Brassinosteroids regulate plasma membrane anion channels in addition to proton pumps during expansion of Arabidopsis thaliana cells

Brassinosteroids (BRs) are involved in numerous physiological processes associated with plant development and especially with cell expansion. Here we report that two BRs, 28-homobrassinolide (HBL) and its direct precursor 28-homocastasterone (HCS), promote cell expansion of Arabidopsis thaliana suspension cells. We also show that cell expansions induced by HBL and HCS are correlated with the amplitude of the plasma membrane hyperpolarization they elicited. HBL, which promoted the larger cell expansion, also provoked the larger hyperpolarization. We observed that membrane hyperpolarization and cell expansion were partially inhibited by the proton pump inhibitor erythrosin B, suggesting that proton pumps were not the only ion transport system modulated by the two BRs. We used a voltage clamp approach in order to find the other ion transport systems involved in the PM hyperpolarization elicited by HBL and HCS. Interestingly, while anion currents were inhibited by both HBL and HCS, outward rectifying K+ currents were increased by HBL but inhibited by HCS. The different electrophysiological behavior shown by HBL and HCS indicates that small changes in the BR skeleton might be responsible for changes in bioactivity.     

Effect of temperature on plasma membrane and tonoplast ion channels in Arabidopsis thaliana

The temperature dependence of the activity of ion channels was investigated, by means of the patch-clamp technique in the 'whole-cell' configuration, using protoplasts and vacuoles isolated form Arabidopsis thaliana L. cultured cells. The effect of temperature changes in the range 11–22°C was tested on the hyperpolarization and depolarization-activated K⁺ currents in the plasma membrane and on the hyperpolarization-activated K⁻ currents in the tonoplast (vacuolar membrane). All 3 kinds of currents were unaffected by increasing temperature up to 15°C and were activated between 15 and 20°C.     

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana.

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana have been studied at the single-channel level using the patch-clamp technique. The Ca2+ channel in the plasma membrane opened for extracellular Ca2+ influx. The Ca2+ channel in the vacuolar membrane opened for cytoplasmic Ca2+ influx.     

Characterization of a nitrate-permeable channel able to mediate sustained anion efflux in hypocotyl cells from Arabidopsis thaliana

We have characterized a new anionic current in Arabidopsis hypocotyl cells. This current, activated by membrane depolarization, has slow activation and deactivation kinetics in the 10 sec range. It presents many distinct properties from the rapid-type anion current already described on the same membrane. The slow-type channel is highly permeable to nitrate with a PNO3-/PCl- close to 20, but totally impermeable to sulphate. Activation of the channel requires cytosolic ATP and the slow current is partially inhibited by staurosporin, suggesting that channel regulation involves protein phosphorylation. The slow anion channel displays a unique pharmacological profile different from that of the rapid channel: the slow channel is inhibited by DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid) with an IC50 of 26 microM. The slow and rapid anion channels are probably dedicated to specific functions: the first is able to mediate sustained anion efflux, while the second is a good candidate to be involved in fast electrical signalling.     

Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.     

Distinct pools of epithelial sodium channels are expressed at the plasma membrane

The epithelial Na+ channel (ENaC) is assembled in the endoplasmic reticulum from three structurally related subunits (alpha, beta, and gamma). Channel maturation within the biosynthetic pathway involves cleavage of the alpha and gamma subunits by furin and processing of N-linked glycans on alpha, beta, and gamma to complex type. Both mature and immature subunits have been observed at the surface of stably transfected Madin-Darby canine kidney cells. We have examined whether channel maturation is an all-or-none event or whether heterogeneous processing of channel subunits occurs within an individual channel complex. Using an immobilized lectin to isolate proteins with complex type N-glycans, we found that individual channel complexes with mature subunits lack immature subunits. Furthermore, terminal processing of N-glycans on ENaC subunits was not dependent on cleavage of ENaC subunits, and proteolysis of channel subunits was not dependent on prior processing of N-glycans. Our results suggest that processing of subunits within an individual channel complex is an all-or-none event such that channels present on the cell surface contain either all mature or all immature subunits. The presence of immature channel complexes at the plasma membrane provides epithelial cells with a reserve of poorly functional channels that can be activated by proteases in post-Golgi compartments.     

Cation channels in the Arabidopsis plasma membrane

In vivo analyses have identified different functional types of ion channels in various plant tissues and cells. The Arabidopsis genome contains approximately 70 genes for ion channels, of which 57 might be cation-selective channels (K(+), Ca(2+) or poorly discriminating channels). Here, we describe the different families of (putative) cation channels: the Shakers, the two-P-domain and Kir K(+) channels (encoded by the KCO genes), the cyclic-nucleotide-gated channels, the glutamate receptors, and the Ca(2+) channel TPC1. We also compare molecular data with the data obtained in planta, which should lead to a better understanding of the identity of these channels and provide clues about their roles in plant nutrition and cell signalling.     

Substrate regulation of single potassium and chloride ion channels in Arabidopsis plasma membrane

Patch clamp measurements of excised inside-out patches of Arabidopsis thaliana plasma membrane reveal at least two ion channels which conduct either potassium or chloride. The conductance of the potassium channel ranged from 5 to 70 picosiemens depending on KCl concentration. The conductance increased linearly with increasing cytoplasmic-side [KCl]; the extent of this dependence declined as extracytoplasmic-side [KCl] was increased. This indicates that substrate regulation of the potassium channel is a consequence of the molecular architecture of the channel: in particular, multi-ion binding sites within the channel pore. The chloride channel conductance (ranging from 5-40 picosiemens) was independent of cytoplasmic-side [KCl] until a threshold concentration of about 300 millimolar was reached. Such behavior is expected only if the channel is allosterically regulated by cytoplasmic-side K⁺ and/or Cl⁻. The median open times of either channel (about 200 milliseconds for the potassium channel and 20 milliseconds for the chloride channel) were unaffected by substrate concentrations.     

Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells.

Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides. Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state. Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s). We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.     

Malate-induced feedback regulation of plasma membrane anion channels could provide a CO2 sensor to guard cells.

Plants have developed strategies to circumvent limitations in water supply through the adjustment of stomatal aperture in relation to the photosynthetic capacity (water-use efficiency). The CO2 sensor of guard cells, reporting on the metabolic status of the photosynthetic tissue, is, however, as yet unknown. We elucidated whether extracellular malate has the capability to serve as a signal metabolite in regulating the membrane properties of guard cells. Patch-clamp studies showed that slight variations in the external malate concentration induced major alterations in the voltage-dependent activity of the guard cell anion channel (GCAC1). Superfusion of guard cell protoplasts with malate solutions in the physiological range caused the voltage-gate to shift towards hyperpolarized potentials (Km(mal) = 0.4 mM elicits a 38 mV shift). The selectivity sequence of the anion channel NO3- (4.2) > or = I- (3.9) > Br- (1.9) > Cl- (1) >> mal (0.1) indicates that malate is able to permeate GCAC1. The binding site for shifting the gate is, however, located on the extracellular face of the channel since cytoplasmic malate proved ineffective. Single-channel analysis indicates that extracellular malate affects the voltage-dependent mean open time rather than the unitary conductance of GCAC1. In contrast to malate the rise in the extracellular Cl- concentration increases the unit conductance of the anion efflux channel. We suggest that stomata sense changes in the intercellular CO2 concentration and thus the photosynthetic activity of the mesophyll via feedback regulation of anion efflux from guard cells through malate-sensitive GCAC1.     

Combined action of guard cell plasma membrane rapid- and slow-type anion channels in stomatal regulation

Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO₂, the double mutant lacking SLAC1 and QUAC1 was nearly insensitive to these stimuli, indicating the need for coordinated activation of both R- and S-type anion channels in stomatal closure.

Combined impairment of guard cell slow and rapid anion channels results in increased stomatal conductance and complete stomatal insensitivity to abscisic acid, darkness, and elevated CO₂.     

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall

The heptapeptide Tyr-Gly- Arg-Gly-Asp- Ser-Pro containing the sequence Arg-Gly-Asp (RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K_d1 1 nM, and K_d2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.     

Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyl

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.     

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO4(2-) as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of -180 mV exhibiting a maximum peak inward current (I(peak)) at approximately -130 mV. These currents reversed at potentials close to the equilibrium potential for SO4(2-), indicating that the inward currents represented SO4(2-) efflux. Replacing intracellular SO4(2-) with Cl- or NO3(-) resulted in inward currents exhibiting similar properties to the SO4(2-) efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO4(2-) was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.     

Nucleotides provide a voltage-sensitive gate for the rapid anion channel of arabidopsis hypocotyl cells

The rapid anion channel of Arabidopsis hypocotyl cells is highly voltage-dependent. At hyperpolarized potentials, the channel is closed, and membrane depolarization is required for channel activation. We have previously shown that channel gating is regulated by intracellular nucleotides. In the present study, we further analyze the channel gating, and we propose a mechanism to explain its regulation by voltage. In the absence of intracellular nucleotides, closure at hyperpolarized voltages is abolished. Structure-function studies of adenyl nucleotides show that the apparent gating charge of the current increases with the negative charge carried by nucleotides. We propose that the fast anion channel is gated by the voltage-dependent entry of free nucleotides into the pore, leading to a voltage-dependent block at hyperpolarized potentials. In agreement with this mechanism in which intracellular nucleotides need to be recruited to the channel pore, kinetic analyses of whole-cell and single-channel currents show that the rate of closure is faster when intracellular nucleotide concentration is increased, whereas the rate of channel activation is unchanged. Furthermore, decreasing the concentration of extracellular chloride enhances the intracellular nucleotide block. This result supports the hypothesis of a mechanism in which blocking nucleotides and permeant anions interact within the channel pore.     

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10⁻⁹ M of the gibberellin GA₄₊₇, whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA₄₊₇ has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA₄₊₇ than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process. Received: 27 February 1999 / Accepted: 21 May 1999     

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.     

Homer regulation of native plasma membrane calcium channels in A431 cells

Homers are adapter proteins that play a significant role in the organization of calcium signaling protein complexes. Previous functional studies linked Homer proteins to calcium influx in nonexcitable cells. These studies utilized calcium imaging or whole-cell current recordings. Because of limited resolution of these methods, an identity of Homer-modulated ion channels remained unclear. There are several types of plasma membrane calcium influx channels in A431 cells. In the present study, we demonstrated that Homer dissociation resulted in specific activation of I(min) channels but not of I(max) channels in inside-out patches taken from A431 cells. In contrast, inositol 1,4,5-trisphosphate activated both I(min) and I(max) channels in inside-out patches. Short (1a) and long (1c) forms of Homer had different effects on I(min) channel activity. Homer 1a but not Homer 1c activated I(min) in the patches. This study indicates that I(min) channels are specifically regulated by Homer proteins in A431 cells.