Role of calcium and reactive oxygen species in wounding-induced stomatal closure triggered by long-distance signaling in Arabidopsis thaliana

Plants have evolved sophisticated signaling mechanisms to detect and respond to environmental stresses, ensuring their survival under adverse conditions. Among these mechanisms, long-distance signaling plays a crucial role in coordinating systemic responses to mechanical injury, such as leaf or root wounding. This signaling relies on the propagation of calcium (Ca²⁺) and reactive oxygen species (ROS) waves, electrical signals, hydraulic pressure changes, and phytohormones, which together regulate plant physiological responses to stress. One critical aspect of the plant stress response is stomatal closure, an acclimative mechanism mediated by Ca²⁺ and ROS signaling pathways that minimizes water loss and prevents pathogen entry. This study investigates the specific roles of the Ca²⁺-ATPases ACA8 and ACA10, as well as the ROS-producing enzymes AtCuAOβ and RBOHD, in regulating stomatal responses to mechanical injury and to the wound-associated phytohormone methyl-jasmonate (MeJA) at both wounded and distal sites. Loss-of-function aca8 and aca10 mutants exhibited impaired stomatal closure, while ACA8 overexpression also disrupted the response, suggesting that balanced Ca²⁺ extrusion is essential. Interestingly, ACA8 and ACA10 function downstream of ROS accumulation, as mutants displayed wild-type ROS levels despite defective Ca²⁺ signaling. RBOHD emerged as a key player in systemic stomatal closure. While local responses remained unaffected in rbohd mutants, stomatal closure in unwounded distal tissues was significantly impaired, indicating that RBOHD-mediated ROS production is crucial for systemic responses. This was further supported by experiments using a RBOH inhibitor, which confirmed that no other RBOHs contribute to this process. The interplay between AtCuAOβ and RBOHD in systemic ROS production after wounding was also highlighted in stomata guard cells: while Atcuaoβ mutants lacked ROS accumulation in guard cell of both wounded and distal cotyledons, rbohd mutants showed reduced ROS levels only in distal tissues, reinforcing their distinct but complementary roles in ROS signaling. This study further explored the involvement of ACA8, ACA10, AtCuAOβ, and RBOHD in Ca²⁺ mediated long-distance signaling. Ca²⁺ wave propagation analysis demonstrated that ACA8 and ACA10 are essential for transmitting injury-induced Ca²⁺ signals to distal tissues. Mutants failed to propagate the Ca²⁺ wave beyond the injury site, indicating their crucial role in rapid signal transmission. Furthermore, RBOHD—but not AtCuAOβ—was implicated in amplifying Ca²⁺ signaling, as rbohd mutants exhibited altered Ca²⁺ propagation patterns. Finally, a temporal analysis of systemic whole-plant ROS accumulation over 45 minutes revealed that AtCuAOβ and RBOHD contribute to different phases of ROS production. Atcuaoβ mutants exhibited low ROS levels throughout, while rbohd mutants displayed reduced ROS accumulation only in distal tissues, confirming its role in systemic ROS amplification. Notably, ACA8 and ACA10 did not influence late ROS accumulation, suggesting that Ca²⁺ signaling and long-term ROS production operate through distinct mechanisms. Overall, this study provides a comprehensive analysis of the roles of ACA8, ACA10, AtCuAOβ, and RBOHD in wound-induced Ca²⁺ and ROS signaling. By elucidating their distinct contributions to local and systemic stress responses, this research enhances our understanding of the molecular networks governing plant rapid acclimation to environmental challenges.