Arabidopsis (Arabidopsis thaliana) cyclic nucleotide-gated ion channels (CNGCs) form a large family consisting of 20 members and have been implicated in Ca 2+ signaling related to various physiological processes, such as pathogen defense, development, and thermotolerance. The null mutant of AtCNGC2, defense, no death (dnd1), exhibits autoimmune phenotypes, while it is impaired in mounting the hypersensitive response, which is a hallmark of effector-triggered (i.e. RESISTANCE-gene mediated) resistance. It has been suggested that AtCNGC2 is involved in defense responses and likely other aspects of physiology through its role as a Ca 2+ -conducting channel. However, the downstream signaling components and its relation with AtCNGC4, which is the closest paralog of AtCNGC2, remain elusive. Despite the fact that cngc4 mutants display almost identical phenotypes to those seen in cngc2 mutants, not much is known about their relationship. Here, we report the identification and characterization of the Arabidopsis mutant repressor of defense no death1 (rdd1), obtained from a suppressor screen of a transfer DNA insertion knockout mutant of AtCNGC2 in order to identify downstream components of dnd1-mediated signal transduction. rdd1 suppressed the majority of dnd1-mediated phenotypes except Ca 2+ hypersensitivity. In addition, rdd1 also suppressed the dnd1-mediated lateflowering phenotype that was discovered in this study. Our genetic analysis conducted to elucidate the relationship between AtCNGC2 and AtCNGC4 indicates that RDD1 is also involved in AtCNGC4-mediated signal transduction. Lastly, bimolecular fluorescence complementation analysis suggests that AtCNGC2 and AtCNGC4 are likely part of the same channel complex.

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structurefunction study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth b-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K + -uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the a-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.

The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure–function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the αC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 °C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the αC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire αC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the αC-helix is functionally important in plant CNGCs.

Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. These channels have so far been reported to be involved in a diverse range of physiological phenomena. For example, AtCNGC18 was reported to play an important role in pollen tube growth, while AtCNGC2, 4, 11, and 12 were implicated in mediating pathogen defence. To identify additional functions for AtCNGC11 and 12, various physiological aspects were analysed using both AtCNGC11 and 12 single knockout mutants as well as a double mutant. Although AtCNGC11 and 12 can function as K+ and Ca2+ channels in yeast, it was found that the loss of AtCNGC11 and 12 in Arabidopsis caused increased sensitivity to Ca2+ but not K+, indicating a specific function for these genes in Ca2+ signalling in planta. However, they did not show an alteration in Ca2+ accumulation, suggesting that AtCNGC11 and 12 are not involved in general Ca2+ homeostasis but rather in the endogenous movement of Ca2+ and/or Ca2+ signalling. Furthermore, these channels synergistically contribute to the generation of a Ca2+ signal that leads to gravitropic bending. Finally, AtCNGC11 and 12 gene expression was induced during dark-induced senescence and AtCNGC11 and 12 knockout mutants displayed enhanced chlorophyll loss, which was even more pronounced in the double mutant, also indicating synergistic roles in senescence. The findings indicate that (i) some CNGC family members have multiple physiological functions and (ii) some plant CNGCs share the same biological function and work in a synergistic manner.

The Arabidopsis autoimmune mutant, defense-no death 1 (dnd1) is a null mutant of CYCLIC NUCLEOTIDE-GATED ION CHANNEL2 (AtCNGC2). dnd1 exhibits constitutive pathogen resistance responses including higher levels of endogenous salicylic acid (SA), which is an important signaling molecule for pathogen defense responses. Recently we have reported that dnd1 exhibits a significantly delayed flowering phenotype, indicating the involvement of AtCNGC2 in flowering transition. However, since SA has been known to influence flowering timing as a positive regulator, the delayed flowering phenotype in dnd1 was unexpected. In this study, we have asked whether SA is involved in the dnd1-mediated delayed flowering phenotype. In addition, in order to gain insight into the involvement of SA and CNGCs in flowering transition, we analyzed the flowering transition of cpr22, another CNGC mutant with a similar autoimmune phenotype as dnd1 (including high SA accumulation), and null mutants of several other CNGCs. Our data suggest that dnd1 does not require SA or SA signaling for its delayed flowering phenotype, while SA was responsible for the early flowering phenotype of cpr22. None of the other CNGC mutants besides AtCNGC4 (1) displayed an alteration in flowering transition. This indicates that AtCNGC2 and AtCNGC4 have a unique role controlling flowering timing and this function is independent from its role in pathogen defense.