title: Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds.
abstract: Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix. CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5' and 3' untranslated regions. Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide. However, the three proteins activated in the same way the CaM-dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.
authors.affiliation: UMR 1191 Physiologie Moléculaire des Semences, LRPV, 16 bd Lavoisier, 49045 Angers Cedex 01, France.
authors.forename: Frédéric D
authors.initials: FD
authors.lastname: Duval
authors.forename: Michelle
authors.initials: M
authors.lastname: Renard
authors.forename: Michel
authors.initials: M
authors.lastname: Jaquinod
authors.forename: Valérie
authors.initials: V
authors.lastname: Biou
authors.forename: Françoise
authors.initials: F
authors.lastname: Montrichard
authors.forename: David
authors.initials: D
authors.lastname: Macherel
mesh_terms: D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D018506:Gene Expression Regulation, Plant; D018525:Germination; D008958:Models, Molecular; D005810:Multigene Family; D009154:Mutation; D018532:Pisum sativum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D002147:Calmodulin; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins