LAUSR

Significance Ca2+ signaling is closely linked to the triggering of plant immune responses. We show that when the bacterial elicitor peptide flg22 is used to trigger immune responses in mutants missing tonoplast Ca2+ pumps, it causes disrupted Ca2+ signals that are higher in amplitude than expected from wild-type plants. Relocalization of a Ca2+ pump normally found in the plasma membrane to the tonoplast rescues both the tonoplast pump knockout mutant’s immune-related phenotypes and aberrant Ca2+ signaling dynamics. These observations support the notion that tonoplast-localized pumps are critical for the proper maintenance of Ca2+ dynamics in innate immune responses but that any regulatory systems specific to the endogenous tonoplast pumps may not be required to support these signaling events. One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.