Phylogenetic Analyses and Expression Studies Reveal Two Distinct Groups of Calreticulin Isoforms in Higher Plants

Abstract

Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.Calreticulin (CRT) is a highly conserved protein mainly localized to the endoplasmic reticulum (ER) in plants and to the ER/sarcoplasmic reticulum in mammals (for review, see Crofts and Denecke, 1998;Michalak et al., 1999;Hadlington and Denecke, 2000;Johnson et al., 2001). CRT is a multifunctional protein, suggested to be involved in over 40 intra-and extracellular processes in mammalian cells. However, the main focus has been on its role in calcium signaling (Camacho and Lechleiter, 1995;Nakamura et al., 2001;Arnaudeau et al., 2002) and as a chaperone (Hebert et al., 1996;Saito et al., 1999;Nakamura et al., 2001). CRT comprises three major subdomains: a highly conserved N domain, a high-affinity but low-capacity Ca 2ϩ -binding P domain, and a lowaffinity but high-capacity Ca 2ϩ -binding C domain ending with an ER retention signal (Michalak et al., 1999).Although the role of CRTs as chaperone-like proteins and in calcium signaling is well established in mammals, the functions of CRT have been elusive in plants until recently. Plant CRTs have been shown to bind calcium with similar characteristics as their mammalian homologs (Chen et al., 1994;Hassan et al., 1995;Navazio et al., 1995;Coughlan et al., 1997;Li and Komatsu, 2000) and recently also to have calcium-storing functions in the ER of plant cells (Persson et al., 2001;Wyatt et al., 2002). In contrast to most animal CRTs, glycosylation of CRTs is generally observed in plants (Navazio et al., 1995(Navazio et al., , 2002Pagny et al., 2000). Plant CRTs are up-regulated in response to a variety of stress-mediated stimuli, e.g. pathogenrelated signaling molecules (Denecke et al., 1995;Jaubert et al., 2002) and gravistimulation (Heilmann et al., 2001), and are highly expressed during mitosis (Denecke et al., 1995), embryogenesis (Borisjuk et al., 1998), and in floral tissues (Chen et al., 1994;...

Molecular cloning of a new wheat calreticulin gene TaCRT1 and expression analysis in plant defense responses and abiotic stress resistance

“…Through function-known proteins in group I and II, it can be concluded that plant calreticulin protein conserved highly in its structure evolution, but with diversity in biological functions, thus promoting plant adaption abilities to environmental stresses. In addition, AtCRT3 and TaCRT in group III were identified to play roles in plant response to salt and drought stresses respectively (Persson et al, 2003;Jia et al, 2008). TaCRT1, a member of group III, is 97.2% identical to TaCRT, and so it may play certain roles in plant defense responses or stress resistance.…”

“…Wheat gene TaCRT enhanced tobacco drought tolerance whereas it was overexpressed in tobacco (Jia et al, 2008). AtCRT3 expression was inducible by high salt treatment (Persson et al, 2003). In addition, several calreticulins were found to play important roles in plant resistance to virus.…”

“…In Group II, tobacco ACH72686 and papaya CpCRT participate in plant defense responses (Lu et al, 2010;Shen et al, 2010), while tobacco NtRPL60 and caster bean AAB71420 play roles in plant growth and development (Denecke et al, 1995;Coughlan et al, 1997). In Group III, only Arabidopsis AtCRT3 and wheat TaCRT have been demonstrated to function in plant response to salt and drought stress (Persson et al, 2003;Jia et al, 2008). Therefore, the members in each group possess diverse biological functions (Figure 2).…”

The function of calreticulin in plant immunity

“…13 Phylogenetic studies revealed two distinct isoforms of CRT in higher plants. 14 Unlike the highly acidic C-domains in the AtCRT1/2 isoform and their mammalian counterpart HsCRT1 (Fig. 1B), the electrically neutral C-domain of the AtCRT3 isoform loses its high capacity Ca 2+ -binding ability.…”

Calreticulins are not all the same

“…However, in tunicamycin-treated Arabidopsis cell cultures CRT3 was induced after 30 min and returned to normal levels after 4 h, with CRT1/2 having an opposite behavior. CRT3 may therefore be specifically subjected to early UPR response (4). UPR can function by two waves of induction, and only the second one seems to be triggered by accumulation of misfolded proteins in the ER (12).…”