Sylvia Lindberg

Cytosolic calcium and pH signaling in plants under salinity stress

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pHcyt, either individually, or in coordination with changes in cytosolic Ca2+ concentration, [Ca2+]cyt, evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca2+ and protons. The variety and complexity of Ca2+ and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca2+]cyt concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca2+]cyt and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca2+]cyt and pHcyt at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.

A new perspective on auxin perception.

An important question in modern plant biology concerns the mechanisms of auxin perception. Despite the recently discovered soluble receptor, the F-box protein TIR1, there is no doubt that another type of signal perception exists, and is linked to the plasma membrane. Two models for the receptor have been suggested: either the receptor includes a protein kinase, or it is coupled with a G-protein. We propose a third model, acting through Ca(2+)-channels in the plasma membrane. The model is based on the revealed rapid auxin-induced reactions, including changes in the membrane potential, shifts in cytosol concentration of Ca(2+) and H(+) and modulation of cell sensitivity to hormones by the external Ca(2+) concentration. Detailed inhibitor analysis with both living cells and isolated plasma membranes show that auxin might directly stimulate Ca(2+) transport through the plasma membrane. A hypothetical scheme of auxin perception at the plasma membrane is suggested together with further transduction events. In addition, comparative analyses of auxin and serotonin perceptions are provided.

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.

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

AbstractMain conclusionThe heterologous expression ofAtPCS1in 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.

Auxin-induced Cytosol Acidification in Wheat Leaf Protoplasts Depends on External Concentration of Ca2+

Summary The present investigation focuses on the rapid cytoplasmic acidification of wheat leaf protoplasts upon auxin treatment and how this process depends on the external concentration of calcium. The tetra [acetoxymethyl] ester of the carboxyfluorescein BCECF and fluorescence microscopy were used to determine the cytosolic pH of the protoplasts from wheat leaves. Both at external pHs 6.0 and 7.0, the physiologically active synthetic auxin analogue naphthalene-1-acetic acid (1-NAA) induced a rapid acidification in the cytosol of the protoplasts, but physiological non-active naphthalene-2-acetic acid (2-NAA) did not. The acidification depended on the concentration of 1-NAA. The maximum cytoplasmic shift in pH (ΔpH = 0.55) in the presence of external pH 7.0 and 0.1 mmol/L Ca2+ was obtained upon addition of 0.1 μmol/L 1-NAA. At external pH 6.0, the dependence of cytoplasmic acidification on 1-NAA concentration was not changed but the amplitude decreased. It may be concluded that the cytoplasmic acidification is a rapid mechanism of auxin action, and not a simple reflection of 1-NAA influx as a weak acid. In the presence of 1 μmol/L CaCl2, compared with 0.1 μmol/L, the maximum cytoplasmic acidification (ΔpH 0.6 units) at both pH values was obtained upon addition of a 10 times lower concentration of 1-NAA (0.01 μmol/L). Verapamil (1 μmol/L), an inhibitor of plasma membrane Ca2+-channels, significantly decreased the cytoplasmic acidification. The 4-bromo-calcium ionophor (A 23187) induced a rapid cytoplasmic acidification. Preincubation of the protoplasts with vanadate decreased the auxin- and ionophoredependent acidification and led to a transient inhibition of plasma membrane H+-ATPase. Therefore, the rapid auxin-induced acidification of the cytosol is supposed to depend on activation of plasma membrane Ca2+ channels.

Calcium Signalling in Plant Cells Under Environmental Stress

A change of intracellular calcium concentration is an early event in a large array of biological processes in plants, such as cell division, polarity, growth and development at normal conditions and under adaptation to abiotic and biotic stresses. This chapter focuses on calcium signalling induced by different types of abiotic stresses, such as salt, cold, anoxia, aluminium and heavy metal stress, while a minor part deals with biotic stress signalling. Most investigations, so far, concerned Ca2+ signalling in the cytosol; however, signalling in the nucleus and other cell compartments such as mitochondria, ER and cell wall have also been reported. The specific “signature” of calcium, including duration, amplitude and frequency of the signalling, induced by different stresses is essential for a change of the physiological function. Different stores for calcium take part in the signalling under various types of stress. Of special interest is a comparison of signalling in tolerant and sensitive species and cultivars.

Effects of ABA on the Distribution of Sucrose and Protons Across the Plasmalemma of Pea Mesophyll Protoplasts. — Suggesting a Sucrose/Proton Symport

Summary The effects of abscisic acid (10 −9 −10 −6 mol/L) were studied on light induced CO 2 -dependent O 2 evolution, sucrose synthesis, efflux of sucrose and changes in cytosolic proton concentration in mesophyll protoplasts of Pisum sativum L. cv. Fenomen. Photosynthesis did not change with increased apoplastic ABA or sucrose concentrations although sucrose synthesis decreased in the presence of ABA. In the absence of apoplastic sucrose the sucrose effiux increased and was most pronounced at 10 −7 mol/L ABA. The sucrose efflux also increased in the presence of ABA and sucrose together and was most pronounced at 10 −9 mol/L ABA and 20 mmol/L apoplastic sucrose. The cytosolic proton concentration decreased upon addition of ABA with or without sucrose, but increased upon addition of p -chloromercuribenzenesulfonic acid, a sucrose transport inhibitor. The cytosolic proton concentration decreased in the presence of 10 -9 mol/L ABA in combination with various apoplastic sucrose concentrations. In the presence of 10 -7 -10 -6 mol/L ABA in combination with apoplastic sucrose levels higher than 1 mmol/L, the cyrosolic concentration of proton increases. The results indicate that there is a relationship between the external environment and the sucrose effiux and cytosolic proton concentration in pea mesophyll protoplasts and that there is a sucrose/proton symport at the plasmalemma of pea mesophyll cells. They also show that sucrose increases the sensitivity to ABA in its action on sucrose effiux and cytosolic proton concentration.

In-situ determination of intracellular concentrations of K+ in barley (Hordeum vulgare L. cv. Kara) using the K+-binding fluorescent dye benzofuran isophthalate

The tetra[acetoxymethyl] ester of the K+-binding fluorescent dye benzofuran isophthalate (PBFI-AM) was used to determine changes in intracellular potassium (K+) concentrations and to measure net transport of K+ in barley (Hordeum vulgare L. cv. Kara) root and leaf protoplasts. When this dye binds to free K+ inside the cytoplasm, the fluorescence intensity ratio 340/380 nm increases in direct relation to the K+ concentration. Because of a delay in the uptake of dye into the vacuoles, it is possible to determine K+ concentrations in the vacuoles and transport of K+ from the cytoplasm into the vacuole. The uptake of PBFI-AM in root and leaf protoplasts of barley differed in the absence or presence of external K+ and was faster at pH 5.5 than at pH 7.0. The fluorescence intensity of the dye was stable for at least 20 h when the protoplasts were kept at 4°C. In the presence of nigericin, the fluorescence intensity of both cells and protoplasts was linearly related to the external concentration of K+ (up to 100 mM).

Effects of pH and Mineral Nutrition Supply on K+(86Rb+) Influx and Plasma Membrane ATPase Activity in Roots of Sugar Beets

Summary Sugar beet seedlings ( Beta vulgaris L. cv. Primahill) were cultivated at varying pH and nutrient levels addition rates. Hydrolyzing ATPase activity of root plasma membranes and K + ( 86 Rb + ) flux into roots of intact plants were determined after the different treatments. Relatively high pH (6.5), high concentration of nutrients and a high sodium: potassium (Na: K) ratio in the root medium increased the specific Na + − and (Na + + K + )-activation of the ATPase. In contrast, a low relative addition rate of nutrients and low pH (5.3) enhanced the specific K + -activation. The 2,4-dinitrophenol (DNP)-sensitive (metabolic) influx of K + ( 86 Rb + ) increased > 3 times when the seedlings were raised at pH 5.0, as compared with pH 6.0, and was higher after a relatively low addition rate of nutrients than after a higher rate, or high concentration of nutrients during the cultivation. Thus the specific K + -activation of the ATPase and the K + ( 86 Rb + ) influx were increasing under the same conditions. If K + influx depends on the activity of the H + -ATPase, these results suggest that the activity of the H + -pump increases at low pH and limited supply of nutrients. At high pH and high Na + : K + ratio in the nutrient solution, the increased activation of the ATPase by Na + , and less activation by K + , indicates a larger capacity to extrude Na + than to extrude protons and take up K + .

Effects of pH and Mineral Nutrition Supply on Lipid Composition and Protein Pattern of Plasma Membranes from Sugar Beet Roots

Summary Plasma membranes (PM) were isolated by two-phase-partitioning from roots of 21-day-old sugar beet ( Beta vulgaris L. cv. Monohill) seedlings cultivated under different conditions. The seedlings were either grown in a complete nutrient solution with a high concentration of nutrients (H-s roots) and high (6.5) or low (5.3) pH, or in a low concentration medium (L-s roots) with a 15% daily relative addition rate of nutrients at pH 5.3. The protein pattern, the phospholipids, the glycolipids and the free sterols were analysed. The protein pattern of plasma membranes did not change, but the composition of lipids changed under the different conditions of cultivation. After cultivation at a low concentration of nutrients and low pH, the free sterols dominated while after cultivation at a high concentration of nutrients, the phospholipids were the dominant lipids. The Δ 7 -sterols were the most abundant free sterols representing more than 60% of the free sterols in PM of roots growing in a low concentration of nutrients and more than 70 to 80% in plasma membranes of roots growing in a high concentration. Other free sterols present were stigmasterol, sitosterol, campesterol and brassicasterol. The ratio of more planar/to less planar sterols increased with high salt treatment. Phosphatidylethanolamine and phosphatidylcholine, which were the major phospholipids, increased with high salt concentration in the growth medium. The glycolipid levels remained substantially unchanged. However, the ratio of phospholipids to glycolipids increased with high salt and high pH. The relative distribution of fatty acids in the lipid classes also changed after cultivation of the seedlings under different conditions. The results show that the different growth conditions used in these experiments caused important changes to the plasma membrane lipid composition, which were well correlated with differences in ATPase activities and K + ( 86 Rb + ) influx observed under the same experimental conditions.