Abstract
Calcium (Ca(2+)) is a key second messenger in eukaryotes where it regulates a diverse array of cellular processes in response to external stimuli. An important Ca(2+) sensor in both animals and plants is calmodulin (CaM). In addition to evolutionarily conserved CaM, plants possess a unique family of CaM-like (CML) proteins. The majority of these CMLs have not yet been studied, and investigation into their physical properties and cellular functions will provide insight into Ca(2+) signal transduction in plants. Here we describe the characterization of CML42, a 191-amino acid Ca(2+)-binding protein from Arabidopsis. Ca(2+) binding to recombinant CML42 was assessed by fluorescence spectroscopy, NMR spectroscopy, microcalorimetry, and CD spectroscopy. CML42 displays significant alpha-helical secondary structure, binds three molecules of Ca(2+) with affinities ranging from 30 to 430 nm, and undergoes a Ca(2+)-induced conformational change that results in the exposure of one or more hydrophobic regions. Gene expression analysis revealed CML42 transcripts at various stages of development and in many cell types, including the support cells, which surround trichomes (leaf hairs) on the leaf surface. Using yeast two-hybrid screening we identified a putative CML42 interactor; kinesin-interacting Ca(2+)-binding protein (KIC). Because KIC is a protein known to function in trichome development, we examined transgenic CML42 knockout plants and found that they possess aberrant trichomes with increased branching. Collectively, our data support a role for CML42 as a Ca(2+) sensor that functions during cell branching in trichomes.
Highlights
Sponses appropriate for the perceived stimulus [1]
The results shown represent GUS-staining patterns in and overall these observations are consistent with the sequence- at least five independent transgenic Arabidopsis lines. based prediction that NtermCML42 comprises only one Ca2ϩ- CML42::GUS was expressed at the hypocotyl-root interface binding site (Fig. 1) [9]
CML42 displays characteristics reminiscent of a relay Ca2ϩ sensor, including the physical properties associated with Ca2ϩinduced conformational changes and Ca2ϩ affinity
Summary
CaM, calmodulin; CML, calmodulin-like; KIC, kinesin-interacting calcium-binding protein; ITC, isothermal titration calorimetry; ANS, 8-anilino-1-naphthalenesulfonate; HSQC, heteronuclear single-quantum coherence; KCBP, kinesin-like calmodulin-binding protein; GUS, -glucuronidase; TnC, troponin C; GST, glutathione S-transferase. We examined the biochemical properties of CML42 as a Ca2ϩ sensor and identified a role for CML42 in trichome morphology. Trichomes (leaf hairs) are specialized, epidermal cells that are often branched in many plant species and are thought to play a variety of roles, including herbivore deterrence, UV irradiation protection, and reduction in transpiration (water loss) [14, 15]. Due to their uniquely branched structure, trichomes are often studied as a model for cell morphology in plants. The present study provides new insight into the biochemical properties of CML42 and demonstrates a novel function for this protein in the Ca2ϩ-mediated regulation of trichome morphology
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