Overexpression of tonoplast Ca2+-ATPase in guard cells synergistically enhances stomatal opening and drought tolerance.

Stomata play a crucial role in plants by controlling water status and responding to drought stress. However, simultaneously improving stomatal opening and drought tolerance has proven to be a significant challenge. To address this issue, we employed the OnGuard quantitative model, which accurately represents the mechanics and coordination of ion transporters in guard cells. With the guidance of OnGuard, we successfully engineered plants that overexpressed the main tonoplast Ca2+-ATPase gene, ACA11, which promotes stomatal opening and enhances plant growth. Surprisingly, these transgenic plants also exhibited improved drought tolerance due to reduced water loss through their stomata. Again, OnGuard assisted us in understanding the mechanism behind the unexpected stomatal behaviors observed in the ACA11 overexpressing plants. Our study revealed that the overexpression of ACA11 facilitated the accumulation of Ca2+ in the vacuole, thereby influencing Ca2+ storage and leading to an enhanced Ca2+ elevation in response to abscisic acid. This regulatory cascade finely tunes stomatal responses, ultimately leading to enhanced drought tolerance. Our findings underscore the importance of tonoplast Ca2+-ATPase in manipulating stomatal behavior and improving drought tolerance. Furthermore, these results highlight the diverse functions of tonoplast-localized ACA11 in response to different conditions, emphasizing its potential for future applications in plant enhancement.

Arabidopsis SNARE SYP132 impacts on PIP2;1 trafficking and function in salinity stress.

In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H+-ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals.

Analyzing Protein-Protein Interactions Using the Split-Ubiquitin System.

The split-ubiquitin technology was developed over 20 years ago as an alternative to Gal4-based, yeast-two-hybrid methods to identify interacting protein partners. With the introduction of mating-based methods for split-ubiquitin screens, the approach has gained broad popularity because of its exceptionally high transformation efficiency, utility in working with full-length membrane proteins, and positive selection with little interference from spurious interactions. Recent advances now extend these split-ubiquitin methods to the analysis of interactions between otherwise soluble proteins and tripartite protein interactions.

SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis.

The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane (PM) H+-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity. To study SYP132 and associated trafficking of PM H+-ATPase 1 (AHA1) and PATHOGENESIS-RELATED PROTEIN1 (PR1) during pathogenesis, we used the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria for infection of Arabidopsis (Arabidopsis thaliana) plants. SYP132 overexpression suppressed bacterial infection in plants through the stomatal route. However, bacterial infection was enhanced when bacteria were infiltrated into leaf tissue to bypass stomatal defenses. Tracking time-dependent changes in native AHA1 and SYP132 abundance, cellular distribution, and function, we discovered that bacterial pathogen infection triggers AHA1 and SYP132 internalization from the plasma membrane. AHA1 bound to SYP132 through its regulatory SNARE Habc domain, and these interactions affected PM H+-ATPase traffic. Remarkably, using the Arabidopsis aha1 mutant, we discovered that AHA1 is essential for moderating SYP132 abundance and associated secretion of PR1 at the plasma membrane for pathogen defense. Thus, we show that during pathogenesis SYP132 coordinates AHA1 with opposing effects on the traffic of AHA1 and PR1.

Unusual Roles of Secretory SNARE SYP132 in Plasma Membrane H+-ATPase Traffic and Vegetative Plant Growth.

The plasma membrane proton (H+)-ATPases of plants generate steep electrochemical gradients and activate osmotic solute uptake. H+-ATPase-mediated proton pumping orchestrates cellular homeostasis and is a prerequisite for plastic cell expansion and plant growth. All evidence suggests that the population of H+-ATPase proteins at the plasma membrane reflects a balance of their roles in exocytosis, endocytosis, and recycling. Auxin governs both traffic and activation of the plasma membrane H+-ATPase proteins already present at the membrane. As in other eukaryotes, in plants, SNARE-mediated membrane traffic influences the density of several proteins at the plasma membrane. Even so, H+-ATPase traffic, its relationship with SNAREs, and its regulation by auxin have remained enigmatic. Here, we identify the Arabidopsis (Arabidopsis thaliana) Qa-SNARE SYP132 (Syntaxin of Plants132) as a key factor in H+-ATPase traffic and demonstrate its association with endocytosis. SYP132 is a low-abundant, secretory SNARE that primarily localizes to the plasma membrane. We find that SYP132 expression is tightly regulated by auxin and that augmented SYP132 expression reduces the amount of H+-ATPase proteins at the plasma membrane. The physiological consequences of SYP132 overexpression include reduced apoplast acidification and suppressed vegetative growth. Thus, SYP132 plays unexpected and vital roles in auxin-regulated H+-ATPase traffic and associated functions at the plasma membrane.

Dual Sites for SEC11 on the SNARE SYP121 Implicate a Binding Exchange during Secretory Traffic.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis (Arabidopsis thaliana) plasma membrane is subject to the Sec1/Munc18 protein SEC11, which, along with plasma membrane K+ channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. How SEC11 binding is coordinated with the K+ channels is poorly understood, as both SEC11 and the channels are thought to compete for the same SNARE binding site. Here, we identify a second binding motif within the N terminus of SYP121 and demonstrate that this motif affects SEC11 binding independently of the F9xRF motif that is shared with the K+ channels. This second, previously unrecognized motif is centered on residues R20R21 of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R20R21 motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K+ uptake associated with the F9xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11Δ149 fragment. We conclude that the R20R21 motif represents a secondary site of interaction for the Sec1/Munc18 protein during the transition of SYP121 from the occluded to the open conformation that leads to SNARE complex assembly.

SNAREs SYP121 and SYP122 Mediate the Secretion of Distinct Cargo Subsets.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion and contribute to homoeostasis, pathogen defense, cell expansion, and growth in plants. In Arabidopsis (Arabidopsis thaliana), two homologous Qa-SNAREs, SYNTAXIN OF PLANTS121 (SYP121) and SYP122, facilitate the majority of secretory traffic to the plasma membrane, and the single mutants are indistinguishable from wild-type plants in the absence of stress, implying a redundancy in their functions. Nonetheless, several studies suggest differences among the secretory cargo of these SNAREs. To address this issue, we conducted an analysis of the proteins secreted by cultured wild-type, syp121, and syp122 mutant Arabidopsis seedlings. Here, we report that a number of cargo proteins were associated differentially with traffic mediated by SYP121 and SYP122. The data also indicated important overlaps between the SNAREs. Therefore, we conclude that the two Qa-SNAREs mediate distinct but complementary secretory pathways during vegetative plant growth.

Gating control and K+ uptake by the KAT1 K+ channel leaveraged through membrane anchoring of the trafficking protein SYP121.

Vesicle traffic is tightly coordinated with ion transport for plant cell expansion through physical interactions between subsets of vesicle-trafficking (so-called SNARE) proteins and plasma membrane Kv channels, including the archetypal inward-rectifying K+ channel, KAT1 of Arabidopsis. Ion channels open and close rapidly over milliseconds, whereas vesicle fusion events require many seconds. Binding has been mapped to conserved motifs of both the Kv channels and the SNAREs, but knowledge of the temporal kinetics of their interactions, especially as it might relate to channel gating and its coordination with vesicle fusion remains unclear. Here, we report that the SNARE SYP121 promotes KAT1 gating through a persistent interaction that alters the stability of the channel, both in its open and closed states. We show, too, that SYP121 action on the channel open state requires SNARE anchoring in the plasma membrane. Our findings indicate that SNARE binding confers a conformational bias that encompasses the microscopic kinetics of channel gating, with leverage applied through the SNARE anchor in favour of the open channel.

VAMP721 Conformations Unmask an Extended Motif for K+ Channel Binding and Gating Control.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a major role in membrane fusion and contribute to cell expansion, signaling, and polar growth in plants. The SNARE SYP121 of Arabidopsis thaliana that facilitates vesicle fusion at the plasma membrane also binds with, and regulates, K+ channels already present at the plasma membrane to affect K+ uptake and K+-dependent growth. Here, we report that its cognate partner VAMP721, which assembles with SYP121 to drive membrane fusion, binds to the KAT1 K+ channel via two sites on the protein, only one of which contributes to channel-gating control. Binding to the VAMP721 SNARE domain suppressed channel gating. By contrast, interaction with the amino-terminal longin domain conferred specificity on VAMP721 binding without influencing gating. Channel binding was defined by a linear motif within the longin domain. The SNARE domain is thought to wrap around this structure when not assembled with SYP121 in the SNARE complex. Fluorescence lifetime analysis showed that mutations within this motif, which suppressed channel binding and its effects on gating, also altered the conformational displacement between the VAMP721 SNARE and longin domains. The presence of these two channel-binding sites on VAMP721, one also required for SNARE complex assembly, implies a well-defined sequence of events coordinating K+ uptake and the final stages of vesicle traffic. It suggests that binding begins with VAMP721, and subsequently with SYP121, thereby coordinating K+ channel gating during SNARE assembly and vesicle fusion. Thus, our findings also are consistent with the idea that the K+ channels are nucleation points for SNARE complex assembly.

Commandeering Channel Voltage Sensors for Secretion, Cell Turgor, and Volume Control.

Control of cell volume and osmolarity is central to cellular homeostasis in all eukaryotes. It lies at the heart of the century-old problem of how plants regulate turgor, mineral and water transport. Plants use strongly electrogenic H+-ATPases, and the substantial membrane voltages they foster, to drive solute accumulation and generate turgor pressure for cell expansion. Vesicle traffic adds membrane surface and contributes to wall remodelling as the cell grows. Although a balance between vesicle traffic and ion transport is essential for cell turgor and volume control, the mechanisms coordinating these processes have remained obscure. Recent discoveries have now uncovered interactions between conserved subsets of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that drive the final steps in secretory vesicle traffic and ion channels that mediate in inorganic solute uptake. These findings establish the core of molecular links, previously unanticipated, that coordinate cellular homeostasis and cell expansion.

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane.

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K(+) channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K(+) channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K(+) channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a "hand-off" in channel control between the two SNARE proteins that is woven together with vesicle fusion.

Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

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

The Kv-like (potassium voltage-dependent) K(+) channels at the plasma membrane, including the inward-rectifying KAT1 K(+) channel of Arabidopsis (Arabidopsis thaliana), 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-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (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 countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the 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.

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.

SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites.

BACKGROUND: Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. RESULTS: We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest. CONCLUSIONS: The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.