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).
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 (CCR) 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 PBL2UMP, which is then recruited by RKS1/ZRK1 to form a ZAR1-RKS1-PBL2UMP 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-PBL2UMP complex is:
cation (out) ⇋ cation (in)
Plant Ca2+ channel protein, Mid1 complementary activity 1, MCA1 (Iida et al. 2013). MCA1 and MCA2 each forms a homotetramer and exhibit Ca2+-permeable mechanosensitive channel activity. Both are single-pass type I transmembrane proteins with their N-termini located extracellularly and their C-termini located intracellularly. An EF hand-like motif, coiled-coil motif, and Plac8 motif may all be in the cytoplasm, suggesting that the activities of both channels can be regulated by intracellular Ca2+ and protein interactions (Kamano et al. 2015). However, hydropathy plots suggest that the Plac8 domain may be transmembrane with 3 TMSs. mca1 but not mca2 mutants show defects in root entry into hard agar, whereas mca2 but not mca1 mutants are defective in Ca2+ uptake in A. thaliana roots (Hamilton et al. 2015). Root growth reduction in response to mechanical stress involves MCA1 tgether with WDL5 (Q94C48) subject to ethylene-mediated regulation) and the co-receptor BAK1 (Q94F62) (Okamoto et al. 2021).
MCA1 of Arabidopsis thaliana
Plant Ca2+ channel protein, Mid1 complementary activity 2, MCA2 (Iida et al. 2013). Catalyzes mechanical stress-induced Ca2+ influx. It is tetrameric with a small transmembrane domain and a large cytoplasmic domain (Shigematsu et al. 2014). MCA1 and MCA2 both have their N-termini located extracellularly and their C-termini located intracellularly. An EF hand-like motif, coiled-coil motif, and Plac8 motif may all be in the cytoplasm, suggesting that the activities of both channels can be regulated by intracellular Ca2+ and protein interactions (Kamano et al. 2015). However hydropathy plots suggest that the Plac8 domain may be transmembrane with 3 TMSs. mca1 but not mca2 mutants show defects in root entry into hard agar, whereas mca2 but not mca1 mutants are defective in Ca2+ uptake in A. thaliana roots (Hamilton et al. 2015).
MCA2 of Arabidopsis thaliana
MCA1 isoform X2 of 377 aas with one N-terminal TMS and possibly 3 or 4 C-terminal TMSs.
MCA1 of Solanum pennellii (Lycopersicon pennellii)
PLAC8 family protein of 385 aas with MID1-complementing activity.
PLAC8 family protein of Theobroma cacao
Mid1 complementing activity 1 of 154 aa
MCA1 of Vigna radiata
Receptor protein kinase of 567 aas. The first 140 aas are homologous to the N-terminal domains of MCA1 and 2; residues 240 - 430 are homologous to ser/thr protein kinases of 9.A.15.1.1, 9.B.45.1.3 and 9.B.106.3.1.
Receptor protein kinase of Zea mays
Uncharacterized leucine-rich repeat domain-containing proteins of 387 aas and putative protein kinase of 399 aas, respectively, each with one TMS, the first of these proteins at the N-terminus, and the second near its C-terminus. These two proteins are most similar to different parts of the other proteins in TC subclass # 1.A.87.2.
UPs of Desulfosarcina alkanivorans
Leucine-rich repeat (LRR) receptor-like serine/threonine-protein kinase, ERECTA, of 966 aas and 2 TMSs, one at the N-terminus of the protein, and one at residues 580 - 600. Oterh peaks of hydrophobicity may also be transmembrane. It is a receptor kinase that, together with ERL1 and ERL2,
regulates aerial architecture, including inflorescence (e.g. shoot
apical meristem-originating organ shape, elongation of the internode and
pedicels, and adaxial-abaxial polarity), and stomatal patterning (e.g.
density and clustering), probably by tuning cell division and expansion. It regulates canalization as well as cell wall composition and structure, and it confers resistance to the pathogenic bacteria Ralstonia solanacearum and
to the necrotrophic fungi Plectosphaerella cucumerina and Pythium
irregulare. It is required for callose deposition upon infection. (Torii et al. 1996).
ERL2 of Arabidopsis thaliana
Receptor-like kinase 1, RKL1, of 655 aas and 2 TMSs, one N-terminal and one centrally located. These receptor-like kinases directly regulate the functions of membrane transport proteins in plants (Li et al. 2022).
RKL1 of Arabidopsis thaliana
Transmembrane kinase receptor of 942 aas and 2 TMSs, one at the N-terminus of the protein and the second at residue 490 (Chang et al. 1992). It phosphorylates only serine and threonine residues (Schaller and Bleecker 1993) and is involved in auxin signal transduction and cell expansion as well as proliferation regulation (Dai et al. 2013). With ABP1, it is a cell surface auxin perception complex that activates ROP signaling pathways (Xu et al. 2014). It is required for auxin promotion of pavement cell interdigitation and promotes the formation of the ABP1-TMK1 protein complex (Xu et al. 2014).
TMK1 of Arabidoopsis thaliana
Nod-factor receptor 1a, NFR1, of 621 aas and about 6 TMSs in an estimated 2 + 1 + 1 + 1 + 1 TMS topology. This protein plus NFR5 constitutes the Lotus japonicus core receptor complex in root nodule symbiosis that initiates the cortical root nodule organogenesis program (Rübsam et al. 2023).
NFR1 of Lotus japonicus
Nod-factor receptor 5, NFR5, of 595 aas and about possibly 3 TMSs, one at the N-terminus, one at residue 250, and one at residue 470. This protein plus NFR1 (TC# 1.A.87.2.14) constitutes the Lotus japonicus core receptor complex in root nodule symbiosis that initiates the cortical root nodule organogenesis program (Rübsam et al. 2023).
NFR5 of Lotus japonicus
G-type lectin S-receptor-like serine/threonine-protein kinase, SRK, of 853 aas with possibly 4 TMSs, one at the N-terminus of the protein, and 3 more at residues 450, 580 and 710 (Zhou et al. 2023).
SRK of Arabidopsis thaliana
Pseudokinase (serine/threonine protein kinase), ZRK1, of 351 aas and 2 strongly hydrophobic TMSs (at residues 130 and 260) (Bi et al. 2021).
ZRK1 of Arabidopsis thaliana
The ZAR1-RKS1-PBL2UMP complex which transports cations including Ca2+ (Bi et al. 2021).The cations transported include Na+, K+, Cs+, Mg2+ and Ca2+. All three proteins included in this complex are homologous to the proteins in TC family 1.A.87.
The ZAR1-RKS1-PBL2UMP complex of Arabidopsis thaliana:
ZAR1 of 716 aas (Q9ZU46)
RKS1 of 833 aas (Q9ZT07)
PBL2UMP of 426 aas (O49839)
Protein kinase domain protein of 522 aas.
PKD protein of Oryza sativa
Receptor for extracellular ATP which functions in plant growth, development and stress responses; lectin receptor kinase 1.9; DORN1. Binds ATP with high affinity (46nM) and is required ofr ATP-induced calcium response, mitogen-activated protein kinase activation and normal gene expression (Choi et al. 2014).
DORN1 of Arabidopsis thaliana
GHR1 (GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1) transmembrane receptor-like protein of 1053 aas and 1 - 3 TMSs. Regulates the SLAC1 protein (2.A.16.5.1) (Wang et al. 2017). The C-terminus shows extensive sequence similarity with members of this family, but the N-terminus shows similarity with members of family 3.A.20 (Leucine repeat proteins).
GHR1 of Arabidopsis thaliana
Uncharacterized protein with an ATP binding domain of 629 aas and 2 TMSs.
UP of Arabidopsis thaliana
Protein BRASSINOSTEROID INSENSITIVE 1, BRI1, of 1196 aas and 2 or 3 TMSs. Receptor with kinase activity
acting on both serine/threonine- and tyrosine-containing substrates. In response to brassinosteroid binding, it regulates a signaling cascade
involved in plant development, including expression of light- and
stress-regulated genes, promotion of cell elongation, normal leaf and
chloroplast senescence, and flowering. It binds brassinolide, and less
effectively, castasterone (Oh et al. 2009).
BRI1 of Arabidopsis thaliana
The phytosulfokine receptor, PSKR1, of 1008 aas with both a serine/threonine-protein kinase activity and a guanylate cyclase activity (Kwezi et al. 2011). In response to phytosulfokine binding, it activates a signaling cascade involved in plant cell differentiation, organogenesis, somatic embryogenesis, cellular proliferation and plant growth. It is also involved in plant immunity, with antagonistic effects on bacterial and fungal resistances (Mosher et al. 2013). CNGC17 and AHAs form a functional cation-translocating unit that is activated by PSKR1/BAK1 and possibly other BAK1/RLK complexes (Ladwig et al. 2015). PSKR is a transmembrane LRR-RLK family protein with a binding site for the small signalling peptide, phytosulfokine (PSK). There are 15 members in rice (Orysa sativa), induced under different conditions in different plant tissues (Nagar et al. 2020). PSKR1 and PSYR1 mediate a signaling pathway in response to two distinct ligands, which redundantly contribute to cellular proliferation and plant growth (Amano et al. 2007).
PSKR1 of Arabidopsis thaliana (Mouse-ear cress)
Plant cadmium resistance, PCR, protein of 164 aas. Shows homology to the C-terminal PLAC8 domain of MCA1 and 2.
Cadmium resistance protein of Solanum lycopersicum (Tomato) (Lycopersicon esculentum)
Fruit-weight 2.2 protein of 197 aas and 3 TMSs. May be involved in Cd2+ resistance as well as translocation of Cd2+ from roots to shoots (Xiong et al. 2018). May form homooligomeric structures in the membrane.
FWL protein of Medicago truncatula (Barrel medic) (Medicago tribuloides)
Fruit-weight 2.2 protein of 161 aas and 4 TMSs. May be involved in Cd2+ resistance as well as translocation of Cd2+ from roots to shoots (Xiong et al. 2018). May form homooligomeric structures in the membrane.
FWL protein of Medicago truncatula (Barrel medic) (Medicago tribuloides)
Fruit Weight 2.2 (FW2.2) protein of 163 aas and possibly 3 TMSs. See family description for details (Beauchet et al. 2021).
FW2.2 of Solanum lycopersicum (Tomato) (Lycopersicon esculentum)
Plant Cadmium Resistance (PCR) protein. This protein corresponds to the C-terminal PLAC8 domain of MCA1 (TC# 1.A.87.1.1) (Song et al., 2011). The plant cadmium resistance (PCR) gene family has been characterized in Brassica napus and one member, has been functionally analyzed: BnPCR10.1 is involved in cadmium and copper tolerance( (Liu et al. 2023).
PLAC8 family protein of Arabidopsis thaliana
Sea squirt membrane protein of 110 aas
Membrane protein of Ciona intestinalis
Uncharacterized protein of 161 aas
UP of Capsella rubella
Plant cadmium resistance 6 protein, CadR6, of 224 aas.
CadR6 of Arabidopsis thaliana
Uncharacterized protein of 186 aas
UP of Glycine max
Plant cadmium resistance 1 protein of 151 aas and 2 TMSs. PCR1. Involved in glutathione-independent cadmium resistance. Reduces cadmium uptake rather than activating efflux, but is not closely coupled to calcium transport (Song et al. 2011).
PCR1 of Arabidopsis thaliana
Plant cadmium resistance 2 (PCR2) protein. Zinc ion exporter (Song et al. 2010; Song et al. 2011). Involved in glutathione-independent cadmium resistance. Reduces cadmium uptake rather than activating efflux, but is not closely coupled to calcium transport.
PCR2 of Arabidopsis thaliana
FW2.2-like (FWL) protein of 180 aas and 2 or 3 TMSs. It is involved in plant and fruit development, and possibly in calcium transport (Libault and Stacey 2010). See family description for details about its possible functions in the tomato (Beauchet et al. 2021).
FWL of Persea americanan (Avocado)
Ubiquitin protein ligase with the first 250 aas homologous to MCA2.
Ubiquitin ligase of Physcomitrella patens
U box containing protein 15
U box protein of Solanum lycopersicum (Tomato) (Lycopersicon esculentum)
Protein kinase_Tyr of 657 aas with N-terminal domain similar to that of MCA1, with N-terminal TMS containing a conserved aspartyl residue.
PKinase-Tyr of Phanerochaete carnosa