A new function for the Arabidopsis thaliana SNARE SYP121

Eukaryotic cells maintain a compartmental cellular organization of membrane-enclosed organelles that communicate with each other through the exchange of trafficking vesicles. Members of a superfamily of membrane proteins, the so-called SNAREs, are essential for the necessary fusion of vesicle membranes to the membrane of target organelles. SNAREs are needed to overcome the energy barrier that prevents spontaneous membrane fusion events. A number of studies from the past decade indicated that SNARE proteins might fulfill a function beyond merging membranes. The mammalian plasma membrane SNARE Syntaxin1A was shown to directly interact with and through this interaction modify the activity of, for example, a calcium ion channel and a potassium ion channel. In its classical function as SNARE protein, Syntaxin1A mediates specialized vesicle fusion events such as synaptic transmission in neurons or secretion of insulin from pancreatic cells. These specialized vesicle fusion events require precise timing that is controlled by intracellular signaling events. These intracellular signaling events involve the coordinated action of members from different families of ion channels. Current models suggest that the dual functions of a SNARE protein in ion channel regulation and membrane fusion serve to fine-tune highly regulated vesicle fusion events. This thesis provides evidence for the first direct interaction between a SNARE protein and an ion channel from plants and suggests a function for this interaction in Arabidopsis potassium nutrition. Three different protein-protein interaction assays for full-length membrane proteins that comprised a yeast mating based split-ubiquitin assay, co-immunoprecipitation after expression in insect cells and bi-molecular fluorescence complementation after transient Arabidopsis root transformation, confirm that the Arabidopsis plasma membrane SNARE SYP121 interacts in vitro and in vivo with the Shaker ion channel subunit KC1. Furthermore, the interaction between KC1 and SYP121 is specific over the closest homologue of Syp121, namely SYP122. Shaker channels are plasma membrane proteins with four subunits that transport the essential macronutrient potassium in response to changes in membrane voltage. The KC1 subunit is unique among the Shaker channels. It can only act as a regulatory subunit that modifies channel properties when forming heterotetramers with other Shaker subunits such as AKT1, not as functional homotetramer. AKT1 is expressed predominantly in the root epidermis, i.e. root hairs, where it overlaps with the more broadly expressed KC1 and SYP121. Previous publications showed that a low external potassium concentration combined with high levels of ammonium that is used to block all root potassium uptake systems apart from AKT1, causes akt1 null mutants to display strongly reduced main root length as well as whole plant potassium content compared to wild type plants. It is shown here that the phenotype of both syp121 and kc1 null mutants is identical to the akt1 mutant under these growth conditions. The design of new antibodies against native AKT1 and KC1 and an optimized protocol for root plasma membrane protein enrichment and solubilisation allowed for the first time visualization of native Arabidopsis AKT1 protein. This technical advance made it possible to confirm that both Shaker channel subunits are present in equal amounts in the plasma membrane of roots cells from syp121 mutant and wild type plants. It is concluded that the potassium uptake phenotype of the syp121 mutant is not caused by the absence of channel proteins from the plasma membrane due to a disruption of the vesicle trafficking function of the SNARE SYP121. An alternative function for SYP121 in potassium nutrition that involves direct interaction with AKT1-KC1 heterotetrameric channels is supported by electrophysiological measurements after heterologous expression in Xenopus leavis oocytes. SYP121 modifies the voltage-dependent potassium uptake characteristics of AKT1-KC1 heterotetramers in a way most easily understood in context of a conformational change within the voltage sensing protein parts of the Shaker channel that are caused by the direct interaction with the SNARE protein. It is concluded that the identical potassium uptake phenotype of the akt1, kc1 and syp121 mutants is caused by the inability to form a functional tripartite complexes. As KC1 is able to form heterotetrameric channels with several different Shaker channel subunits, for example KAT1 that is highly expressed in guard cells, it is likely that this novel interaction between KC1 and SYP121 might modulate channel activities in different tripartite complexes to affect various cellular functions

Bioenergy modelling for Southern Africa - benchmarking Namibia and South Africa

Namibia and South Africa as part of Southern Africa are focussing on new technologies which on the one hand have the capacity to address energy shortages, particularly to increase power generation capacity; and on the other hand fulfil socio-economic development goals with minimal negative environmental impact. Bio-oil as a product from fast pyrolysis lends itself towards bioenergy production; to serve as a liquid fuel both for heat production and/or to fuel stationary engines or power generating equipment. Fast pyrolysis is a relatively new technology globally; and not yet introduced to Southern Africa. This research therefore describes bioenergy production via fast pyrolysis systems. The potential of the bioenergy so produced is investigated in terms of its potential to fill energy gaps, particularly power, as well as to fulfil socio-economic and environmental conservation targets in Namibia and South Africa. Namibia and South Africa possess vast wood-based biomass resources which can be converted to bioenergy via fast pyrolysis. This research models the wood-based biomass resources available for bioenergy production in Namibia and South Africa respectively; describes their physical and chemical properties and provides information on where they are located within, and how they can be harvested in a sustainable manner in Namibia and South Africa. The analysis to introduce fast pyrolysis into the Namibia and South Africa is based on an in-depth review of past experiences with pyrolysis technologies and the types of products successfully sold from various pyrolysis operations. The results of biomass modelling and description are used to model a bioenergy production system via fast pyrolysis. In Namibia fast pyrolysis operations are focusing on power generation in the Otjiwarongo and Okakarara farmland area, with a capacity of up to 20MW over a 20-year period. The power so generated is based on wood from bush encroachment only. In South Africa, the wood-based resource, i.e. alien plant species and bush encroachment, could provide communities in three provinces with at least 1MW but not more than 5MW power respectively over a period of at least 20 years. However, the introduction of new technologies and their products, such as fast pyrolysis and bio-oil for bioenergy production to Namibian and South African markets would be cumbersome. Technical and non-technical as well regulatory barriers have been identified; these need to be overcome before fast pyrolysis is accepted in the market

Biodesalination: an emerging technology for targeted removal of Na+and Cl−from seawater by cyanobacteria

Although desalination by membrane processes is a possible solution to the problem of freshwater supply, related cost and energy demands prohibit its use on a global scale. Hence, there is an emerging necessity for alternative, energy and cost-efficient methods for water desalination. Cyanobacteria are oxygen-producing, photosynthetic bacteria that actively grow in vast blooms both in fresh and seawater bodies. Moreover, cyanobacteria can grow with minimal nutrient requirements and under natural sunlight. Taking these observations together, a consortium of five British Universities was formed to test the principle of using cyanobacteria as ion exchangers, for the specific removal of Na+ and Cl− from seawater. This project consisted of the isolation and characterisation of candidate strains, with central focus on their potential to be osmotically and ionically adaptable. The selection panel resulted in the identification of two Euryhaline strains, one of freshwater (Synechocystis sp. Strain PCC 6803) and one of marine origin (Synechococcus sp. Strain PCC 7002) (Robert Gordon University, Aberdeen). Other work packages were as follows. Genetic manipulations potentially allowed for the expression of a light-driven, Cl−-selective pump in both strains, therefore, enhancing the bioaccumulation of specific ions within the cell (University of Glasgow). Characterisation of surface properties under different salinities (University of Sheffield), ensured that cell–liquid separation efficiency would be maximised post-treatment, as well as monitoring the secretion of mucopolysaccharides in the medium during cell growth. Work at Newcastle University is focused on the social acceptance of this scenario, together with an assessment of the potential risks through the generation and application of a Hazard Analysis and Critical Control Points plan. Finally, researchers in Imperial College (London) designed the process, from biomass production to water treatment and generation of a model photobioreactor. This multimodal approach has produced promising first results, and further optimisation is expected to result in mass scaling of this process

Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor

The Kv-like K+ channels at the plasma membrane, including the inward-rectifying KAT1 K+ channel of Arabidopsis, are important targets for manipulating K+ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K+ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-mutagenesis to explore residues that are thought to form two electrostatic counter-charge centers either side of a conserved Phe residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the counter-charge centers favored the open channel. Modelling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in context of the effects on hydration of amino-acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel

Clustering of the K⁺ channel GORK of Arabidopsis parallels its gating by extracellular K⁺

GORK is the only outward-rectifying Kv-like K<sup>+</sup> channel expressed in guard cells. Its activity is tightly regulated to facilitate K<sup>+</sup> efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K<sup>+</sup> channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K<sup>+</sup> concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K<sup>+</sup> channel blocker Ba<sup>2+</sup>. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K<sup>+</sup>, a characteristic that ensures the channel facilitates only K<sup>+</sup> efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K<sup>+</sup> above 1 mM. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K<sup>+</sup>

Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation

A chloroplast retrograde signal, 3’phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.Wannarat Pornsiriwong, Gonzalo M Estavillo, Kai Xun Chan, Estee E Tee, Diep Ganguly, Peter A Crisp, Su Yin Phua, Chenchen Zhao, Jiaen Qiu, Jiyoung Park, Miing Tiem Yong, Nazia Nisar, Arun Kumar Yadav, Benjamin Schwessinger, John Rathjen, Christopher I Cazzonelli, Philippa B Wilson, Matthew Gilliham, Zhong-Hua Chen, Barry J Pogso

Ion transport, membrane traffic and cellular volume control

Throughout their development, plants balance cell surface area and volume with ion transport and turgor. This balance lies at the core of cellular homeostatic networks and is central to the capacity to withstand abiotic as well as biotic stress. Remarkably, very little is known of its mechanics, notably how membrane traffic is coupled with osmotic solute transport and its control. Here we outline recent developments in the understanding of so-called SNARE proteins that form part of the machinery for membrane vesicle traffic in all eukaryotes. We focus on SNAREs active at the plasma membrane and the evidence for specialisation in enhanced, homeostatic and stress-related traffic. Recent studies have placed a canonical SNARE complex associated with the plasma membrane in pathogen defense, and the discovery of the first SNARE as a binding partner with ion channels has demonstrated a fundamental link to inorganic osmotic solute uptake. Work localising the channel binding site has now identified a new and previously uncharacterised motif, yielding important clues to a plausible mechanism coupling traffic and transport. We examine the evidence that this physical interaction serves to balance enhanced osmotic solute uptake with membrane expansion through mutual control of the two processes. We calculate that even during rapid cell expansion only a minute fraction of SNAREs present at the membrane need be engaged in vesicle traffic at any one time, a number surprisingly close to the known density of ion channels at the plant plasma membrane. Finally, we suggest a framework of alternative models coupling transport and traffic, and approachable through direct, experimental testin