Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.