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;...

BackgroundCalreticulin (CRT) is a ubiquitous ER protein involved in multiple cellular processes in animals, such as protein folding and calcium homeostasis. Like in animals, plants have evolved divergent CRTs, but their physiological functions are less understood. Arabidopsis contains three CRT proteins, where the two CRTs AtCRT1a and CRT1b represent one subgroup, and AtCRT3 a divergent member.Methodology/Principal FindingsThrough expression of single Arabidopsis family members in CRT-deficient mouse fibroblasts we show that both subgroups have retained basic CRT functions, including ER Ca2+-holding potential and putative chaperone capabilities. However, other more general cellular defects due to the absence of CRT in the fibroblasts, such as cell adhesion deficiencies, were not fully restored. Furthermore, in planta expression, protein localization and mutant analyses revealed that the three Arabidopsis CRTs have acquired specialized functions. The AtCRT1a and CRT1b family members appear to be components of a general ER chaperone network. In contrast, and as recently shown, AtCRT3 is associated with immune responses, and is essential for responsiveness to the bacterial Pathogen-Associated Molecular Pattern (PAMP) elf18, derived from elongation factor (EF)-Tu. Whereas constitutively expressed AtCRT1a fully complemented Atcrt1b mutants, AtCRT3 did not.Conclusions/SignificanceWe conclude that the physiological functions of the two CRT subgroups in Arabidopsis have diverged, resulting in a role for AtCRT3 in PAMP associated responses, and possibly more general chaperone functions for AtCRT1a and CRT1b.