Expression and Ion Transport Activity of Rice OsHKT1;1 Variants

OsHKT1;1 in rice, belongs to the high-affinity K+ Transporter family, has been found to be involved in salt tolerance. OsHKT1;1 in japonica rice (Nipponbare) produces mRNA variants, but their functions remain elusive. In salt tolerant rice, Pokkali, eight OsHKT1;1 variants (V1-V8) were identified in addition to the full-length OsHKT1;1 (FL) cDNA. Absolute quantification by qPCR revealed that accumulation of OsHKT1;1-FL mRNA is minor in contrast to that of OsHKT1;1-V1, -V2, -V4, and -V7 mRNAs, all of which are predominant in shoots, while only V1 and V7 mRNAs are predominant in roots. Two electrode voltage clamp (TEVC) experiments using Xenopus laevis oocytes revealed that oocytes-expressing OsHKT1;1-FL from Pokkali exhibited inward-rectified currents in the presence of 96 mM Na+ as reported previously. Further TEVC analyses indicated that six of eight OsHKT1;1 variants elicited currents in a Na+ or a K+ bath solution. OsHKT1;1-V6 exhibited a similar inward rectification to the FL protein. Contrastingly, however, the rests mediated bidirectional currents in both Na+ and K+ bath solutions. These data suggest possibilities that novel mechanisms regulating the transport activity of OsHKT1;1 might exist, and that OsHKT1;1 variants might also carry out distinct physiological roles either independently or in combination with OsHKT1;1-FL

Salinity tolerance mechanisms in glycophytes: An overview with the central focus on rice plants

Elevated Na+ levels in agricultural lands are increasingly becoming a serious threat to the world agriculture. Plants suffer osmotic and ionic stress under high salinity due to the salts accumulated at the outside of roots and those accumulated at the inside of the plant cells, respectively. Mechanisms of salinity tolerance in plants have been extensively studied and in the recent years these studies focus on the function of key enzymes and plant morphological traits. Here, we provide an updated overview of salt tolerant mechanisms in glycophytes with a particular interest in rice (Oryza sativa) plants. Protective mechanisms that prevent water loss due to the increased osmotic pressure, the development of Na+ toxicity on essential cellular metabolisms, and the movement of ions via the apoplastic pathway (i.e. apoplastic barriers) are described here in detail.ArticleRICE. 5:11 (2012)journal articl

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status

Changes in expression level of OsHKT1;5 alters activity of membrane transporters involved in K+ and Ca2+ acquisition and homeostasis in salinized rice roots

In rice, the OsHKT1;5 gene has been reported to be a critical determinant of salt tolerance. This gene is harbored by the SKC1 locus, and its role was attributed to Na+ unloading from the xylem. No direct evidence, however, was provided in previous studies. Also, the reported function of SKC1 on the loading and delivery of K+ to the shoot remains to be explained. In this work, we used an electrophysiological approach to compare the kinetics of Na+ uptake by root xylem parenchyma cells using wild type (WT) and NIL(SKC1) plants. Our data showed that Na+ reabsorption was observed in WT, but not NIL(SKC1) plants, thus questioning the functional role of HKT1;5 as a transporter operating in the direct Na+ removal from the xylem. Instead, changes in the expression level of HKT1;5 altered the activity of membrane transporters involved in K+ and Ca2+ acquisition and homeostasis in the rice epidermis and stele, explaining the observed phenotype. We conclude that the role of HKT1;5 in plant salinity tolerance cannot be attributed to merely reducing Na+ concentration in the xylem sap but triggers a complex feedback regulation of activities of other transporters involved in the maintenance of plant ionic homeostasis and signaling under stress conditions

AtHKT1;1 Mediates Nernstian Sodium Channel Transport Properties in Arabidopsis Root Stelar Cells

The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na+) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na+ and K+ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K+ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na+ over K+ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na+ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na+ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na+ channel transport properties

A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interactions

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs