How plant cells sense the outside world through hydrogen peroxide

Chemically reactive, oxygen-containing molecules called reactive oxygen species (ROS) are central to cell function. Plant cells generate various ROS, including hydrogen peroxide (H₂O₂), which has a key role in cell signalling. It is produced in an extracellular space between the plasma membrane and cell wall called the apoplast, in response to a range of factors, including stressors, plant hormones such as abscisic acid, and physical or chemical changes outside the cell¹. But whether and how this extracellular H₂O₂ (eH₂O₂) is sensed at the cell surface is unknown. Writing in Nature, Wu et al.² identify the first known cell-surface H₂O₂ receptor in plants.

The apoplast and cell wall act as a dynamic interface between plant cells and the outside world, with all its threats, challenges and opportunities. Some eH₂O₂ moves from the apoplast into the cytoplasm through channel proteins called aquaporins³. However, unlike the cytoplasm, the apoplast contains relatively few molecules that counteract oxidation¹ — and so ROS, including H₂O₂, can survive for much longer in the apoplast than in the cytoplasm. This is a compelling reason to suspect that there is a sensor for eH₂O₂ in the apoplast.

Although little is known about the initial target of eH₂O₂, the consequences of its production are much better defined⁴. It is clear that eH₂O₂ triggers an influx of calcium ions (Ca²⁺) into the cell, which then leads to the systemic transmission of signals between cells in waves, activating processes such as pathogen resistance or acclimation to stress across the entire plant⁵. In addition, eH₂O₂ signals regulate the polarized growth of pollen tubes and root hairs⁶, and control the opening and closing of stomata³ — pores on the outer layer of the leaf formed by two guard cells. Stomata enable the free passage of molecules such as carbon dioxide and oxygen into the plant when open, and can close to prevent water loss from the plant.

Wu et al. set out to identify cell-surface receptors for eH₂O₂ that trigger Ca²⁺ signalling, using a ‘forward’ genetic-screen approach. They treated seeds of the plant Arabidopsis thaliana with a chemical that induces DNA mutations, then screened the resulting plants to identify mutants that showed low Ca²⁺ influxes in response to H₂O₂. They named these mutant plants hydrogen-peroxide-induced Ca²⁺ increases 1 (hpca1).

The authors then identified the HPCA1 protein. They report that HPCA1 is a membrane-spanning enzyme of a protein family known as leucine-rich repeat (LRR) receptor kinases. The group also showed that HPCA1 has two special pairs of cysteine (Cys) amino-acid residues in its extracellular domain. The thiol groups of Cys residues are known⁷ to be a target for oxidation by H₂O₂. The authors demonstrate that the presence of eH₂O₂ leads to oxidation of the extracellular Cys residues of HPCA1 in guard cells. This modification activates HPCA1’s intracellular kinase activity, triggering Ca²⁺-channel activation and Ca²⁺ influx, followed by stomatal closure (Fig. 1).

In the absence of eH₂O₂, the hpca1 seedlings showed no differences from wild-type seedlings. However, their guard cells were less sensitive to eH₂O₂ than were those of the wild-type seedlings, showing lower than wild-type levels of Ca²⁺ influx in response to eH₂O₂. HPCA1 is therefore required to convert the eH₂O₂ signal into a physiological response. Moreover, the abscisic acid-dependent production of eH₂O₂ by guard cells was defective in the hpca1 mutants. Of note, the function of HPCA1 in eH₂O₂ signalling was not limited to guard cells, and the authors provided evidence that eH₂O₂ signalling helps to transmit environmental signals to the nucleus of various cell types to regulate gene expression.

Oxidation of Cys by H₂O₂ leads to the formation of a sulfenic acid (SOH), which is at the heart of reduction–oxidation (redox) signalling. Sulfenic acids are rather unstable intermediates that can be further oxidized to sulfinic (SO₂H) and sulfonic (SO₃H) acid, or can undergo ‘exchange reactions’ to form disulfide bonds. For HPCA1 to function properly as a receptor for eH₂O₂, the Cys oxidation process must be readily reversible, re-forming thiol residues that can be oxidized again. However, the factors that mediate reduction of the oxidized HPCA1 are unknown. One candidate is a membrane-bound electron-transport system, such as the one that reduces an oxidized form of the antioxidant molecule ascorbic acid in the apoplast⁸. Membrane-bound and apoplastic thioredoxin-like proteins are also putative candidates, given that thioredoxin is a well-characterized reducing agent for oxidized Cys residues of proteins.

Wu and colleagues have uncovered a receptor-kinase-mediated eH₂O₂ sensing mechanism that does not resemble any known eH₂O₂ receptors or sensors reported in other organisms. Nonetheless, HPCA1 might be part of a much wider portfolio of sensors used by plants to perceive and respond to environmental changes through ROS signals. The identification of such receptors has proved challenging, not least because likely candidates are members of very large protein families. Sophisticated screens, such as that used by Wu et al., will be required to tease out the family members that have ROS sensing and signalling roles. Once these sensors have been identified, it should be relatively easy to manipulate their properties to produce model plants and crops that have, for example, increased or depressed sensitivity to environmental H₂O₂ signals, and so show altered tolerance to environmental threats.

Stomatal closure is not regulated just by H₂O₂; it is also a response to elevated atmospheric CO₂ levels^3,9. It will be intriguing to see how proteins such as HPCA1 function in redox signalling networks that are likely to prepare plants for life in a future high-CO₂ world. High CO₂ levels can stimulate photosynthesis and depress photorespiration; changes in the photosynthesis:respiration ratio have a wide-ranging impact on cellular redox balance, because photorespiration generates a molecule of H₂O₂ in one organelle, the peroxisome, for every oxygen molecule assimilated in another organelle, the chloroplast, during photosynthesis. Perhaps other H₂O₂ sensors act together with HPCA1 to transmit organelle-specific redox messages to the nucleus, along with messages from the external face of the plasma membrane.