A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K + channel subunit that assembles with different inward-rectifying K + channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K + channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K + channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inwardrectifying K + current in Arabidopsis root epidermal protoplasts as well as K + acquisition and growth in seedlings when channel-mediated K + uptake was limiting. That SYP121 should be important for gating of a K + channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE-ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K + uptake coordinately with membrane expansion for cell growth.

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca 2؉ -sensitive ion channels of Vicia guard cells by promoting Ca 2؉ release from intracellular stores to raise cytosolic-free [Ca 2؉ ]. NO-sensitive Ca 2؉ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca 2؉ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca 2؉ channels and Ca 2؉ -insensitive K ؉ channels, and NO scavengers failed to block the activation of these K ؉ channels evoked by ABA. These results place NO action firmly within one branch of the Ca 2؉ -signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.cGMP-mediated signaling ͉ stress physiology ͉ cyclic ADP ribose ͉ cytosolic-free [Ca 2ϩ ] elevation ͉ Vicia

SummaryVesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1-YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants.

SummaryRecent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant-pathogen defence, and are a prerequisite for drought and abscisic acid (ABA)