Detecting Touch-Induced Calcium Dynamics With Live-Cell Imaging in Torenia Stigma

Background

Plants constantly perceive mechanical stimuli throughout their life cycle, from wind-blown leaves and probing roots to pollen hydration and seed germination. Indeed, a plant's existence can be viewed as a continuous history of responding to such mechanical cues. Upon stimulation, plants upregulate touch-related TOUCH/TCH genes, which mediate contact-induced morphogenesis by integrating hormonal pathways involving auxin, cytokinin, ethylene, and jasmonic acid [1,2]. Beyond these slower morphological changes, some plants exhibit rapid movements in response to touch. Leaves of species like the Venus flytrap and Mimosa pudica execute rapid movements for insect capture or herbivory avoidance, respectively [3,4]. Thus, external mechanical forces fundamentally shape a plant's adaptive strategies.

Calcium ions (Ca²⁺) serve as one of the most important second messengers within plant cells. Advances in genetically encoded fluorescent indicators now allow real-time observation of Ca²⁺ dynamics in living tissues. Studies utilizing these tools have demonstrated that diverse stimuli trigger pronounced fluctuations in cytosolic Ca²⁺ ([Ca²⁺]_cyt) concentrations [5–7]. Mechanical stimuli are particularly effective inducers of such Ca²⁺ signals. In Arabidopsis, touching the leaf tip generates a calcium signal at the site of contact, which subsequently spreads through the vascular bundles to the petiole [5]. When a single trichome is touched, the resulting calcium signal radiates outward from the trichome as a center [6]. In the touch-sensitive plant Mimosa pudica, touching causes the leaflets to close within seconds. During this process, [Ca²⁺]_cyt levels rise from the veins to the pulvinus, significantly amplifying in that location [4]. Shortly thereafter, the signal transmits hierarchically to the next pulvini, ultimately causing the entire compound leaf to fold [4]. Notably, the speed of calcium signal transmission in Mimosa is much faster than in Arabidopsis leaves. Similarly, in the Venus flytrap, after the first touch, [Ca²⁺]_cyt concentration rises starting at the touched trigger hair and spreads in the leaf [3]. Following a second touch, the [Ca²⁺]_cyt concentration reaches the threshold to trigger closure [3]. Interestingly, this signal remains confined to the stimulated trap and does not propagate via the vasculature or to adjacent leaves [3]. These findings establish that mechanical touch directly initiates Ca²⁺ fluctuation and propagation, which is essential for activating downstream stress-response genes or driving rapid cellular deformations.

Although different plants employ Ca²⁺ as a messenger to transduce external mechanical cues into internal responses, the specific pathways and transduction mechanisms exhibit notable variation. Deciphering the Ca²⁺ signatures elicited by touch across different species is therefore key to understanding the conservation and specificity within this critical signaling network. Here, we have identified the touch-responsive behavior of stigmas in Torenia fournieri, a classical model in the study of plant reproduction [9,10]. The bilobed stigma of T. fournieri closes within seconds after mechanical stimulation, and we have demonstrated that the generation and propagation of touch-induced Ca²⁺ signals are essential for this stigma movement [11]. In this study, we present an integrated experimental protocol that details how to trigger stigma movement through controlled mechanical stimulation and how to visualize the associated [Ca²⁺]_cyt signal on living stigmas during this rapid response. Given that this protocol allows stimulation of a single papilla cell on the inner epidermal surface of the Torenia fournieri stigma, it may offer a valuable reference for mechanical stimulation of single-cell structures in plants, including leaf trichomes of Arabidopsis.