1.A.87 The Mechanosensitive Calcium Channel (MCA) Family

Mechano-sensitive channels of plants sense increases in tension induced by mechanical stimuli, such as touch, wind, turgor pressure and gravitation. Plant homologues of MscS bacterial mechano-sensitive channels are known which are gated by membrane tension. Two of them have been shown to be involved in the protection of osmotically stressed plastids in Arabidopsis thaliana (see TC# 1.A.23.4.4). Membrane tension is not a mediator of long-range intracellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains (Shi et al. 2018). Lipid bilayer tensiometers have been used for the study of mechanosensitive ion channels (Pérez-Mitta and MacKinnon 2023). Heavy metal tolerance mechanisms of Brassica species have been reviewed (Shehzad et al. 2023).

Iida et al. (2013) identified another group of candidates for mechano-sensitive channels in Arabidopsis, named MCA1 and MCA2, whose homologues are exclusively found in plant genomes. MCA1 and MCA2 are composed of 421 and 416 amino acyl residues, respectively, share 73% identity in their amino acid sequences, and are not homologous to any other known ion channels or transporters. A structural study revealed that the N-terminal region (~173 amino acids) of both proteins is necessary and sufficient for Ca²⁺ influx activity. This region has one putative transmembrane segment containing an Asp residue whose substitution mutation abolished activity.Their physiological study suggested that MCA1, expressed at the root tip, is required for sensing the hardness of the agar medium or soil. In addition, MCA1 and MCA2 were shown to be responsible for hypo-osmotic shock-induced increases in [Ca²⁺]_cyt . Thus, both proteins appear to be involved in the process of sensing mechanical stresses. Iida et al. (2013) discussed the possible roles of both proteins in sensing mechanical and gravitational stimuli.  Several homologues may serve as receptors and regulatory proteins rather than ion channels, and several of these are included in this family in TCDB.  Their roles as mechanosensitive plasma membrane Ca²⁺-permeable channels, such as OsMCA1and OsMCA2 in rice seems to allow them to play roles in the generation of reactive oxygen species and in hypo-osmotic signaling (Kurusu et al. 2012; Kurusu et al. 2012; Kurusu et al. 2012).

MCA proteins show various topologies.  Several show a 1 + 3 TMS topology (subfamily 1) while others (subfamily 2) appear to have a 1 + 3 + 3 TMS topology, and still others have just 3 TMSs (subfamily 3).  The 3 TMSs in these last mentioned proteins appear to correspond to the last 3 TMSs in subfamilies 1 and 2. The topologies of subfamilies 4 and 5 are not clear.  There may be additional topological variations.

The FW2.2 gene is associated with the major Quantitative Trait Locus (QTL) governing fruit size in the tomato, and it acts by negatively controlling cell division during fruit development. FW2.2 belongs to a multigene family named the CELL NUMBER REGULATOR (CNR) family (Beauchet et al. 2021). The CNR proteins harbour the uncharacterized PLAC8 motif made of two conserved cysteine-rich domains separated by a variable region that are predicted to be transmembrane segments, and indeed FW2.2 localizes to the plasma membrane.  Beauchet et al. 2021 reviewed the knowledge on PLAC8-containing CNR/FWL proteins in plants, which participate in plant organogenesis and the regulation of organ size, especially in fruits, and in cadmium resistance, ion homeostasis and/or Ca²⁺ signalling. Within the plasma membrane, FW2.2 and some CNR/FWL proteins are localized in microdomains. Hence FW2.2 and CNR/FWL could be involved in a transport function of signalling molecules across membranes, thus influencing organ growth via a cell-to-cell trafficking mechanism (Beauchet et al. 2021).

The generalized reaction reported to be catalyzed by MCA1 and MCA2 is:

Ca²⁺(out)  →  Ca²⁺ (in)

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. Bi et al. 2021 employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. The authors showed that activation of ZAR1 in the plant cell led to Glu11-dependent Ca²⁺ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. Cations transported include The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.

The plant innate immune system is composed of cell surface receptors, which perceive immunogenic molecular patterns derived from invading pathogens, and intracellular nucleotide-binding, leucine-rich repeat receptors (NLRs), which sense pathogen effectors that are delivered into the host cell intended to promote pathogenesis. Plant NLRs can be classified based on their variable N-terminal domains, thus those carrying a coiled coli (CC) domain are called CNLs, those carrying a Toll-interleukin 1 receptor (TIR) domain are referred to as TNLs, and those carrying an RPW8-like CC domain (CC_R) are called RNLs. NLRs are also important innate immune sensors in animals. Upon activation, NLRs often form oligomeric complexes referred to as inflammasomes in animals and resistosomes in plants. Animal inflammasomes activate caspases to promote the maturation of gasdermin proteins, which form pores in the plasma membrane (PM) to trigger pyroptosis and immune responses.  Activation of plant NLRs also leads to regulated cell death called the hypersensitive response (HR). ZAR1 is a CNL with a canonical CC domain that acts both as a sensor for pathogen effectors and an executor for signaling. ZAR1 is an ancient NLR that emerged more than 100 million years ago and is able to sense a growing number of pathogen effector proteins (Bi et al. 2021).

ZAR1 senses diverse effector proteins by associating with a class of homologous pseudokinases called ZRKs and a second class of kinases, PBLs. Prior to pathogen infection, ZAR1 interacts with various ZRKs in the resting state. Upon pathogen delivery of effectors into the plant cell, ZRKs in the pre-formed complexes further recruit PBL proteins that have been post-translationally modified by effectors to form ternary complexes.  For example, AvrAC uridylylates the Arabidopsis PBL2 protein, giving rise to PBL2^UMP, which is then recruited by RKS1/ZRK1 to form a ZAR1-RKS1-PBL2^UMP complex which transports cations including Ca²⁺ (Bi et al. 2021). The channel is permeable to Na⁺, K⁺, Cs⁺, Mg²⁺ and Ca²⁺. The three proteins that comprise this complex are all members of TC family 1.A.87 as is ZRK1 of 351 aas and 2 TMSs (see TC# 1A 87.2.17).

The reaction catalyzed by the ZAR1-RKS1-PBL2^UMP complex is:

cation (out) ⇋ cation (in)