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71.
Michael Sandmann Kamil Sk?odowski Pawel Gajdanowicz Erwan Michard Marcio Rocha Judith L Gomez-Porras Wendy González Luiz Gustavo Guedes Corraa Santiago J Ramírez-Aguilar Tracey Ann Cuin Joost T van Dongen Jean-Baptiste Thibaud Ingo Dreyer 《Plant signaling & behavior》2011,6(4):558-562
Potassium (K+) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K+ in plant homeostasis was shown. It was demonstrated that K+ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K+ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K+ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K+ channel.Key words: potassium, channel, potassium channel, AKT2, phloem (re)loading, post-translational modifications, potassium batteryPotassium (K+) is the most abundant mineral element in plants, and together with nitrogen and phosphorous, is limiting for plant production in many natural and agricultural habitats. Voltage-gated K+ channels are key players in the acquisition of K+ ions from the soil and in its redistribution within the plant.1 Structurally, these channels result from the assembly of four so-called α-subunits. The subunits are encoded by nine genes in Arabidopsis and both homo- and hetero-tetramers are expressed.2,3 The K+ channel α-subunits can be categorized into four different subfamilies, based on the voltage-gating characteristics of the exogenous K+ conductance when expressed in an appropriate heterologous expression system. Kin α-subunits form hyperpolarization-activated channels that mediate K+ uptake.4–7 Kout α-subunits form depolarization-activated channels that mediate K+ release from cells.8–10 Ksilent subunits appear unable to yield functional homomeric channels, but can combine with Kin subunits and fine-tune the K+-uptake properties of the resulting heteromeric channels.11–14 Finally, Kweak α-subunits form channels with complex voltage-gating; they allow both K+ uptake and release.15–19 In Arabidopsis, a single member is found in this subfamily, AKT2, and this channel can assemble in heteromeric channels with the Kin subunit KAT2.20To date, only scarce and speculative information has been obtained for the function of Kweak channels. When expressed in heterologous expression systems, two different subpopulations of AKT2 channels differing in their sensitivity to voltage were found.21 Channels of the first type showed gating properties and currents analogous to that of Kin channels, while the other sort enabled a non-rectified (leak-like) current; they were open over the entire physiological voltage range.A given channel can be converted from one type to the other by post-translational modifications.21 A voltage-dependent phosphorylation was found to be an essential step for this switch,22,23 although the kinase responsible for this conversion still needs to be uncovered.24 In biophysical studies, mutant versions of the Arabidopsis Kweak channel subunit AKT2 have been created that showed impaired gating mode settings.22,23 Recently, Gajdanowicz et al. generated transgenic Arabidopsis thaliana plants that express these mutant AKT2 channels in the background of the akt2-1 null-allele plant.25 The major conclusion from analyses of these mutants is that the status switching of AKT2 from an inward-rectifying to a non-rectifying channel is crucial for plants to overcome energy-limiting conditions. This function of AKT2 could be correlated to its expression in phloem tissues. Selective expression of AKT2 under the control of the phloem companion cell-specific AtSUC2 promoter rescued the akt2-1 line, but conversely, selective expression of AKT2 under the control of the guard cell-specific GC1 promoter,26 resulted in further impairment of plant growth (Fig. 1). By combining diverse experimental approaches with mathematical simulation methods, an existing model for phloem (re)loading18,27 was fundamentally improved. This allowed the uncovering of a novel and interesting role of K+ in phloem physiology: K+ gradients present between the sieve element/companion cell (SE/CC) complex and the apoplast can serve as an energy source in phloem (re)loading processes. This “potassium battery” can be tapped by means of AKT2 regulation. This clarifies the observation of Deeken et al.28 that in AKT2 loss-of-function mutant plants, assimilates leaking away from the sieve tube were not efficiently reloaded into the main phloem stream.Open in a separate windowFigure 1AKT2 expressed only in guard cells delays plant development. (A–C) Representative wild-type, akt2-1 and akt2-1+pGC1:AKT2 complementation plants grown for 7 weeks (A), 9 weeks (B) and 12 weeks (C) under 12-h day/12-h night conditions at normal light intensity (150 µmol m−2 s−1). (D) akt2-1+pGC1:AKT2 developed a similar number of leaves as the akt2-1 knock out plants, but bolting-time was delayed. (B and E) After 9 weeks, wild-type plants were at an advanced bolting stage, akt2-1 plants had started bolting, but only initial signs of bolting were visible in akt2-1+pGC1:AKT2 plants. (C and F) At 12 weeks, akt2-1 plants had caught up with the wild-type and akt2-1+pGC1:AKT2 was just starting to bolt, although rosette-leaves were showing clear signs of senescence. For the generation of akt2-1+pGC1:AKT2, the AKT2 cDNA was fused to the guard cell-specific GC1 promoter26 kindly provided by J.I. Schroeder, San Diego. The pGC1:AKT2 construct was cloned into pGreen0229-35S by replacing the 35S promoter and then transformed into the akt2-1 knockout plant. All seeds were cold-treated for 24 h at 4°C. Plants were grown on artificial substrate (type GS-90, Einheitserde). After 2 weeks, seedlings were transferred to single pots. Plants were grown in 60% relative humidity at 21°C during the day and 18°C at night. Phenotypical analyses were done in the middle of the day. Data are shown as means ± SD of n ≥ 9 plants. Statistical analyses using Student''s t test: (D, WT/akt2-1: p < 2e-08; D, WT/pGC-AKT2: p < 2e-08; D, akt2-1/pGC-AKT2: p < 5e-03; E, WT/akt2-1: p < 4e-06; E, WT/pGC-AKT2: p < 1e-10; E, akt2-1/pGC-AKT2: p < 5e-04; F, WT/akt2-1: p = 0.51; F, WT/pGC-AKT2: p < 1e-10; F, akt2-1/pGC-AKT2: p < 1e-10).AKT2 expression is especially abundant in phloem tissues and the root stele, both of which are characterized by a poor availability of oxygen.29,30 This local internal hypoxia impairs respiratory activity of the vascular tissue and concomitantly, respiratory ATP production is reduced.31 As a consequence, phloem transport is very susceptible to decreasing oxygen supply to the plant.29,32 It is therefore comprehensible that the above mentioned support by the K+ driving force for sucrose retrieval is especially relevant in the phloem. Indeed Gajdanowicz et al.25 showed that transgenic plants lacking the AKT2 K+ channel were severely impaired in growth when exposed to mild hypoxia (10% v:v), whereas growth of wild-type plants was unaffected by this treatment. These observations illustrate the importance of biochemical flexibility in plant cells to cope with the energetic consequences of the steep oxygen concentration gradients that generally occur in plant stems and roots.In fact, the role of K+ gradients in driving sugar, amino acid and organic acid transport across plant cell membranes was first suggested several decades ago.33,34 Experimental evidence for this concept was provided by various tests in which pieces of plant tissue were incubated in solutions with different K+ concentrations and pH levels.33,34 Unfortunately, at that time the lack of genetic information to support this hypothesis (e.g., identifying transporter proteins that could provide a molecular mechanism to explain the working mechanism of substrate transport driven by a K+-motive force) resulted in this idea falling into oblivion. Indeed, the unequivocal experimental observation of this new role of K+ gradients in phloem reloading is extremely challenging. Under normal experimental conditions, K+ fluxes and sucrose fluxes are coupled during phloem loading in source tissues and unloading in sink tissues. Nonetheless, computational simulations predict that under certain conditions, a local K+/Suc antiport is also thermodynamically possible. In this antiport system, the energy from the K+ gradient is used to transport Suc into the phloem. This process is only transient; flooding the apoplast with K+ will decrease the K+ gradient. However, the gradient can be maintained for longer if surrounding cells take up the apoplastic K+ for their own use. A K+/Suc antiport will not occur in obvious sink or source tissues since the energy balances in such cells are fundamentally different. Consequently, in these tissues only the coupled symport of K+ and Suc can be observed. However, the computational predictions allowed the identification of the experimental conditions under which the effect of the K+/Suc antiport system is empirically observable at the whole plant level.An essential role in the regulation of AKT2 is played by (de)phosphorylation events of serine residues at positions S210 and S329. The replacement of both serines by asparagine (AKT2-S210N-S329N) resulted in a K+-selective leak that is locked in a continuously open mode when the channels are expressed in Xenopus oocytes. Under certain conditions, plants expressing the AKT2-S210N-S329N mutation showed growth benefits over wild-type plants; akt2-1+AKT2-S210N-S329N plants reach the generative state faster, possess an increased number of leaves and increased fresh weight (Fig. 2). Intuitively, one would expect a continuously open channel to cause severe problems for the plant, not a benefit as was observed here. We therefore have to postulate that phosphorylation at residues AKT2-S210 and AKT2-S329 is insufficient for converting AKT2 from an inward-rectifying into a non-rectifying channel; other, as yet unknown mechanisms, must contribute to the switch in the AKT2 gating mode. Such a concept would correspond to results that would otherwise be hard to explain. For instance, when both serine residues were replaced by glutamate, the mutant AKT2-S210E-S329E still showed wild-type characteristics.22 The S to E substitution is expected to mimic the phosphorylated state better than the S to N replacement. Furthermore, position AKT2-K197 has a fundamental influence on the AKT2 gating mode.23 AKT2 mutants with that particular lysine substituted with a serine are far less sensitive towards (de)phosphorylation; they display the characteristics of a pure inward-rectifying K+ channel,23 and transgenic Arabidopsis plants expressing AKT2 channels with this substitution showed the characteristics of akt2-1 knock-out plants.25 Initially, it was proposed that the positive charge is important for sensitizing AKT2 to phosphorylation. However, the charge-conserving mutant AKT2-K197R is similar to the charge inverting mutant AKT2-K197D,23 a purely inward-rectifying channel (Fig. 3). We therefore need to take into account that in plants, K197 may also be a target of post-translational modification.35 At present, we can explain the beneficial effect of the AKT2-S210N-S329N mutant on plant growth only by a multiple step regulation of AKT2 (Fig. 4). The double-N mutation would then bypass the phosphorylation step, but AKT2-S210N-S329N could still be deregulated into an inward-rectifying channel. Thus, AKT2 can be considered as a highly specialized Kin channel that can be converted into a leak-like channel by a cascade of post-translational modification steps.Open in a separate windowFigure 2Plants expressing the AKT2-S210N-S329N mutant reach the generative state faster than wild-type plants. The mutant channel AKT2-S210N-S329N was expressed under the control of the native AKT2 promoter in the akt2-1 knock-out background. (A) Photos of representative Arabidopsis thaliana plants grown 7 weeks under short day conditions (12-h day/12-h night, light intensity = 150 µE m−2s−1). Seven weeks after sowing, plants expressing only AKT2-S210N-S329N mutant channels (n = 22) differed significantly (Student''s t test, p < 4e-05) from wild-type plants (n = 20) in the height of the main inflorescent stalk (B) and fresh weight (C). At later time points, these differences decrease.25Open in a separate windowFigure 3The mutant AKT2-K197R channel is inward-rectifying. Steady-state current-voltage characteristics measured at the end of activation voltage steps. Currents were normalized to the current values measured at −145 mV in 10 mM K+ and are shown as means ± SD (n = 6).Open in a separate windowFigure 4Minimal model for AKT2 gating-mode regulation. To switch AKT2 from an inward-rectifying into a non-rectifying channel, at least two post-translational steps are postulated. (1) Phosphorylation at residues AKT2-S210 and AKT2-S329 (transitions [1]→[2] and [3]→[4]) and (2) a yet unknown modification that most likely involves the residue AKT2-K197 (transitions [1]→[3] and [2]→[4]). Only after both modifications will AKT2 allow the efflux of K+ (state [4]). 相似文献
72.
Dennis H. Lau Nicholas J. Shipp Darren J. Kelly Shivshankar Thanigaimani Melissa Neo Pawel Kuklik Han S. Lim Yuan Zhang Karen Drury Christopher X. Wong Nicholas H. Chia Anthony G. Brooks Hany Dimitri David A. Saint Lindsay Brown Prashanthan Sanders 《PloS one》2013,8(8)
Background
Both ageing and hypertension are known risk factors for atrial fibrillation (AF) although the pathophysiological contribution or interaction of the individual factors remains poorly understood. Here we aim to delineate the arrhythmogenic atrial substrate in mature spontaneously hypertensive rats (SHR).Methods
SHR were studied at 12 and 15 months of age (n = 8 per group) together with equal numbers of age-matched normotensive Wistar-Kyoto control rats (WKY). Electrophysiologic study was performed on superfused isolated right and left atrial preparations using a custom built high-density multiple-electrode array to determine effective refractory periods (ERP), atrial conduction and atrial arrhythmia inducibility. Tissue specimens were harvested for structural analysis.Results
Compared to WKY controls, the SHR demonstrated: Higher systolic blood pressure (p<0.0001), bi-atrial enlargement (p<0.05), bi-ventricular hypertrophy (p<0.05), lower atrial ERP (p = 0.008), increased atrial conduction heterogeneity (p = 0.001) and increased atrial interstitial fibrosis (p = 0.006) & CD68-positive macrophages infiltration (p<0.0001). These changes resulted in higher atrial arrhythmia inducibility (p = 0.01) and longer induced AF episodes (p = 0.02) in 15-month old SHR. Ageing contributed to incremental bi-atrial hypertrophy (p<0.01) and atrial conduction heterogeneity (p<0.01) without affecting atrial ERP, fibrosis and arrhythmia inducibility. The limited effect of ageing on the atrial substrate may be secondary to the reduction in CD68-positive macrophages.Conclusions
Significant atrial electrical and structural remodeling is evident in the ageing spontaneously hypertensive rat atria. Concomitant hypertension appears to play a greater pathophysiological role than ageing despite their compounding effect on the atrial substrate. Inflammation is pathophysiologically linked to the pro-fibrotic changes in the hypertensive atria. 相似文献73.
Marita Westhrin Minli Xie Magnus ?. Older?y Pawel Sikorski Berit L. Strand Therese Standal 《PloS one》2015,10(3)
Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering. 相似文献
74.
Turkseven S Kruger A Mingone CJ Kaminski P Inaba M Rodella LF Ikehara S Wolin MS Abraham NG 《American journal of physiology. Heart and circulatory physiology》2005,289(2):H701-H707
Increased heme oxygenase (HO)-1 activity attenuates endothelial cell apoptosis and decreases superoxide anion (O2-) formation in experimental diabetes by unknown mechanisms. We examined the effect of HO-1 protein and HO activity on extracellular SOD (EC-SOD), catalase, O2-, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) levels and vascular responses to ACh in control and diabetic rats. Vascular EC-SOD and plasma catalase activities were significantly reduced in diabetic compared with nondiabetic rats (P < 0.05). Upregulation of HO-1 expression by intermittent administration of cobalt protoporphyrin, an inducer of HO-1 protein and activity, resulted in a robust increase in EC-SOD but no significant change in Cu-Zn-SOD. Administration of tin mesoporphyrin, an inhibitor of HO-1 activity, decreased EC-SOD protein. Increased HO-1 activity in diabetic rats was associated with a decrease in iNOS but increases in eNOS and plasma catalase activity. On the other hand, aortic ring segments from diabetic rats exhibited a significant reduction in vascular relaxation to ACh, which was reversed with cobalt protoporphyrin treatment. These data demonstrate that an increase in HO-1 protein and activity, i.e., CO and bilirubin production, in diabetic rats brings about a robust increase in EC-SOD, catalase, and eNOS with a concomitant increase in endothelial relaxation and a decrease in O2-. These observations in experimental diabetes suggest that the vascular cytoprotective mechanism of HO-1 against oxidative stress requires an increase in EC-SOD and catalase. 相似文献
75.
76.
77.
Alpha-melanocyte stimulating hormone (alpha-MSH) and ghrelin play significant yet opposite roles in the regulation of feeding: alpha-MSH inhibits, whereas ghrelin stimulates consumption. The two peptidergic systems may interact in the process of food intake control. A single report published thus far has shown that a synthetic agonist of the melanocortin receptors, MTII, injected in the hypothalamic paraventricular nucleus (PVN) decreases feeding generated by ghrelin. We found that very low doses of alpha-MSH and MTII administered ICV significantly reduced ghrelin-dependent hyperphagia. However, an endogenous molecule, alpha-MSH, infused in the PVN did not exert an inhibitory effect on ghrelin-induced consumption, whereas the effective dose of PVN MTII exceeded that necessary to decrease short-term deprivation-induced feeding. We conclude that it is likely that in feeding regulation alpha-MSH and ghrelin "interact" at the central nervous system level, but the involvement of the PVN in this interaction appears questionable. 相似文献
78.
Grzywacz P Plum LA Sicinski RR Clagett-Dame M DeLuca HF 《Archives of biochemistry and biophysics》2007,460(2):274-284
The discovery of 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) as a bone selective and bone anabolic form of vitamin D has stimulated an investigation of structure/function of bone selectivity. Four new 2-substituted-19-norvitamin D analogs 3-6 have been developed to study the structure-activity relationship at C-25. As predicted, removing the 25-hydroxy group (compound 3) from the very potent analog 2MD and its 2-methyl derivatives (5 and 6) dramatically reduces in vitro activities, but biological potency is nearly fully restored in vivo likely due to in vivo 25-hydroxylation. The introduction of a methyl group at C-25 (compound 4) that blocks in vivo 25-hydroxylation reduces biological activity both in vitro and in vivo. However, analog 4 retains bone selectivity making it interesting as a possible therapeutic for bone loss diseases. 相似文献
79.
80.