A natural single-nucleotide polymorphism in the CAMTA3 transcription factor regulates its function and transcription of its target genes

Abstract

CAMTA3, a Ca²⁺/calmodulin-binding transcription factor, is a key regulator of plant immunity in Arabidopsis. Here, we identified a novel naturally occurring single-nucleotide polymorphism that results in a missense nonconservative mutation (CAMTA3_H386D) in many Arabidopsis ecotypes. This region of CAMTA3 is not part of any previously characterized regulatory domains. To study the consequence of this change on the function of CAMTA3, we introduced the CAMTA3_H386D into camta3, a loss-of-function mutant that exhibits a constitutive cell death phenotype, chlorotic lesions on leaves, and reduced plant size. Phenotypic and molecular analysis of these lines indicated that the expression of CAMTA3_H386D in the camta3 mutant did not complement the mutant phenotypes. Also, the ecotypes containing the CAMTA3_H386D exhibited camta3 phenotypes. Marker genes associated with salicylic acid biosynthesis and pathogen response were upregulated in the CAMTA3_H386D lines and the Arabidopsis accessions 7127 (Est-1) and 9941 (Fei-0), as in camta3, indicating that H386D mutation alters CAMTA3 activity in regulating the expression of known target genes. In Nicotiana benthamiana transient expression assays, CAMTA3_H386D failed to induce the expression of a luciferase reporter gene driven by the rapid stress-responsive elements (RSRE) that contain the known binding sites of CAMTA3, suggesting that CAMTA3_H386D mutation impairs its ability to activate its target genes. Transgenic lines and tested natural accessions expressing CAMTA3_H386D showed enhanced levels of H₂O₂ and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Collectively, our results indicate that the H386D mutation in a previously unknown regulatory region of CAMTA3 is essential for its function.