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.

Cyclic Nucleotide Gated Ion Channels (CNGCs) have been firmly established as Ca2+-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca2+ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca2+ signals in response to different triggers. In this study we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca2+ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid (IAA), indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TAA–YUC-dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca2+ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

Cyclic Nucleotide Gated Ion Channels (CNGCs) are non-selective cation channels that are involved in regulating responses to both biotic and abiotic stresses in plants.CNGC2 has been implicated in plant immunity and Ca 2+ signaling through the study of the autoimmune phenotypes exhibited by the null mutant, defense, no death1 (dnd1).However, dnd1 also shows additional phenotypes that are unique among autoimmune mutants. This suggests that CNGC2 plays multiple biological roles beyond pathogen defense. In this study, we cloned the gene that encodes the first suppressor of dnd1 (cngc2), REPRESSOR OF DEFENSE, NO DEATH1 (RDD1), which encodes an auxin biosynthesis gene, YUCCA6 (YUC6). We found that dnd1 (cngc2) is defective in auxinmediated root growth inhibition and gravitropic responses in roots. Consistently with these auxin resistance phenotypes, we found dnd1 shows a dampened response to exogenous auxin compared to wildtype plants using the auxin inducible DR5::GUS and DII:VENUS reporter systems. Finally, auxin-induced Ca 2+ influx was examined using the Förster resonance energy transfer (FRET)-based, genetically encoded Ca 2+ indicator yellow cameleon (YC)-Nano65. We captured severe defects in auxin-induced Ca 2+ increase in dnd1. These defects were rescued by the rdd1 mutation. Our findings highlight the unexpected involvement of CNGC2 in auxin-induced Ca 2+ signaling and calls into question the current interpretation of dnd1 phenotypes in defense signaling. 4Cutadapt software 61 and unpaired reads, as well as those of length < 36 nt, were excluded from all further analyses. Paired reads were aligned against the TAIR10 Col-0 reference genome using BWA 62 and sequence variants were detected with the mpileup function of SAMtools 63 .GUS enzymatic assay. Seedlings were grown on ½ MS, 1% sucrose, and 0.8% (w/v) agar plates for 6 days before being transferred to plates supplemented with 1 µM IAA, or solvent alone, for 24 hours. GUS reporter activity was analyzed at excitation of 365 nm and emission of 455 nm using a TECAN plate reader (every 10 min for 2 hours) in the presence of 4methylumbelliferyl glucuronide (4-MUG). GUS activity was standardized against protein concentration and data was reported as GUS activity in pmol 4-methylumbelliferone (4MU) per µg protein).Pathogen infection. Infection with Hyaloperonospora arabidopsidis isolate Noco, which is virulent to Columbia ecotype of Arabidopsis was performed as described previously with 5 x 10 5 spores per ml 26 .Root growth inhibition. Sensitivity to auxin was analyzed as previously described by 38 .Seedlings were grown on minimal medium of ½ MS, 1% sucrose, and 0.8% agar supplemented with various concentrations of IAA. To analyze the root growth inhibition, the data of root growth on auxin media and minimal media was plotted.Analysis of endogenous Salicylic acid. Endogenous SA was analyzed using the Acinetobacter sp. ADPWH lux-based biosensor as described 64 . 6 mm, Fisher Scientific) for 6 days at 23 °C. The tip of the root was exposed by removing a small window...