The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth

Calcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage-independent rapidly activating Ca2+-permeable non-selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium-permeable NSCCs co-existed with hyperpolarisation-activated Ca2+ channels (HACCs), which activated 40-60 min after forming the whole-cell configuration. HACCs activated at voltages <-130 to -150 mV, showed slow activation kinetics and were regulated by cytosolic Ca2+ ([Ca2+]cyt). Using aequorin-expressing plants, a linear relationship between membrane potential (Vm) and resting [Ca2+]cyt was observed, indicating the involvement of NSCCs. Intact root 45Ca2+ influx was reduced by Gd3+ (NSCC blocker) but was verapamil and TEA+ insensitive. In the root elongation zone, both root net Ca2+ influx (measured by Ca2+-selective vibrating microelectrode) and NSCC activity were increased compared to the mature epidermis, suggesting the involvement of NSCC in growth. A Ca2+ acquisition system based on NSCC and HACC co-existence is proposed. In mature epidermal cells, NSCC-mediated Ca2+ influx dominates whereas in specialised root cells (root hairs and elongation zone cells) where elevated [Ca2+]cyt activates HACCs, HACC-mediated Ca2+ influx predominates.     

Determination of cell electroporation from the release of intracellular potassium ions

When cells are exposed to a strong enough external electric field, transient aqueous pores are formed in the membrane. The fraction of electroporated cells can be determined by measuring the release of intracellular potassium ions. The current work is the first study where such a method was employed successfully not only with cells suspended in the medium with a rather high concentration of potassium (4-5 mM) but also with cells that release some part of intracellular potassium responding, in this way, to the stress caused by manipulation procedures during the preparation of the cell suspension. Experiments were carried out on mouse hepatoma MH-A22 cells exposed to a square-wave electric pulse. The curves showing the dependence of the fraction of the cells that have become permeable to bleomycin, a membrane-impermeable cytotoxic drug, are close to the ones showing the release of intracellular potassium ions.     

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.     

Micro-chemical imaging of cesium distribution in Arabidopsis thaliana plant and its interaction with potassium and essential trace elements

Cesium as an alkali element exhibits a chemical reactivity similar to that of potassium, an essential element for plants. It has been suggested that Cs phytotoxicity might be due either to its competition with potassium to enter the plant, resulting in K starvation, or to its intracellular competition with K binding sites in cells. Such elemental interactions can be evidenced by chemical imaging, which determines the elemental distributions. In this study, the model plant Arabidopsis thaliana was exposed to 1 mM cesium in the presence (20 mM) or not of potassium. The quantitative imaging of Cs and endogenous elements (P, S, Cl, K, Ca, Mn, Fe, and Zn) was carried out using ion beam micro-chemical imaging with 5 microm spatial resolution. Chemical imaging was also evidenced by microfocused synchrotron-based X-ray fluorescence (microXRF) which presents a better lateral resolution (<1 microm) but is not quantitative. Cesium distribution was similar to potassium which suggests that Cs can compete with K binding sites in cells. Cesium and potassium were mainly concentrated in the vascular system of stems and leaves. Cs was also found in lower concentration in leaves mesophyll/epidermis. This late representing the larger proportion in mass, mesophyll/epidermis can be considered as the major storage site for cesium in A. thaliana. Trichomes were not found to accumulate cesium. Interestingly, increased Mn, Fe, and Zn concentrations were observed in leaves at high chlorosis. Mn and Fe increased more in the mesophyll than in veins, whereas zinc increased more in veins than in the mesophyll suggesting a tissue specific interaction of Cs with these trace elements homeostasis. This study illustrates the sensitivity of ion beam microprobe and microfocused synchrotron-based X-ray fluorescence to investigate concentrations and distributions of major and trace elements in plants. It also shows the suitability of these analytical imaging techniques to complement biochemical investigations of metallic stress in plants.     

拟南芥钾离子吸收、转运及低钾胁迫的分子机理研究进展

钾(K)作为植物所需的3种大量元素之一,参与体内诸多的生理和生化过程,对于植物的生长和发育极其重要。目前,国内外学者对植物吸收、运输和利用K⁺的研究已有一定深度,尤其以模式植物拟南芥(Arabidopsis thaliana(L.) Heynh.)为研究对象。其中,与K⁺吸收、转运相关的离子通道和转运蛋白一直都是研究热点。本文综合近年来国内外相关研究进展,主要阐述K⁺通道和转运蛋白,K⁺的吸收和运输,类钙调磷酸酶(Calcineurin B-Like, CBL)-CBL相互作用蛋白激酶(CBL-Interacting protein kinase, CIPK)信号途径,参与该信号转导的一些小信号分子,对K⁺研究方面存在的问题进行了总结,并对未来的研究方向进行了展望。     

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.     

拟南芥养分离子转运蛋白研究进展

养分离子的跨膜转运是细胞获取养分的重要环节,亦是植物在组织和器官水平上进行养分吸收运移的基础。文中综述了拟南芥中养分离子转运蛋白在基因克隆、序列与结构分析、功能鉴定、表达与调控方面的研究进展,其中着重讨论了这些转运蛋白在氮、磷和钾等营养元素吸收、运输、分配中的作用。     

Genetic differences among wild oat lines in potassium uptake and growth in relation to potassium supply

Summary Short-term (10 min) K⁺ (⁸⁶Rb) influxes (ϕK _oc ⁺ ), rates of net K⁺ uptake (ϕK _net ⁺ ) and growth in relation to K⁺ supply were studied in genetically pure lines of wild oat. The wild oat lines employed in this study showed substantial differences in these trais. ϕK _oc ⁺ was higher in the AN lines (AN5 1, AN 474) than CS 40 and SH lines (SH 319, SH 430) in plants grown under both low and high K⁺ conditions. Kinetic constants V_max and K_m for ϕK _oc ⁺ , of selected lines and the rates of change of these constants with root K⁺ concentration ([K⁺]) showed that ϕK _oc ⁺ in AN 51 was consistently higher than in CS 40 and SH 319. ϕK _net ⁺ was also generally higher in AN 51. ϕK _net ⁺ (at different growth stages), unlike ϕK _oc ⁺ , failed to correlate with root [K⁺]; ϕK _net ⁺ values were low during the first two weeks despite lower root [K⁺]. CS 40 showed the highest utilization efficiency, produced the largest amount of biomass, absorbed most K⁺ and flowered earliest (by day 30). AN 51 had not produced flowers at day 42.     

Relationships between physiological and morphological attributes related to phosphate uptake in 25 genotypes of Arabidopsis thaliana

The degree of genetic integration among morphological and physiological characters associated with phosphate uptake in impoverished versus fertile sites was studied among 25 inbred homozygous lines of Arabidopsis thaliana (L.) Heynh. The characters recorded were initial uptake rates, C_min (which reflects the ability to deplete phosphate to low concentrations), SRL (specific root length), and root: shoot allocation variables. Highly significant genotypic differentiation was detected for all of the variables (P<0.001). Cmin was correlated with influx per cm or mg of root; the lines better able to pick up Pi at low concentrations generally had slower initial uptake rates per cm or mg of root. This suggests an evolutionary divergence between a character important for nutrient uptake under fertile conditions, and one important for nutrient uptake under impoverished conditions. This relationship however, may not be valid due to the non-proportional relationship between root length or mass and influx. With a measure of influx that took this non-proportionality into account, the relationship became nonsignificant. SRL was negatively correlated (P<0.05) with influx per unit length of root. This suggests that there is an allocation-based trade-off between root length and diameter among Arabidopsis thaliana genotypes. Lower uptake rates per unit length of root for genotypes with thin roots may simply be a consequence of lower root surface area.     

Orientation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> KAT1 Channel in the Plasma Membrane

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K⁺ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K⁺ channel, we have inserted a flag epitope in the NH₂ terminus or the S3–S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K⁺ channel.     

Mechanisms of potassium absorption by higher plant roots

Potassium, as a plant macronutrient, is accumulated in plant cells from relatively dilute soil solutions and is indispensable for many vital processes. Studies characterising potassium uptake by roots stretch back over many decades. However, it is only with the introduction of modern electrophysiological and molecular techniques that investigations have been possible at a molecular level. Such approaches have confirmed the existence of discrete high and low affinity uptake systems at the root plasma membrane and have greatly enhanced our understanding of the underlying molecular nature of these uptake systems.
High affinity K⁺ uptake from micromolar external K⁺ levels is coupled to H⁺ transport as demonstrated independently by patch clamping of single root protoplasts and by studying the transport system after expression in Xenopus oocytes . The measured coupling ratio between the two ions is 1:1 and is sufficient to account for an accumulation ratio in excess of 10⁶, a value which encompasses experimental observations on K⁺ accumulation.
Low affinity K⁺ uptake activates at relatively high external K⁺ levels in the millimolar range and is 'passive' i.e. down the electrochemical gradient for potassium. In two higher plant species single cell inward potassium currents have been identified which are associated with low affinity potassium uptake. Furthermore, specific ion channels which underlie these potassium influxes and form a major constituent of the low affinity potassium uptake pathway have been identified and characterised.     

Prenylcysteine methylesterase in Arabidopsis thaliana

Prenylated proteins undergo a series of post-translational modifications, including prenylation, proteolysis, and methylation. Collectively, these modifications generate a prenylcysteine methylester at the carboxyl terminus and modulate protein targeting and function. Prenylcysteine methylation is the only reversible step in this series of modifications. However, prenylcysteine -carboxyl methylesterase (PCME) activity has not been described in plants. We have detected a specific PCME activity in Arabidopsis thaliana membranes that discriminates between biologically relevant and irrelevant prenylcysteine methylester substrates. Furthermore, we have identified an Arabidopsis gene (At5g15860) that encodes measurable PCME activity in recombinant yeast cells with greater specificity for biologically relevant prenylcysteine methylesters than the activity found in Arabidopsis membranes. These results suggest that specific and non-specific esterases catalyze the demethylation of prenylcysteine methylesters in Arabidopsis membranes. Our findings are discussed in the context of prenylcysteine methylation/demethylation as a potential regulatory mechanism for membrane association and function of prenylated proteins in Arabidopsis.     

Zinc uptake and radial transport in roots of Arabidopsis thaliana: a modelling approach to understand accumulation

Background and Aims

Zinc uptake in roots is believed to be mediated by ZIP (ZRT-, IRT-like proteins) transporters. Once inside the symplast, zinc is transported to the pericycle, where it exits by means of HMA (heavy metal ATPase) transporters. The combination of symplastic transport and spatial separation of influx and efflux produces a pattern in which zinc accumulates in the pericycle. Here, mathematical modelling was employed to study the importance of ZIP regulation, HMA abundance and symplastic transport in creation of the radial pattern of zinc in primary roots of Arabidopsis thaliana.

Methods

A comprehensive one-dimensional dynamic model of radial zinc transport in roots was developed and used to conduct simulations. The model accounts for the structure of the root consisting of symplast and apoplast and includes effects of water flow, diffusion and cross-membrane transport via transporters. It also incorporates the radial geometry and varying porosity of root tissues, as well as regulation of ZIP transporters.

Key Results

Steady-state patterns were calculated for various zinc concentrations in the medium, water influx and HMA abundance. The experimentally observed zinc gradient was reproduced very well. An increase of HMA or decrease in water influx led to loss of the gradient. The dynamic behaviour for a change in medium concentration and water influx was also simulated showing short adaptation times in the range of seconds to minutes. Slowing down regulation led to oscillations in expression levels, suggesting the need for rapid regulation and existence of buffering agents.

Conclusions

The model captures the experimental findings very well and confirms the hypothesis that low abundance of HMA4 produces a radial gradient in zinc concentration. Surprisingly, transpiration was found also to be a key parameter. The model suggests that ZIP regulation takes place on a comparable timescale as symplastic transport.     

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana

Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.     

Chromosome painting in Arabidopsis thaliana

Chromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C(-1)). Pools of contiguous 113-139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.     

拟南芥(Arabidopsis thaliana)氮、碳离子注入诱变效应分析

采用N +、C +离子注入拟南芥 (Arabidopsisthaliana)种子 ,统计了种子的发芽指数 (发芽率和发芽势 ) ;用改良的RAPD技术对N+离子注入种子植株的DNA进行11个引物的随机片段多态性扩增。结果表明 ,合适剂量的N+、C+离子注入可使种子发芽率提高 ,两种离子注入种子的发芽率峰值 (分别为92.3 %和74.4 % )都在5×1014ions/cm2;分析N +离子注入材料发现 ,在1×1013 -1×1016ions/cm2剂量范围内 ,基因组DNA的变异率与发芽指数的变化趋势基本一致 ,变异率峰值 (9.0 % )在1×1015 ions/cm2。结果提示 ,分析低能N+离子诱变效应的最佳注入剂量在1×1014-5×1015 ions/cm2。对N+、C+离子注入的比较发现 ,一定范围内同等剂量C+离子注入的诱变率高于N +离子注入