Arabidopsis thaliana contains the putative K
efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport ...and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K
transport activity, whereas KEA4 and KEA6 functioned as a K
uptake system. The thylakoid membrane-localized Na
/H
antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na
/H
selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K
transport activity. The first residue in the conserved motif was not a determinant for K
or Na
selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K
transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K
transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K
transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane.
Saccharomyces cerevisiae possesses a transient receptor potential (TRP) channel homolog TRPY1 in its vacuolar membrane, considered to be an ancestral TRP channel. So far, studies have focused on the ...channel properties of TRPY1, but its regulation and physiologic role remained to be elucidated. Here, we investigated TRPY1 channel function in vitro and in vivo. Patch‐clamp recording of TRPY1 in yeast vacuolar membranes showed that Ca2+ on the lumen side inhibited TRPY1‐mediated channel activity, whereas luminal Zn2+ increased the currents. TRPY1 was activated in the presence of a reducing agent, 2‐mercaptoethanol. The cysteine at position 624 was identified as the target for this activating action. This activation was independent of the presence of cytosolic Ca2+. The amplitude of TRPY1‐mediated current was reduced by addition of phosphatidylinositol 3‐phosphate on the cytosolic side but not by phosphatidylinositol (PI) or phosphatidylinositol 3,5‐phosphate. Measurement of the transient Ca2+ increase in response to hyper‐osmotic shock in several yeast mutants defective in different steps of the PI phosphate biogenesis pathway supported this interpretation. Addition of a microtubule inhibitor strongly decreased the transient cytosolic Ca2+ increase upon hyper‐osmotic shock. Taken together, the data indicate that the vacuolar TRPY1 Ca2+ channel mediates the perception of cytosolic signals that were induced by external changes in osmolarity, and participates in the modulation of cytosolic calcium signaling through Ca2+ release from the vacuole to maintain intracellular Ca2+ homeostasis in yeast.
Saccharomyces cerevisiae possesses an ancestral transient receptor potential channel, TRPY1, in its vacuolar membrane. We evaluated the activation/inhibition properties of TRPY1 with respect to channel regulatory sites for sensing reducing reagents, calcium in the cytosol and the vacuole, and phosphatidylinositol phosphates‐mediated channel activation. The multimodal regulation of TRPY1 activity contributes to the maintenance of cytosolic calcium homeostasis during environmental changes.
In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. ...VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca
-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca
-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.
Plant hormones are transported across cell membranes during various physiological events. Recent identification of abscisic acid and strigolactone transporters suggests that transport of various ...plant hormones across membranes does not occur by simple diffusion but requires transporter proteins that are strictly regulated during development. Here, we report that a major glucosinolate transporter, GTR1/NPF2.10, is multifunctional and may be involved in hormone transport in Arabidopsis thaliana. When heterologously expressed in oocytes, GTR1 transports jasmonoyl-isoleucine and gibberellin in addition to glucosinolates. gtr1 mutants are severely impaired in filament elongation and anther dehiscence resulting in reduced fertility, but these phenotypes can be rescued by gibberellin treatment. These results suggest that GTR1 may be a multifunctional transporter for the structurally distinct compounds glucosinolates, jasmonoyl-isoleucine and gibberellin, and may positively regulate stamen development by mediating gibberellin supply.
N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes ...remains unknown.
Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+-regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants.
We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5–CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5–CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes.
Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.
The circadian leaf opening and closing (nyctinasty) of Fabaceae has attracted scientists’ attention since the era of Charles Darwin. Nyctinastic movement is triggered by the alternate swelling and ...shrinking of motor cells at the base of the leaf. This, in turn, is facilitated by changing osmotic pressures brought about by ion flow through anion and potassium ion channels. However, key regulatory ion channels and molecular mechanisms remain largely unknown. Here, we identify three key ion channels in mimosoid tree Samanea saman: the slow-type anion channels, SsSLAH1 and SsSLAH3, and the Shaker-type potassium channel, SPORK2. We show that cell-specific circadian expression of SsSLAH1 plays a key role in nyctinastic leaf opening. In addition, SsSLAH1 co-expressed with SsSLAH3 in flexor (abaxial) motor cells promoted leaf opening. We confirm the importance of SLAH1 in leaf movement using SLAH1-impaired Glycine max. Identification of this “master player” advances our molecular understanding of nyctinasty.
•Nyctinasty is triggered by asymmetrical volume changes of motor cells•Circadian regulation of the anion channel SsSLAH1 underlies nyctinasty•Gene expression of SsSLAH1 shows morning-phased circadian rhythms•The importance of SLAH1 was confirmed by using SLAH1-impaired Glycine max
Observations of the daily opening and closing of leaves (nyctinasty) in the 18th century provided some of the first evidence of a biological clock. Oikawa et al. show that circadian regulation of the anion channel SsSLAH1 in the mimosoid tree is the likely molecular mechanism behind nyctinasty.
Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a ...distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.
Mechanosensitive channels play an important role in the adaptation of cells to hypo-osmotic shock. Among members of this channel family in Escherichia coli, the exact function and physiological role ...of the mechanosensitive channel homolog YbdG remain unclear. Characterization of YbdG's physiological role has been hampered by its lack of measurable transport activity. Using a nitrosoguanidine mutagenesis-aided screen in combination with next-generation sequencing, here we isolated a mutant with a point mutation in ybdG. This mutation (resulting in a I167T change) conferred sensitivity to high osmotic stress, and the mutant cells differed from WT cells in morphology during hyperosmotic stress at alkaline pH. Interestingly, unlike the cells containing the I167T variant, a null-ybdG mutant did not exhibit this sensitivity and phenotype. Although I167T was located near the putative ion-conducting pore in a transmembrane region of YbdG, no change in ion channel activities of YbdG-I167T was detected. Of note, introduction of the WT C-terminal cytosolic region of YbdG into the I167T variant complemented the osmo-sensitive phenotype. Co-precipitation of proteins interacting with the C-terminal YbdG region led to the isolation of HldD and FbaA, whose overexpression in cells containing the YbdG-I167T variant partially rescued the osmo-sensitive phenotype. This study indicates that YbdG functions as a component of a mechanosensing system that transmits signals triggered by external osmotic changes to intracellular factors. The cellular role of YbdG uncovered here goes beyond its predicted function as an ion or solute transport protein.
The physiological effects of caesium (Cs) on living cells are poorly understood. Here, we examined the physiological role of Cs
on the activity of the potassium transporters in E. coli. In the ...absence of potassium (K
), Kup-mediated Cs
uptake partially supported cell growth, however, at a much lower rate than with sufficient K
. In K
-limited medium (0.1 mM), the presence of Cs
(up to 25 mM) in the medium enhanced growth as much as control medium containing 1 mM K
. This effect depended on the maintenance of basal levels of intracellular K
by other K
uptake transporters. Higher amounts of K
(1 mM) in the medium eliminated the positive effect of Cs
on growth, and revealed the inhibitory effect of high Cs
on the growth of wild-type E. coli. Cells lacking Kdp, TrkG and TrkH but expressing Kup grew less well when Cs
was increased in the medium. A kdp mutant contained an increased ratio of Cs
/K
in the presence of high Cs
in the medium and consequently was strongly inhibited in growth. Taken together, under excess Cs
conditions Kup-mediated Cs
influx sustains cell growth, which is supported by intracellular K
supplied by Kdp.
Jasmonates are ubiquitously occurring plant growth regulators with high structural diversity that mediate numerous developmental processes and stress responses. We have recently identified ...12-O-β-D-glucopyranosyljasmonic acid as the bioactive metabolite, leaf-closing factor (LCF), which induced nyctinastic leaf closure of Samanea saman. We demonstrate that leaf closure of isolated Samanea pinnae is induced upon stereospecific recognition of (-)-LCF, but not by its enantiomer, (+)-ent-LCF, and that the nonglucosylated derivative, (-)-12-hydroxyjasmonic acid also displays weak activity. Similarly, rapid and cell type-specific shrinkage of extensor motor cell protoplasts was selectively initiated upon treatment with (-)-LCF, whereas flexor motor cell protoplasts did not respond. In these bioassays related to leaf movement, all other jasmonates tested were inactive, including jasmonic acid (JA) and the potent derivates JA-isoleucine and coronatine. By contrast, (-)-LCF and (-)-12-hydroxyjasmonic acid were completely inactive with respect to activation of typical JA responses, such as induction of JA-responsive genes LOX2 and OPCL1 in Arabidopsis (Arabidopsis thaliana) or accumulation of plant volatile organic compounds in S. saman and lima bean (Phaseolus lunatus), generally considered to be mediated by JA-isoleucine in a COI1-dependent fashion. Furthermore, application of selective inhibitors indicated that leaf movement in S. saman is mediated by rapid potassium fluxes initiated by opening of potassium-permeable channels. Collectively, our data point to the existence of at least two separate JA signaling pathways in S. saman and that 12-O-β-D-glucopyranosyljasmonic acid exerts its leaf-closing activity through a mechanism independent of the COI1-JAZ module.
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