In plants, calcium ions (Ca2+) function as a central signal for diverse stimuli, ranging from internal developmental cues to physical or biological stresses such as infection. However, the transient nature of Ca2+ signals and the enigmatic identities of plant Ca2+ channels have made the role of these ions difficult to study. Moreover, the connection between Ca2+ channels and specific plant responses is often unclear. Writing in Nature, Thor et al.1 now clarify one such connection, and report their finding of a type of Ca2+ channel that is activated during a specific response against infection.
Two specialized, moon-shaped cells, called guard cells, form a leaf pore called a stoma (Fig. 1a). Stomata allow gas exchange, including the entry of carbon dioxide for the energy-generating process of photosynthesis. They are thus essential for plant survival. However, disease-causing microorganisms (pathogens) can use stomata as a gateway for invasion. To limit infection, plants close stomata on recognizing such an attack, in a defence response called stomatal immunity2. The surfaces of the cells of both plants and animals have receptor proteins containing regions called kinase domains, and these proteins can recognize evolutionarily conserved microbial molecular motifs called pathogen-associated molecular patterns (PAMPs) and initiate signalling pathways needed for defence.
In the model plant Arabidopsis thaliana, a receptor protein called FLS2, which has a kinase domain, binds to the bacterial protein flagellin, recognizing a region of this PAMP called flg22. This recognition event causes FLS2 to form an active receptor complex with another cell-surface receptor kinase called BAK1. The complex adds a phosphate group to a cytoplasmic kinase called BIK1. This phosphorylation of BIK1 activates immune responses3, such as the production of reactive oxygen species by the protein RBOHD. BIK1 is required for stomatal immunity4, and if guard cells contain a mutant version of the gene that encodes this kinase, the plant cannot respond to flg22. However, the link between PAMP recognition and Ca2+-mediated stomatal closure regulated by BIK1 has been unclear.
To join the dots, Thor et al. speculated that, through direct phosphorylation, BIK1 controls the Ca2+ channel(s) required for stomatal immunity. The authors focused on an ion channel called OSCA1.3, which is phosphorylated on sensing flg22. Thor and colleagues report that OSCA1.3 is permeable to Ca2+, and that phosphorylation of OSCA1.3 by BIK1 at serine amino-acid residue 54 (in the same type of motif as that phosphorylated by BIK1 in RBOHD) activates this channel on pathogen recognition (Fig. 1b). Furthermore, the authors’ observation that the gene that encodes OSCA1.3 is specifically expressed in stomata is consistent with a role for the channel in stomatal immunity.
The OSCA family of proteins are evolutionarily conserved ion channels, and A. thaliana contains 15 members of this family. Each ion channel is probably formed of two OSCA proteins. The largest group of these proteins, clade 1, includes OSCA1.1, OSCA1.2 (also known as OSCA1) and OSCA1.35–7. OSCA1.1 and OSCA1.2 are Ca2+-permeable channels that are also permeable to several other types of positively charged ion (cations), and they are activated by an ionic imbalance known as osmotic stress5,7.
Thor et al. observed no clear effect on the immune response to flg22 in a mutant plant in which the gene OSCA1.3 was disabled. However, in a plant engineered also to have a mutant version of another clade 1 member — the gene OSCA1.7 — stomatal closure on perceiving flg22 was impaired and susceptibility to bacterial infection was enhanced, compared with the response in the wild-type plant. OSCA1.7 has a similar protein motif to the one phosphorylated on OSCA1.3 by BIK1, and is activated through phosphorylation by BIK1 to generate a Ca2+ influx into cells. Thus, it seems that OSCA1.3 and OSCA1.7 are Ca2+ channels that regulate stomatal immunity and they probably function in a redundant manner, such that if OSCA1.3 is absent, OSCA1.7 can fulfil its role. Whether just one or both of these proteins together form Ca2+ channels that act in stomatal immunity is unknown.
In addition to identifying these Ca2+ channels, Thor et al. explored the role of the plant hormone abscisic acid (ABA), which regulates stomatal closure when the plant senses a water deficit. This hormone also controls stomatal defences, because stomata of ABA-deficient plants do not close effectively on perceiving pathogens2. However, the authors found that a plant with mutations in the genes encoding both OSCA1.3 and OSCA1.7 is fully responsive to ABA, indicating that these channels are not involved in ABA-mediated stomatal closure. This observation corroborates previous evidence4 that the regulation of stomatal immunity by BIK1 does not require ABA. Furthermore, Thor et al. report that the overall Ca2+-signal activation by flg22 in leaves that had mutations in the genes encoding both OSCA1.3 and OSCA1.7 was not impaired; guard cells make up only a small fraction of leaf cells. This result strongly supports the specific role of these channels in stomatal immunity, rather than general immunity, even though BIK1 is required for both types of response.
How changes in Ca2+ concentration deliver stimulus-specific cellular responses is a central question in this area of research. One proposed idea is that stimulus-specific temporal patterns of cytoplasmic Ca2+ levels might provide a key cue, and that these ‘Ca2+ signatures’ might be generated and decoded by specific Ca2+-binding components, such as calmodulin proteins or calcium-dependent protein kinases8. Thor and colleagues’ results suggest instead that the Ca2+ channels themselves might determine specificity, at least for stomatal immunity.
Although OSCA proteins allow stomata to close independently of ABA involvement, closure mediated either by ABA or in response to infection probably involves the same mechanism, which eventually closes stomata through water movement out of guard cells. Therefore, both pathways should converge at some point. The activation of channels that enable negatively charged ions (anions) to exit the cell, such as S-type anion channels, termed SLACs, is a crucial step in stomatal movement9. The protein kinase OPEN STOMATA1, which is a component of an ABA-mediated signalling pathway, activates SLACs and has been proposed2 as a point of convergence for defence responses and ABA signalling. However, some calcium-dependent protein kinases also activate SLACs10, and such Ca2+-signal decoders, or perhaps even the anion channels themselves, might be the convergence point instead.
An emerging theme in studies of plant Ca2+ channels is their regulation by phosphorylation. Previous studies11,12 reported that BIK1 and BAK1 phosphorylate members of another group of plant Ca2+ channels, the cyclic nucleotide-gated ion channels, to regulate their function or stability. The phosphorylation of OSCA1.3 and OSCA1.7 by BIK1 underscores the connection between receptor kinases and Ca2+ channels, presumably to generate stimulus-specific Ca2+ signals. It will be interesting to determine whether OSCA-family proteins interact with other components on the surface of cells to form a structure called a channelosome — a group of signalling molecules surrounding an ion channel13.
OSCA proteins have so far been linked mostly to the sensing of osmotic stress. They are categorized as a type of mechanosensing channel, one that converts physical forces into biochemical signals14,15. Are OSCA1.3 and OSCA1.7 activated by osmotic stress or mechanical stimulation, in addition to their activation by BIK1? Did the two proteins evolve a defence-specific role, or do they also have other functions in stomata? Understanding the biological function of each OSCA and the Ca2+ signals they generate will shed light on stomatal biology. Such insights could be crucial for the bioengineering of plants to meet future challenges in crop production.