Plants sense environmental conditions and respond by changing development and physiology. Calcium (Ca2+) is a second messenger thought to play a critical role in plant responses to developmental and environmental stimuli. Calmodulin (CaM) is the prototypical Ca2+ sensor that is highly conserved among eukaryotes. The Arabidopsis genome encodes 50 CaM-like (CML) proteins in addition to CaM. CML functions remain largely unknown. CML24 expression is strongly upregulated by diverse stimuli. The encoded protein shares characteristics with CaM, including primary sequence similarity, predicted tertiary structure, and Ca2+-induced conformational change. Plants with epigenetically silenced CML24 are delayed in the transition to flowering and have altered ion sensitivity.
To further understand CML24 function, I employed genetic, biochemical, cellular, and physiological approaches. Mass spectroscopy analyses suggest that CML24 cysteines may form disulfides; therefore, CML24 may have the capability of transducing oxidative, in addition to Ca2+, signals. Apparent loss- and gain-of-function cml24 point mutants were identified and determined to have alterations in the timing of flowering induction. Despite the physiological consequences of the cml24 mutant amino acid substitutions, the mutant proteins retain the ability to undergo Ca2+-dependent conformational changes. cml24 mutants are defective in expression of the regulatory genes, CONSTANS and FLOWERING LOCUS C (FLC) and have aberrant nitric oxide (NO) accumulation. Altered NO levels underlie FLC enhanced expression in the late-flowering cml24 mutants and contribute in part to delayed flowering. NO-associated 1 (NOA1), not arginine-dependent NO synthase or nitrate reductase, is implicated, through an indirect mechanism, in NO accumulation in cml24 mutants. CML24 directly binds and may increase the enzymatic activity of Autophagy 4b (ATG4b), a cysteine protease that regulates autophagy, the cellular "self eating" process by which eukaryotic cells remove damaged and nonessential cytoplasmic components. Fluorescence imaging and western analysis of autophagy markers and seedling growth assays in response to nutrient-limitation indicate that the cml24 mutants may have defects in autophagy regulation.
Together, this work implicates CML24 as a potential Ca2+ and redox sensor in the regulation of NO accumulation and, through direct interaction with ATG4b, in the regulation of autophagy progression.