Sakharam Waghmare

Stomata are pores at the leaf surface that facilitate CO ₂ entry for photosynthesis while controlling transpirational water loss. Stomatal movements are governed by reversible changes in turgor and cell volume, driven by the transport of osmotic solute across the membrane of guard cells surrounding the pore. Membrane transport depends on the activities of transporters and ion channels present at the membrane and also on their abundance, determined by vesicle traffic. Although much is known about how pumps and ion channels in the membrane are regulated, this is not the case for vesicle trafficking and certainly not in relation to CO ₂ . Here, we report on experiments following the traffic of the K ⁺ channel KAT1 that is important for K ⁺ uptake during stomatal opening. We found that elevated CO ₂ triggers changes in channel mobility within the plane of the plasma membrane and its internalization therefrom. CO ₂ ‐sensitive KAT1 traffic depends on SYP121, a vesicle‐trafficking (SNARE) protein previously associated with water stress and abscisic acid (ABA) that also binds the K ⁺ channel to promote its activation. The CO ₂ ‐sensitive pathway parallels vesicle traffic evoked by ABA, but it also shows important differences, with KAT1–SYP121 binding sensitive to CO ₂ . We show that stomatal response to changes in CO ₂ levels is slowed in the syp121 null mutant, affecting shoot growth and whole‐plant water‐use efficiency. Thus, we uncover a new target for CO ₂ regulation in vesicle traffic and a novel perspective on plant responses to climate change.

Secretory trafficking in plant cells is facilitated by SNARE (soluble N-ethylamide-sensitive factor attachment protein receptor) proteins that drive membrane fusion of cargo-containing vesicles. In Arabidopsis, SYNTAXIN OF PLANTS 132 (SYP132) is an evolutionarily ancient SNARE that functions with syntaxins SYP121 and SYP122 at the plasma membrane. Whereas SYP121 and SYP122 mediate overlapping secretory pathways, albeit with differences in their importance in plant-environment interactions, the SNARE SYP132 is absolutely essential for plant development and survival. SYP132 promotes endocytic traffic of the plasma membrane H⁺-ATPase AHA1 and aquaporin PIP2;1, and it coordinates plant growth and bacterial pathogen immunity through PATHOGENESIS-RELATED1 (PR1) secretion. Yet, little else is known about SYP132 cargoes. Here, we used advanced quantitative Tandem Mass Tagging (TMT) mass spectrometry (MS) combined with immunoblot assays to track native secreted cargo proteins in the leaf apoplast. We found that SYP132 supports a basal level of secretion in Arabidopsis leaves, and its overexpression influences salicylic acid (SA) and jasmonic acid (JA) defence-related cargoes including PR1, PR2, and PR5 proteins. Impairing SYP132 function also suppressed defence-related secretory traffic when challenged with the bacterial pathogen Pseudomonas syringae. Thus, we conclude that, in addition to its role in hormone-related H⁺-ATPase cycling, SYP132 influences basal plant immunity.

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.

Cell expansion requires that ion transport and secretory membrane traffic operate in concert. Evidence from Arabidopsis (Arabidopsis thaliana) indicates that such coordination is mediated by physical interactions between subsets of so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which drive the final stages of vesicle fusion, and K⁺ channels, which facilitate uptake of the cation to maintain cell turgor pressure as the cell expands. However, the sequence of SNARE binding with the K⁺ channels and its interweaving within the events of SNARE complex assembly for exocytosis remains unclear. We have combined protein-protein interaction and electrophysiological analyses to resolve the binding interactions of the hetero-oligomeric associations. We find that the RYxxWE motif, located within the voltage sensor of the K⁺ channels, is a nexus for multiple SNARE interactions. Of these, K⁺ channel binding and its displacement of the regulatory protein SEC11 is critical to prime the Qa-SNARE SYP121. Our results indicate a stabilizing role for the Qbc-SNARE SNAP33 in the Qa-SNARE transition to SNARE complex assembly with the R-SNARE VAMP721. They also suggest that, on its own, the R-SNARE enters an anomalous binding mode with the channels, possibly as a fail-safe measure to ensure a correct binding sequence. Thus, we suggest that SYP121 binding to the K⁺ channels serves the role of a primary trigger to initiate assembly of the secretory machinery for exocytosis.

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.

Channel binding depends on coordination between the two binding sites on VAMP721, consistent with the K+ channels as nucleation points for SNARE complex assembl