Apoplastic calcium executes a shut-down function on plant peroxidases

Abstract

Recently it was demonstrated that PO activity is switched by calcium within the typical range of apoplastic free calcium concentrations (Plieth and Vollbehr, Plant Signal Behav 2012;7: 650–660). The heat stability of POs is also dependent on calcium. Here, a scenario is put forward which assigns calcium a switch-off function under heat: Peroxidases are switched off by heat stress-triggered apoplastic calcium depletion. It is assumed that this initiates apoplastic accumulation of reactive oxygen species (ROS) and eventually triggers a self-amplifying cascade of cellular events involving plasma membrane ion transport. Calcium depletion-initiated ROS accumulation (CaDIRA) may also trigger signal percolation and the formation of systemic responses to many different stress factors in plants. This hypothesis can explain some as yet unexplained observations.

Peroxidases Involved In Molecular Signaling

Class III Peroxidases (POs) of higher plants typically reside in the apoplast (http://peroxibase.toulouse.inra.fr/cellular_localisation.php). They are assumed to facilitate plants to withstand biotic stress situations and pathogen attack.¹ But, the whole diversity of their functions is not yet clear.² POs depend on peroxides and calcium. Both, hydrogen peroxide and calcium ions, are signals of plant abiotic stress response.^3-5 Interestingly, POs are the most heat stable enzymes of plants.^6-9 So, a typical questions at this point is: Are POs possibly nodes in signaling networks mediating heat stress response?

Previously we demonstrated that peroxidases (POs) are Ca²⁺ triggered molecular switches.¹⁰ We also showed that heat stability of peroxidases from higher plants is dependent on Ca²⁺ too. However, POs are switched from heat sensitive to heat tolerant in the nM-range of [Ca²⁺] whereas their activity is switched in the µM range. The latter is the typical range of apoplastic free calcium [Ca²⁺]_apo.¹¹ Since H₂O₂, is both, a signaling molecule and a PO substrate, it is assumed that POs play a role in plant innate heat tolerance, heat response, and possibly other stress response routes.

Heat Stress, And Acquired And Innate Heat Tolerance

Often plants are exposed to temperatures above their optimum for several hours a day. In their natural habitat, plants thrive because they employ many different mechanisms to cope with heat stress.^12,13 The current general model is that during heat stress, signaling cascades involving calcium^14-16 and H₂O₂^17,18 as signal molecules initiate the expression of heat shock proteins,^12,19 and other effectors²⁰ which then function as chaperones and in concert regenerate heat-damaged proteins and/or prevent further proteins misfolding.^21,22 This is acquisition of thermotolerance and is based on cellular events resulting from the exposure to high but sublethal temperatures.¹³ Thermotolerance acquisition takes resources but enables the organism to cope better with subsequent periods of heat.

The innate (or basal) thermotolerance has to be distinguished from acquired thermotolerance.²³ It is the ability to withstand high temperatures without heat preconditioning. The primary thermostability of unadapted cellular modules like proteins and membranes thus constitutes innate thermotolerance. Consequently, species from habitats of different maximum day temperature have different innate thermotolerance because evolution maintained molecular modules of appropriate thermostability.

Timing Is Decisive: Heat Shock-Induced Calcium Flux May Cause A Deactivation Of Peroxidases In The Apoplast And Trigger A Self-Amplifying Positive Feed-Back Loop

A movement of calcium ions between apoplast and cytoplasm during periods of heat is controversially reported. Some studies report an increase in cytoplasmic free calcium [Ca²⁺]_cyt during heating^14,16 whereas others do not.²⁴ This is due to experimental timing as has been also shown for cold treatment.^25,26 With rapid temperature changes a [Ca²⁺]_cyt transient can be detected, however with moderate changes, [Ca²⁺]_cyt transients are small or even below detection level. Thus, not the temperature alone but the rapidity of its change is the principal factor eliciting [Ca²⁺]_cyt transients. Ca²⁺ involved in heat signaling is of extracellular (i.e., apoplastic) origin.¹² Due to the high cytoplasmic calcium buffer capacity,²⁷ a detectable increase of free [Ca²⁺]_cyt in the cytoplasm reports a bulk Ca²⁺ ion flux from the apoplast into the cell. Thus, in a heat shock situation the cytoplasmic [Ca²⁺]_cyt increases at the expense of apoplastic [Ca²⁺]_apo. This causes massive Ca²⁺ depletion in the apoplast due to the much lower calcium buffer capacity in this compartment.²⁸ In consequence, a shutdown of apoplastic PO activity occurs as has been demonstrated recently.¹⁰ This, in turn, causes a H₂O₂ accumulation in the apoplast because H₂O₂ consumption by POs is halted (Fig. 1). It is known that H₂O₂ activates cation transporters and favors further cellular calcium ion influx.^29-32 This accelerates apoplastic calcium depletion. Thereby, PO inhibition and ROS accumulation is potentiated. ROS, accumulating in the apoplast, may then spread by diffusion and trigger other cells and serve as messengers for signal cascades. Hence, it is a calcium-depletion-initiated-ROS-accumulation (CaDIRA) in the apoplast (Fig. 1) providing a positive feed back amplification which probably makes an effect and initiates pathways of stress response in general and of thermotolerance acquisition in particular. In other words, calcium 'chairs' stimulus-response coupling and signal amplification in the apoplast through CaDIRA. Other scenarios have been proposed earlier. Among these are also models involving self-amplifying feed-back loops³³ and depletion-refilling of Ca²⁺ stores.³⁴

Figure 1. The calcium-depletion-initiated-ROS-accumulation (CaDIRA) scenario. Calcium 'chairs' stimulus-response coupling and signal amplification in the apoplast through a positive feed-back loop. Color coding: pale blue = apoplast, yellow = cytoplasm, magenta = vacuole, brown = nucleus, red = plasma membrane. (A) In a resting system cytoplasmic calcium [Ca²⁺]_cyt is kept low by active transport. Apoplastic [Ca²⁺]_apo in contrast is high. This promotes peroxidase (PO) activities which keep H₂O₂ concentration low in the apoplast. (B) Upon heat stress ('stimulus') cation channels are activated and Ca²⁺ ions flow downstream, producing a [Ca²⁺]_cyt increase at the expense of extracellular Ca²⁺. The apoplastic Ca²⁺ pool starts to deplete. (C) At low [Ca²⁺]_apo, PO activities are reduced. This, among other processes, causes H₂O₂ accumulation. H₂O₂ in turn promotes Ca²⁺ flux from the apoplast into the cell which causes persisting [Ca²⁺]_apo depletion, reduced PO activity, increased H₂O₂, and finally a burst of extracellular ROS and intracellular [Ca²⁺]_cyt.

The CaDIRA Scenario Can Answer Some Yet Open Questions

This self amplifying mechanism may further deplete calcium from the apoplast, so that finally nM levels of [Ca²⁺]_apo are reached. This in turn switches POs from heat stable to heat sensitive.¹⁰ This way, under continuing heat, POs are irreversibly heat inactivated. Thus, CaDIRA (Fig. 1) can explain the finding that even moderate heat (42°C) is sufficient to reduce PO activity by almost 50%,³⁵ when temperature was increased quickly (i.e., 'heat shock' was applied). A slower increase in temperature probably causes a reduced calcium depletion in the apoplast, since calcium pumps, employed for cytosolic calcium clearance and apoplast refilling, can keep up with heat-induced Ca²⁺ influx. Consequently, with moderate temperature changes, PO activity is reduced, but the heat stability of the enzymes is less affected since ROS fail to accumulate above precarious levels.

Other abiotic and biotic stimuli are known to elicit [Ca²⁺]_cyt transients.^36,37 Some [Ca²⁺]_cyt transients originate from Ca²⁺ release from intracellular stores³⁸ and others from the extracellular space. The latter can influence apoplastic enzymes and plasma membrane ion transport. This is known for chloride transporters for instance, when the plant is under salt stress. Chloride transport into the cells during salt stress is accelerated when Ca²⁺ is depleted outside the cells and conversely, Ca²⁺ addition outside the cell reduces chloride transport and alleviates salt stress.³⁹ A direct interdependence between [Ca²⁺]_cyt and [Ca²⁺]_apo has been demonstrated.⁴⁰ When salt is withdrawn from salt-adapted root cells then [Ca²⁺]_apo decreases in parallel to a [Ca²⁺]_cyt increase. This suggests that such an effect can also be seen with heat stress. Evidently, more detailed investigations are needed.

Often, physiological responses take place away from the primary site of stimulation. This requires cell-to-cell communication and a systemic spread of signals.⁴¹ Here, the CaDIRA scenario depicted above (Fig. 1) can put ideas across and provide explanations: With a local stimulation (e.g., attack of a phytophagous insect or local heat treatment) few cells around the site of stimulation undergo CaDIRA and switch to an 'excited state'. Since membrane depolarization and calcium flux are involved, apoplastic calcium depletion quickly occurs at the other end of the excited cell with almost no time lag and initiates CaDIRA at neighboring cells. This way, the signal could percolate through the plant tissue with velocities not limited to diffusion rates in the apoplast.

The notion of apoplastic calcium signaling is in line with other studies showing that [Ca²⁺]_apo also controls other extracellular enzymes like cell wall phosphatases.⁴² In addition, apoplastic calmodulin plays an important role in signal transduction.^43-45

Plieth C, Vollbehr S. Calcium promotes activity and confers heat stability on plant peroxidases. Plant Signal Behav. 2012;7:650–660. doi: 10.4161/psb.20065.