Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guidance signals from female cells are being identified; however, how pollen responds to those cues is largely unknown. Here, we show that two predicted cation/proton exchangers (CHX) in Arabidopsis thaliana, CHX21 and CHX23, are essential for pollen tube guidance. Male fertility was unchanged in single chx21 or chx23 mutants. However, fertility was impaired in chx21 chx23 double mutant pollen. Wild-type pistils pollinated with a limited number of single and double mutant pollen producing 62% fewer seeds than those pollinated with chx23 single mutant pollen, indicating that chx21 chx23 pollen is severely compromised. Double mutant pollen grains germinated and grew tubes down the transmitting tract, but the tubes failed to turn toward ovules. Furthermore, chx21 chx23 pollen tubes failed to enter the micropyle of excised ovules. Green fluorescent protein-tagged CHX23 driven by its native promoter was localized to the endoplasmic reticulum of pollen tubes. CHX23 mediated K + transport, as CHX23 expression in Escherichia coli increased K + uptake and growth in a pH-dependent manner. We propose that by modifying localized cation balance and pH, these transporters could affect steps in signal reception and/or transduction that are critical to shifting the axis of polarity and directing pollen growth toward the ovule.

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K+-uptake-system deficient E.coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese Hamster Ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K+ channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

f Photoautotrophic bacteria have developed mechanisms to maintain K ؉ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K ؉ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K ؉ (Kdp). The contributions of each of these K ؉ transport systems to cellular K ؉ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K ؉ uptake, albeit with lower rates than were conferred by Ktr. An onchip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K ؉ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K ؉ depletion. Correspondingly, Kdp-mediated K ؉ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K ؉ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K Living cells have developed specific responses to hyperosmotic shock. Upon exposure to this stress, cells initially lose water and their volume shrinks. In all living cells, K ϩ is the major intracellular cation used for the maintenance of turgor pressure, cytosolic osmolarity, protein structuring, and membrane potential (1-3). In contrast to animals, Na ϩ /K ϩ ATP pumps are generally missing in bacteria and plants. Hence, these cells possess K ϩ uptake transporters to supply K ϩ to the cells. Particularly after hyperosmotic stress, cells quickly take up K ϩ from the medium to increase the intracellular osmolarity, which prevents water efflux from the cell. Data from genetic and biochemical experiments indicate that the activity and the expression of these transporters respond to hyperosmotic stress. In the later phase of acclimation to hyperosmotic stress, cells also induce the synthesis of osmoprotective molecules, such as glutamate, trehalose, proline, and glucosylglycerol (4, 5). Despite an increasing amount of data on cellular osmoregulation involving ion flux across the membrane, direct evidence for the involvement of specific transporters in the cellular response to osmotic up-shock is lacking for photoautotrophic organisms.The cyanobacterium Synechocystis sp. strain PCC 6803 (hereinafter referred to as Synechocystis) is a frequently used unicellular photosynthetic prokaryote that can survive under a wide range of environmental conditions (6). Unlike Escherichia coli, Synechocystis possesses an internal thylakoid membrane system, which...

This study aims to develop learning devices, especially in the evaluation section of science learning in junior high school, which in turn after being implemented in the classroom is expected to develop 21st century skills of students. In particular, this study aims to, 1) identify science learning evaluation tools made by teachers so that they obtain a clear picture of the profile of the evaluation tool, 2) develop a junior high school science learning evaluation tool that supports 21st century skills. This research includes development research in four stages, namely: decide, design, develop, and evaluate. The results of this study indicate that the tools developed by the teacher are in complete category, the learning objectives are clear, although the stages in part still need to be improved. Learning evaluation instruments developed by the teacher have met the principles of assessment, the presentation is in accordance with the curriculum, the sentence in the question is quite communicative, uses good and correct language and does not cause multiple interpretations. However, the majority of questions made by teachers still measure low-level thinking skills. This is because teachers have not been trained in developing questions that measure high-level thinking skills. An evaluation tool that was then developed together with the teacher was intended to perfect the shortcomings of the previous evaluation toolKeywords: 21st Century Skills, Learning Evaluation Tools, Middle School Science

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Mutation of a conserved His-157 in the second pore loop of KtrB drastically reduced the activity of the K ؉ transporter from Synechocystis sp. strain PCC 6803. This result suggests that His-157 plays an essential role in the K ؉ transport activity of the transporter system.

The Na ؉ -dependent K ؉ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K ؉ -translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K ؉ uptake and in modulating Na ؉ dependency. Expression of KtrB alone in a K ؉ uptake-deficient Escherichia coli strain conferred low K ؉ uptake activity that was not stimulated by Na ؉ . Coexpression of both KtrA and KtrE with KtrB increased the K ؉ transport activity in a Na ؉ -dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K m for K ؉ in all cases. However, none of the mutations eliminated the Na ؉ dependency of Ktr-mediated K ؉ transport. Mutations of residues on the cytoplasmic side had larger effects on K ؉ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K ؉ uptake rate and conferred Na ؉ dependency.