Shin Hamamoto

Sodium transport system in plant cells.

Since sodium, Na, is a non-essential element for the plant growth, the molecular mechanism of Na+ transport system in plants has remained elusive for the last two decades. The accumulation of Na+ in soil through irrigation for sustainable agricultural crop production, particularly in arid land, and by changes in environmental and climate conditions leads to the buildup of toxic level of salts in the soil. Since the latter half of the twentieth century, extensive molecular research has identified several classes of Na+ transporters that play major roles in the alleviation of ionic stress by excluding toxic Na+ from the cytosol or preventing Na+ transport to the photosynthetic organs, and also in osmotic stress by modulating intra/extracellular osmotic balance. In this review, we summarize the current knowledge of three major Na+ transporters, namely NHX, SOS1, and HKT transporters, including recently revealed characteristics of these transporters.

The jasmonate-responsive GTR1 transporter is required for gibberellin-mediated stamen development in Arabidopsis

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.

Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts

The tonoplast K+ membrane transport system plays a crucial role in maintaining K+ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K+ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased ∼2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K+ over Na+. NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca2+, but a cytosolic concentration of 45 μm Ca2+ resulted in a 2-fold increase in the amplitude of the K+ current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K+ currents. These results show that NtTPK1 is a novel tonoplast K+ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.

12-Hydroxyjasmonic Acid Glucoside Is a COI1-JAZ-Independent Activator of Leaf-Closing Movement in Samanea saman

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.

HKT transporters mediate salt stress resistance in plants: from structure and function to the field.

Plant cells are sensitive to salinity stress and do not require sodium as an essential element for their growth and development. Saline soils reduce crop yields and limit available land. Research shows that HKT transporters provide a potent mechanism for mediating salt tolerance in plants. Knowledge of the molecular ion transport and regulation mechanisms and the control of HKT gene expression are crucial for understanding the mechanisms by which HKT transporters enhance crop performance under salinity stress. This review focuses on HKT transporters in monocot plants and in Arabidopsis as a dicot plant, as a guide to efforts toward improving salt tolerance of plants for increasing the production of crops and bioenergy feedstocks.

The phosphoinositide PI(3,5)P 2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P2. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P2 did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P2 elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P2 was applied to hTPC2 expressed in baker’s yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P2-mediated activation and Na+ selectivity of mammalian TPC2.

Defining membrane spanning domains and crucial membrane-localized acidic amino acid residues for K⁺ transport of a Kup/HAK/KT-type Escherichia coli potassium transporter

Potassium (K(+))-uptake transport proteins present in prokaryote and eukaryote cells are categorized into two classes; Trk/Ktr/HKT, K(+) channel, and Kdp belong to the same superfamily, whereas the remaining K(+)-uptake family, Kup/HAK/KT has no homology to the others, and neither its membrane topology nor crucial residues for K(+) uptake have been identified. We examined the topology of Kup from Escherichia coli. Results from the reporter fusion and cysteine labeling assays support a model with 12 membrane-spanning domains. A model for proton-coupled K(+) uptake mediated by Kup has been proposed. However, this study did not show any stimulation of Kup activity at low pH and any evidence of involvement of the three His in Kup-mediated K(+) uptake. Moreover, replacement of all four cysteines of Kup with serine did not abolish K(+) transport activity. To gain insight on crucial residues of Kup-mediated K(+) uptake activity, we focused on acidic residues in the predicted external and transmembrane regions, and identified four residues in the membrane regions required for K(+) uptake activity. This is different from no membrane-localized acidic residues essential for Trk/Ktr/HKTs, K(+) channels and Kdp. Taken together, these results demonstrate that Kup belongs to a distinct type of K(+) transport system.

Organelle-localized potassium transport systems in plants

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K(+) transport systems allowing K(+) to move across the membrane. K(+) transport systems in plant organelles act coordinately with the plasma membrane intrinsic K(+) transport systems to maintain cytosolic K(+) concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K(+) channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K(+) homeostasis of the cytoplasm. The initial electrophysiological measurements of K(+) transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K(+) transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K(+) transport system has been isolated from cyanobacteria, which may add to our understanding of K(+) flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K(+) transport proteins.

Molecular bases of multimodal regulation of a fungal transient receptor potential (TRP) channel

Background: Multimodality of TRP channels underlies their diverse physiological functions. Results: We identified a fungal multimodal TRP channel whose cytosolic domain (CTD) mediates various channel regulation. Conclusion: CTD has an oligomerization module critical for osmoreception, yet its flexible structure allows dynamic regulations with other functional modalities. Significance: This work proposes structural and biophysical principles for multimodality of a TRP channel family member. Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Characterization of the role of a mechanosensitive channel in osmotic down shock adaptation in Synechocystis sp PCC 6803.

Synechocystis sp strain PCC 6803 contains one gene encoding a putative large conductance mechanosensitive channel homolog [named SyMscL (slr0875)]. However, it is unclear whether SyMscL contributes to the adaptation to hypoosmotic stress in Synechocystis. Here we report the in vivo characteristics of SyMscL. SyMscL was mainly expressed in the plasma membrane of Synechocystis. Cell volume monitoring using stopped-flow spectrophotometry showed that ΔsymscL cells swelled more rapidly than wild-type cells under hypoosmotic stress conditions. Expression of symscL was under circadian control, and its peak corresponded to the beginning of subjective night. These results indicate that SyMscL functioned as one component of the osmotic homeostatic regulatory system of the cell coordinating the response of Synechocystis to daily metabolic osmotic fluctuations and environmental changes.