Calcium-Mediated Modulation of GC Switch Regulates Peroxisomal H₂O₂ Levels in Response to Wounding in Plants

Abstract

Ca²⁺ and H₂O₂ interact with each other to regulate plant systemic responses. However, their precise mechanism is not fully understood. A recent study revealed that the Ca²⁺ regulates the glycolate oxidase-catalase (GC) switch-mediated photorespiratory H₂O₂ during wounding. Glutamate-receptor-like (GLR) Ca²⁺ channels (GLR 3.3 and GLR3.6) are responsible for Ca²⁺ influx during injury for regulation of the GC switch. Mechanical injury quickly shifts the GC switch to a highly interactive state in the systemic leaves that ultimately results in the reduced peroxisomal H₂O₂. However, the mechanism of H₂O₂ reduction in peroxisome remains elusive.

1. Introduction

Plants are consistently challenged by adverse environmental conditions such as light stress, thermal stress, mechanical injury and pathogen attack. In these circumstances, the local tissues detect the stimuli and transmit signals to other plant tissues through rapid systemic signaling. Plant systemic signaling results in systemic acquired acclimation (SAA), which helps in their survival during stress conditions [1,2]. Hydrogen peroxide (H₂O₂) and calcium (Ca²⁺) ions are key signaling molecules that interplay to mediate plant systemic signaling and the systemic wound response (SWR). H₂O₂ is produced in various compartments of a cell in response to different environmental stimuli and acts as a signaling molecule to trigger downstream responses. Photorespiratory H₂O₂ produced in the peroxisome has a significant role in activating defense mechanisms in plants. Photorespiratory H₂O₂ is generated during the oxidation of glycolate to glyoxylate by enzyme glycolate oxidase (GLO). H₂O₂ is further catalyzed by catalase (CAT) into water (H₂O) and oxygen (O₂). GLO and CAT together serve as a dynamic molecular switch called a GC switch, which balances photorespiratory H₂O₂ during mechanical injury [3].

Ca²⁺ signaling plays a significant role in mitigating a variety of stress conditions. It is involved in a wide range of physiological processes including gene expression, cell division, and differentiation as well as biotic and abiotic stress responses [4,5]. Plants respond to mechanical injury by generating a rapid and systemic increase in cytosolic calcium [Ca²⁺]_cyt concentration [6]. [Ca²⁺]_cyt fluctuations are among the primary events associated with the rapid systemic signaling of plants [7]. Ca²⁺ signals travel long distances during mechanical stress to propagate and modify local signals into a systemic defense response system [8].

Ca²⁺ and H₂O₂ interact with each other to regulate plant systemic responses. However, the exact mechanisms have not been fully revealed. Li et al. [9] in a recent study revealed a partial mechanism by which plants respond to mechanical injury through Ca²⁺-H₂O₂ crosstalk (Figure 1). The study mainly focused on the importance of GC switch-mediated modulation in photorespiratory H₂O₂ during wounding. The authors shed light on the role of Ca²⁺ in modulating photorespiratory H₂O₂ for systemic adaptation by employing different sets of experiments and analyses in Arabidopsis and rice. Understanding these mechanisms is crucial for developing strategies to improve plant growth and stress tolerance, which is of great importance for sustainable agriculture and food security.

2. GC Switch Facilitates Peroxisomal H₂O₂ Decrease during Wounding

The key photorespiration enzymes in Arabidopsis are GOX1, GOX2, and CAT2, which are analogous to GLO1, GLO4, and CATC in rice [10]. Arabidopsis GOXs also interact with CAT, equivalent to the GLO-CAT complex in rice. GLO and CAT interactions dynamically modulate H₂O₂ levels through metabolic pathways [3,11]. The GC switch primarily exists in three states: highly dissociated, intermediate and highly interactive [3]. Wounding triggers the GC switch to change to a highly interactive state, resulting in peroxisomal H₂O₂ reduction. Li et al. [9] performed a bimolecular fluorescence complementation (BiFC) assay to investigate the interaction between GLO and CAT by fusing them to the N- and C-terminal of a yellow fluorescent protein (YFP) and expressing them in Arabidopsis protoplasts. The results demonstrated that GLO and CAT interact with each other, which is further confirmed by split nano luciferase (Nluc) complementation (SLC) and co-immunoprecipitation (Co-IP) assays. Additionally, mechanical injury generates an immediate accumulation of jasmonic acid (JA), which is assisted by the activation of JA-related defense genes. Moreover, the Nluc-based SLC assay demonstrated that methyl jasmonic acid (MeJA) shifts the GC switch to a more interactive state [9].

The authors used ratiometric redox-sensitive green fluorescent protein 2 (roGFP2) fused with peroxisomal target signal 1 (SKL) as a substitute for the detection of photorespiratory H₂O₂ dynamics [10,12,13]. Additionally, two peroxisome-specific H₂O₂ reporter genes HSP17 and SAP12 were also employed to detect peroxisomal H₂O₂ reduction. The extent of decline of peroxisomal H₂O₂ during wounding is significantly reduced by a-hydroxyl-2-pyridinemethanesulfonic acid (HPMS), a GLO activity inhibitor [3]. Moreover, wounding contributes to an elevation in apoplastic H₂O₂ produced by the respiratory burst oxidase homolog (RBOH) [14]. The zinc finger reporter protein ZAT12 and 20,70 -dichlorofluorescein diacetate (H₂DCFDA) [15] are used to detect changes in the RBOH-derived accumulation of H₂O₂ in systemic leaves. The results indicated a rise in apoplastic H₂O₂ levels in response to mechanical injury. Additionally, an inhibition assay using diphenyleneiodonium (DPI) demonstrated that RBOH-derived H₂O₂ has no effect on peroxisomal H₂O₂ reduction. The RBOH-derived H₂O₂ is sensed by H₂O₂-induced Ca²⁺ increase 1 (HPCA1). HPCA1 induces systemic cell-to-cell Ca²⁺ signals and elevates [Ca²⁺]_cyt by activating Ca²⁺ influx channels [16,17], which might be responsible for the Ca-ROS hub that elevates systemic signaling [18]. These findings suggest that the GC switch quickly shifts to a highly interactive state upon local injury and triggers a reduction in peroxisomal H₂O₂ levels compared to a burst of RBOH-derived H₂O₂. These results suggest that mechanical injury activates two different H₂O₂ signaling pathways at the same time: (i) the GC switch-mediated peroxisomal H₂O₂ reduction, and (ii) the RBOH-derived apoplastic H₂O₂ burst. However, the functional significance of GC switch-mediated photorespiratory H₂O₂ needs to be investigated further. In addition, the status of H₂O₂ in the cytoplasm remains elusive, which would be interesting to investigate in future research.

3. GLR-Mediated Ca²⁺ Influx Regulates the GC Switch

It has been well established that the long-distance transmission of a Ca²⁺ signal plays a crucial role in regulating systemic adaptations in plants [19]. Mechanical injury triggers a rapid influx of apoplastic Ca²⁺ into the cytosol, resulting in the elevation of [Ca²⁺]_cyt [20]. The authors found that the elevated [Ca²⁺]_cyt further leads to the transition of GC switch to a highly interactive state by increasing peroxisomal Ca²⁺ ([Ca²⁺]_per). However, the exact mechanism of Ca²⁺ entry into peroxisomes is not fully understood. Li et al. [9] analyzed [Ca²⁺]_per using the fluorescence probe fluo-4/AM and further validated the process with the peroxisome-targeted Ca²⁺ sensor GCaMP3-SKL. The inductive effect of [Ca²⁺]_cyt on the GC switch was also confirmed by a surface plasmon resonance (SPR/Biacore) assay, a Glutathione S-transferase (GST) pull-down assay, a Co-IP assay and exogenous Ca²⁺ treatment. In addition, the authors also used lanthanum chloride (LaCl₃) to block the influx of Ca²⁺ ions into the cytosol. The results revealed that both the GC switch transition and reduction in peroxisomal H₂O₂ levels almost disappeared suggesting that Ca²⁺ influx is required for both the processes. Moreover, it has previously been reported that the increased cellular Ca²⁺ concentration during local wounding does not have any impact on CAT activity in systemic leaves [20]. Hence, the reduction in peroxisomal H₂O₂ levels can not be related to the CAT activity. Therefore, it is still unclear how peroxisomal H₂O₂ declines without an elevation in CAT activity, which would be interesting to investigate in future studies.

GLR3.3 and GLR3.6 are glutamate-receptor-like (GLR) Ca²⁺ channels present on the plasma membrane which are involved in increasing [Ca²⁺]_cyt upon wounding [1,14]. To validate this, Li et al. [9] generated a double-knockout mutant for glr3.3 and glr3.6 in the GCR-Arabidopsis and Col-0 backgrounds. GCR-Arabidopsis shows real-time states of the GC switch, providing a non-invasive method for monitoring plant signaling. The authors reported that the knocking out of glr3.3 and glr3.6 inhibited the influx of [Ca²⁺]_cyt and the systemic wound response of the GC switch, thereby causing a consequent reduction in peroxisomal H₂O₂ levels. The result suggests that GC switch-mediated H₂O₂ reduction is dependent on GLR-dependent Ca²⁺ signaling. Moreover, it is imperative to study the impact of other Ca²⁺ channels using overexpression or knockout approaches because apoplastic H₂O₂-mediated activation of HPCA1 triggers some other Ca²⁺ channels for Ca-ROS hub-mediated systemic signaling [17,18,21].

4. Conclusions and Future Perspectives

This study provides valuable insights into the molecular mechanisms regulating plant systemic signaling in response to wounding by elucidating the interplay between Ca²⁺ ions and H₂O₂. Ca²⁺ and H₂O₂ form a feedback loop in systemic parts of the plant during wounding. Mechanical injury increases [Ca²⁺]_cyt influx in systemic leaves via GLR3.3/GLR3.6 Ca²⁺ channels in contrast to an increase in RBOH-derived apoplastic H₂O₂. Enhanced [Ca²⁺]_cyt further elevates [Ca²⁺]_per that shifts the GC switch to an interactive state, which further leads to a decrease in peroxisomal H₂O₂. The interplay between the above two processes, apoplastic H₂O₂ outburst and peroxisomal H₂O₂ reduction, form an optimized H₂O₂ wave that prepare the plants for biotic and abiotic stress tolerance. This study provides insights into how Ca²⁺ regulates the GC switch in peroxisomes, however, the precise mechanism remains elusive. The mechanism of Ca²⁺ entry into peroxisomes is also not explained. How does peroxisomal H₂O₂ decline without an elevation in CAT activity demands further research. It must be determined whether the CAT has been transported to the cytoplasm or if its production has been reduced. In addition, the role of the GC switch in other stresses and the involvement of other Ca²⁺ channels in GC switch modulation as well as in wounding remain to be addressed in future studies.