Jean Guern

Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [3H]NAA and [14C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

The vacuolar membrane protein alpha‐TIP is a seed‐specific protein of the Major Intrinsic Protein family. Expression of alpha‐TIP in Xenopus oocytes conferred a 4‐ to 8‐fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha‐TIP forms water channels and is thus a new aquaporin. alpha‐TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha‐TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha‐TIP in oocytes, suggesting that phosphorylation of alpha‐TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8‐bromoadenosine 3′,5′‐cyclic monophosphate, forskolin and 3‐isobutyl‐1‐methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha‐TIP by 80‐100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha‐TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP‐dependent increase in the Pf of oocytes expressing alpha‐TIP, as well as for in vitro phosphorylation of alpha‐TIP. These findings demonstrate that the alpha‐TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Intracellular pH in Plant Cells

Publisher Summary This chapter reviews the basic principles of the techniques used for intracellular pH measurement in the main cell compartments—namely, cytoplasm and vacuole. Alongwith being both substrate and product in numerous metabolic reactions, protons fulfill the regulatory role of coordinating the activities of enzyme-catalyzed pathways, membrane transport, and other regulators. The protons connect cellular compartments and also play important roles in intercellular traffic. The sudden pH shifts may impose critical loads on the cells. The technical improvements that have been brought about since the previously published reviews are described in the chapter. The technique involves the extraction of cell sap and the measurement of its pH with a glass electrode. It is used for various types of plant materials. The principle of this technique is based on three requirements: the probe molecule is metabolically inert, only the uncharged form is membrane permeant, and the probe is not to change the pH of the respective compartment. The distribution of protons within a plant cell appears as a critical element of cell organization and function.

Cloning and functional expression of a plant voltage-dependent chloride channel.

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.

Cytoplasmic acidification as an early phosphorylation-dependent response of tobacco cells to elicitors

Elicitor-induced cytoplasmic pH changes of tobacco (Nicotiana tabacum L. cv. Xanthi) cells grown in suspension cultures were explored under a variety of conditions by using a flexible technique based on the distribution of [14C] benzoic acid between the intracellular and extracellular compartments. Comparison of data obtained by this technique and by 31P-nuclear magnetic resonance spectrometry qualifies the benzoic acid distribution method as a convenient and reliable way to probe cytoplasmic pH variations. Various elicitors shown to induce several defense-related responses in tobacco cells, namely oligogalacturonides of degree of polymerization 7–20, pectolyase from Aspergillus japonicus, Phytophthora megasperma crude elicitors and purified cryptogein, triggered cytoplasmic acidifications differing in intensity and kinetics according to the signal molecule. In contrast, no changes in cytoplasmic protons and external pH were observed in cells treated with short galacturonide oligomers, or with soybean-specific hepta β-glucoside from P. megasperma, which are devoid of elicitor activity in tobacco cells. The oligogalacturonide-induced cytoplasmic acidification was inhibited by two structurally unrelated protein kinase inhibitors, staurosporine and 6-dimethylaminopurine, which both reduced the external alkalinization response to the elicitor. The protein phosphatase inhibitor calyculin A alone behaved as an elicitormimicking molecule in triggering cytoplasmic acidification, again associated with extracellular alkalinization. These results indicate that the increase in the cytoplasmic concentration of protons may be considered as a common early intracellular response of tobacco cells to elicitors, associated with the extracellular alkalinization response and controlled by protein phosphorylation.

Comparative studies on the electrical properties of the H+ translocating ATPase and pyrophosphatase of the vacuolar-lysosomal compartment

The electrical properties of the vacuolar‐lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole‐cell configuration of the patch‐clamp technique. Both pumps, the proton‐translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophosphate (MgPP(i)), transport protons into the vacuole and polarize the membrane potential (positive inside the vacuole). Accumulation of protons in the lumen of vacuole vesicles was monitored by absorbance changes of the pH probe, acridine orange. The electrochemical gradient provided by both the ATPase and pyrophosphatase stimulates effectively the uptake of various metabolites such as malate, citrate and sucrose. The maximal current density produced by the ATPase was about 2.5 microA/cm2 and about 0.5 microA/cm2 for the pyrophosphatase. K(m)ATP was 0.6 mM; K(m)PPi was 15‐20 microM with progressive inhibition above 150 microM. At a cytoplasmic pH of 7.5 both enzymes were capable of pumping protons against a 10,000‐fold concentration gradient (pH 3.5 inside the vacuole). Proton current produced by the ATPase was blocked reversibly by extravacuolar NO(3)‐ only.

Alterations of Auxin Perception in rolB-Transformed Tobacco Protoplasts (Time Course of rolB mRNA Expression and Increase in Auxin Sensitivity Reveal Multiple Control by Auxin)

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB transformed protoplasts may account for their increased auxin sensitivity.

Temperature response of Catharanthus roseus cells cultivated in liquid medium

Abstract Suspension cultures of Catharanthus roseus G. Don cells were grown at various temperatures from 16°C to 40°C. The doubling time of cell populations was strongly increased by a fall in temperature from 27°C to 16°C. The cell cycle parameters of cell suspensions grown at 20°C and 27°C were analysed by the fraction of labelled mitoses method and compared. All cell cycle phases were affected by the shift in temperature with a higher sensitivity of the G1 phase. Doubling times of cell populations and total cell cycle times were about the same at 20°C or 27°C. Consequently under these conditions all cells contributed to the growth of the populations. A strong influence of temperature on the amount per cell of the crude alkaloid fraction or on the specific content of serpentine and ajmalicine was observed. Lowering temperature from 27°C to 16°C resulted in a much higher alkaloid content, conversely when temperature was increased from 27°C to 38°C, the alkaloid content was strongly reduced.

Auxin regulates the promoter of the root-inducing rolB gene of Agrobacterium rhizogenes in transgenic tobacco.

SummaryThe regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 TL-DNA was studied by using the β-glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolBGUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.

Calcium channel antagonists induce direct inhibition of the outward rectifying potassium channel in tobacco protoplasts

Verapamil, nifedipine and bepridil, three antagonists of L‐type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacumcv. Xanthi protoplasts with IC50 of 5 μM, 5 μM and 1 μM, respectively. In the outside‐out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacum cv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.