Arabidopsis Calmodulin-Like Proteins, CML15 and CML16 Possess Biochemical Properties Distinct from Calmodulin and Show Non-overlapping Tissue Expression Patterns

Abstract

Calcium ions are used as ubiquitous, key second messengers in cells across eukaryotic taxa. In plants, calcium signal transduction is involved in a wide range of cellular processes from abiotic and biotic stress responses to development and growth. Calcium signals are detected by calcium sensor proteins, of which calmodulin (CaM), is the most evolutionarily conserved and well-studied. These sensors regulate downstream targets to propagate the information in signaling pathways. Plants possess a large family of calcium sensors related to CaM, termed CaM-like (CMLs), that are not found in animals and remain largely unstudied at the structural and functional level. Here, we investigated the biochemical properties and gene promoter activity of two closely related members of the Arabidopsis CML family, CML15 and CML16. Biochemical characterization of recombinant CML15 and CML16 indicated that they possess properties consistent with their predicted roles as calcium sensors. In the absence of calcium, CML15 and CML16 display greater intrinsic hydrophobicity than CaM. Both CMLs displayed calcium-dependent and magnesium-independent conformational changes that expose hydrophobic residues, but the degree of hydrophobic exposure was markedly less than that observed for CaM. Isothermal titration calorimetry indicated two and three calcium-binding sites for CML15 and CML16, respectively, with affinities expected to be within a physiological range. Both CML15 and CML16 bound calcium with high affinity in the presence of excess magnesium. Promoter-reporter analysis demonstrated that the CML16 promoter is active across a range of Arabidopsis tissues and developmental stages, whereas the CML15 promoter activity is very restricted and was observed only in floral tissues, specifically anthers and pollen. Collectively, our data indicate that these CMLs behave biochemically like calcium sensors but with properties distinct from CaM and likely have non-overlapping roles in floral development. We discuss our findings in the broader context of calcium sensors and signaling in Arabidopsis.

Calmodulin-Like (CML) Gene Family in Medicago truncatula: Genome-Wide Identification, Characterization and Expression Analysis

“…CMLs regulate downstream targets in response to various stimuli-induced Ca 2+ fluctuations and signal transduction [ 14 ]. In Arabidopsis , CML15 and CML16 show changes in electrophoretic mobility in the presence of Ca 2+ , which confirms their ability to bind Ca 2+ [ 15 , 16 ]. The roles of CMLs in plant development and stress responses were investigated [ 17 , 18 , 19 ].…”

Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective

“…In the case of CML14, the combination of ITC and NMR techniques has allowed for the demonstration that only one EF-hand can sense Ca 2+ ions, despite the presence of three EF-hand motifs [ 34 ]. Moreover, ITC analysis demonstrated that CML15 and CML16 contain only two and three functional Ca 2+ -binding sites, respectively, out of the four predicted EF-hands [ 35 ]. Therefore, caution should be used in functional predictions based only on sequence analysis ( Table 1 ).…”

“…Therefore, caution should be used in functional predictions based only on sequence analysis ( Table 1 ). In the absence of experimental 3D structures, homology models can be exploited as an alternative to support the presence of functional Ca 2+ -binding sites, as has been done for CML14 [ 34 ], CML15, and CML16 [ 35 ].…”

“…ITC analysis has underscored that Arabidopsis CMLs have a wide range of affinity for Ca 2+ (nM-µM range) [ 30 , 31 , 33 , 35 ]. Thus, the Ca 2+ signaling system might be endowed with greater flexibility as a consequence of the different Ca 2+ binding affinities of the various isoforms of CML.…”

“…Arabidopsis CMLs show heterogeneous behavior towards Mg 2+ ion binding: in CML19 [ 32 ] none of the four EF-hands can bind Mg 2+ , while in CML14 [ 34 ], CML15, and CML16 [ 35 ] the weaker affinity for Ca 2+ in the presence of Mg 2+ indicates that this cation can compete directly for Ca 2+ binding [ 26 , 42 , 43 , 44 , 45 ], thereby reducing the affinity for Ca 2+ by 5-10 fold. For CML16 [ 35 ], Mg 2+ binding seems to impede the binding of Ca 2+ to at least one EF-hand. Mg 2+ also affects the affinity for Ca 2+ in CML36 which possesses two Ca 2+ /Mg 2+ mixed sites with high affinity and two Ca 2+ -specific sites with low affinity [ 33 ].…”