, it is suggested that auxin primarily activates Ca2+-permeable channels in the plasma membrane. In the presence of low external calcium concentration (0.1 mM), 5 mM LiCl almost totally blocked the increase in [Ca2+]cyt, indicating a possible involvement of tonoplast Ca2+-channels in the auxin-induced [Ca2+]cyt shift. Thus, calcium signalling induced by auxin involves both external and internal Ca2+ pools.

Cellular proton dynamics in Elodea canadensis leaves induced by cadmium.

Our earlier investigations showed that Elodea canadensis shoots, grown in the presence of cadmium (Cd), caused basification of the surrounding medium. The present study was aimed to examine the proton dynamics of the apoplastic, cytosolic and vacuolar regions of E. canadensis leaves upon Cd exposure and to establish possible linkage between cellular pH changes and the medium basification. The changes in cytosolic calcium [Ca(2+)]cyt was also investigated as the [Ca(2+)]cyt and [pH]cyt homeostasis are closely linked. The cellular H(+) and Ca(2+) concentrations were monitored by fluorescence microscopy and ion-specific fluorescent dyes. Cadmium concentration of leaf-cell walls was measured after plant cultivation at different fixed levels of starting pH. The protoplasts from E. canadensis leaves were isolated by use of a newly developed enzymatic method. Upon Cd addition, both cytosolic and vacuolar pH of leaf protoplasts increased with a concomitant rise in the cytosolic Ca(2+) concentration. Time course studies revealed that changes in [Ca(2+)]cyt and [pH]cyt followed similar dynamics. Cadmium (0.5 µM) exposure decreased the apoplastic pH by 0.85 units. The maximum cell wall bound Cd-contents were obtained in plants grown at low starting pH. It is concluded that Cd treatment causes apoplastic acidosis in E. canadensis leaves associated with enhanced Cd binding to the cell walls and, consequently, reduced Cd influx into the cytosol.