https://orcid.org/0000-0002-9544-0978
Long-Range Signals Mediate Systemic Defense Responses in Plants
Plants respond to various environmental stresses, such as insect attack or mechanical wounding, with both local and system-wide reactions that prime non-stressed distant regions to mount proactive defenses. For example, the defense hormone jasmonic acid (JA) and its active form, jasmonoyl-isoleucine (JA-Ile), accumulate immediately at the site of wounding and within minutes spread to distant organs (Farmer et al. 2020). JA-Ile induces a set of defense responses ranging from gene expression to shifts in defense metabolism. The precise nature of this rapid, long-range communication system remains incompletely understood in plants. However, events such as the propagation of increases in cytosolic Ca2+ concentration ([Ca2+]cyt), reactive oxygen species (ROS) and electrical signals spatiotemporally correlate with the systemic spread of wound responses (Farmer et al. 2020, Suda and Toyota 2022).
In the vascular plant Arabidopsis thaliana, insect herbivory and mechanical wounding trigger an increase in [Ca2+]cyt and electrical signals (changes in surface or membrane potential) propagating at 1–2 mm/s toward distant unwounded leaves where defense responses, including JA/JA-Ile accumulation, are induced (Mousavi et al. 2013, Toyota et al. 2018). Wound-induced increases in [Ca2+]cyt and electrical signals propagate through the phloem, such as sieve elements, and their pattern of leaf-to-leaf transmission closely matches developmental vascular connections in hypocotyl vascular architecture (Mousavi et al. 2013, Salvador-Recatala et al. 2014, Toyota et al. 2018). Therefore, the vascular tissue acts as a major conduit for long-range leaf-to-leaf communication. This raises the question: what happens in plants that do not possess a true vasculature?
Local and Systemic Ca2+ and Electrical Signals in Bryophytes
Unlike Arabidopsis and other tracheophytes, bryophytes including hornworts, mosses and liverworts lack true vascular tissues. Therefore, as one of the earliest non-vascular land plants, bryophytes are useful models in which to study primitive long-range signaling in land plants particularly because they are also capable of transmitting Ca2+ and electrical signals, albeit with different spatiotemporal kinetics. For example, in the moss Physcomitrium patens, extracellularly applied glutamate and ROS (hydrogen peroxide) trigger systemic propagation of electrical signals, whereas Ca2+ signal propagation is extremely slow (>5 μm/s) or localized to the treated protonema cells (Koselski et al. 2020, 2023). Conversely, osmotic stimuli trigger systemic propagation of Ca2+ signals (∼5 μm/s) throughout the plant body (Storti et al. 2018). In the liverworts Conocephalum conicum and Marchantia polymorpha, long-range electrical signals are induced by various stimuli, such as light and electrical stimuli (Kisnieriene et al. 2022). Surprisingly, the velocities of electrical signals are almost identical to those in vascular plants (Mousavi et al. 2013). However, long-range Ca2+ signals have not yet been visualized in liverworts, and thus the spatiotemporal connection between Ca2+ and electrical signals is yet to be fully elucidated in plants lacking vascular tissues.
A Spatiotemporal Correlation between Ca2+ and Electrical Long-Range Signals in the Liverwort Marchantia
In this issue, Watanabe et al. (2024) simultaneously recorded changes in [Ca2+]cyt and surface potential in M. polymorpha and demonstrated a spatiotemporal coupling between long-range Ca2+ and electrical signals in response to mechanical injury and the underlying molecular machinery conserved in land plants. The experimental configuration used a plastochron 2-stage Marchantia with four branches expressing the genetically encoded GFP-based Ca2+ indicator, GCaMP6f (Chen et al. 2013). Mechanical wounding of thallus branch 1 caused an immediate increase in [Ca2+]cyt in the wounded region, and this [Ca2+]cyt change was transmitted within 1–2 min to distal unwounded branch 2 (Fig. 1). Interestingly, this Ca2+ wave did not extend beyond the intermediate site to branches 3 and 4, originating from distinct meristematic zones (Fig. 1). Furthermore, the velocity of the Ca2+ waves in branches 1 and 2 was 1.51 ± 0.59 mm/s, comparable to that observed in vascular plants, such as Arabidopsis (Toyota et al. 2018) and Mimosa pudica (Hagihara et al. 2022). As mechanical wounding and insect herbivory represent immediate and acute stresses, these conserved velocities might be commonly required to rapidly activate systemic defense responses in land plants (Farmer et al. 2020).
Long-range wound-induced Ca2+ and electrical signals in the non-vascular liverwort M. polymorpha.
Mechanical wounding also induced surface potential changes in both wounded and unwounded branches. The velocity of the electrical signals in branches 1 and 2 was 1.52 ± 0.96 mm/s, almost equivalent to that of the wound-induced Ca2+ waves in Marchantia (Watanabe et al. 2024) and electrical signals in Arabidopsis (Mousavi et al. 2013). Importantly, changes in surface potential did not extend beyond the intermediate site to opposite branches, mirroring the dynamics of Ca2+ waves (Fig. 1). The intermediate site, which differs from other thallus cells and remains dormant, might have lower expression levels of genes related to Ca2+ and electrical long-range signaling.
To further understand the connection between Ca2+ waves and electrical signals, Watanabe et al. (2024) established a system for the simultaneous measurement of changes in [Ca2+]cyt and surface potential in Marchantia. The onset and arrival times of [Ca2+]cyt increases were closely synchronized with those of changes in negative surface potential upon wounding, and the durations of these signals were also highly correlated. Additionally, the velocities of the Ca2+ waves and electrical signals were statistically indistinguishable, suggesting that Ca2+ and electrical signals are spatiotemporally coupled in Marchantia.
Molecular Machinery Underlying Wound-Induced Ca2+ and Electrical Signals in Marchantia
To confirm that the two signaling processes are mechanistically coupled, pharmacological and genetic analyses were performed (Watanabe et al. 2024). Lanthanum chloride (LaCl3) and tetraethylammonium chloride (TEA) are widely used to block Ca2+ and K+ channels, respectively. Marchantia thalli treated extracellularly with LaCl3 or TEA exhibited neither Ca2+ waves nor electrical signals upon wounding, supporting the hypothesis that Ca2+ and electrical signals are interconnected. The findings also suggest that Ca2+ and K+ fluxes through putative Ca2+- and K+-permeable channels are likely involved in the induction and/or propagation of Ca2+ and electrical signals in Marchantia (Fig. 1).
In Arabidopsis, two members of the ionotropic glutamate receptor family, clade 3 GLUTAMATE RECEPTOR–LIKE (GLR) 3.3 and GLR3.6, play major roles in long-range Ca2+ and electrical signals upon wounding (Mousavi et al. 2013, Toyota et al. 2018). Marchantia possesses a single GLR gene (MpGLR) that is phylogenetically close to these Arabidopsis clade 3 GLRs (Simon et al. 2023). To investigate the potential long-range signaling role of MpGLR, Watanabe et al. (2024) generated genome-edited Mpglr-1 mutants in which wound-induced Ca2+ waves and electrical signal propagation were completely abolished, highlighting the indispensable role of MpGLR in long-range Ca2+ and electrical signaling (Fig. 1). The systemic response to wounding was restored to wild-type levels after inducing MpGLR expression, confirming its link to the propagation of Ca2+ and electrical signals in Marchantia. Interestingly, like Arabidopsis, local [Ca2+]cyt increases and surface potential changes were not suppressed at wound sites in Mpglr-1 mutants (Watanabe et al. 2024), suggesting that distinct sensory systems, rather than the GLR ion channel family, mediate the induction of long-range Ca2+ and electrical signaling in plants.
Therefore, Watanabe and coauthors also explored different ion channels that might be potentially involved in wound-induced Ca2+ and electrical signaling in Marchantia. Voltage-gated ion channels of the TWO-PORE CHANNEL (TPC) family are evolutionarily conserved in various species (Hedrich et al. 2023). In Arabidopsis, TPC1 mediates Ca2+ signaling upon salt stress and aphid feeding (Choi et al. 2014, Vincent et al. 2017). Marchantia possesses three TPC genes. MpTPC1, MpTPC2 and MpTPC3 are localized to the vacuolar membrane, with only MpTPC1 showing slow vacuolar channel activity (Hashimoto et al. 2021). However, wound-induced Ca2+ and electrical signals in Mptpc1-1 and Mptpc2-2tpc3-2 double mutants were indistinguishable from the wild type, indicating that MpTPCs are not required for either induction or propagation of wound-induced Ca2+ and electrical signals in Marchantia. Therefore, GLR is the most important family for conducting long-range signaling in land plants, but the induction process at wound sites remains to be explored.
Perspectives
In summary, Watanabe et al. (2024) experimentally demonstrated that the non-vascular liverwort Marchantia propagates Ca2+ and electrical signals in response to wounding, with velocities similar to those observed in vascular plants (i.e. 1–2 mm/s). Pharmacological and genetic analyses suggest that these signals were tightly coupled and required MpGLR but not MpTPC (Fig. 1), supporting the concept that GLR-mediated long-range signaling is widely conserved in land plants. These findings raise important questions about MpGLR, including what mechanisms underpin the activation of these signals? Is MpGLR a ligand-gated Ca2+-permeable channel? Where is the subcellular localization of MpGLR? What are the downstream responses after the arrival of Ca2+ and electrical signals? Finally, how does Marchantia, which lacks the vascular tissues of tracheophytes, achieve these fast velocities and initiate local Ca2+ and electrical signals at the wound sites? To address these questions, powerful imaging technology, such as wide-field, long-term and multi-color fluorescence imaging, that facilitates the spatial and temporal visualization of systemic plant responses will be essential (Toyota and Betsuyaku 2022). With its reduced redundancy in signaling genes compared to higher plants, further study in Marchantia should provide crucial insights into the signaling cascades and regulatory mechanisms governing the dynamic responses of plants to environmental stimuli. This knowledge will establish a foundation for the further exploration of plant long-range signaling.
Data Availability
No new datasets were generated or analyzed in this study.
Disclosures
The author has no conflicts of interest to declare.
