Plant Calreticulins -Calcium-binding proteins with many functions

Abstract: Endoplasmic reticulum (ER) plays an important role in protein synthesis, folding, maturation and transport of newly synthesized proteins, as well as the regulation of cellular Ca2+ homeostasis. A protein that is involved in several of these functions in the ER is calreticulin (CRT), which is a Ca2+-binding chaperone that resides in the lumen of the ER. Animal CRT is a multifunctional protein involved in numerous cellular functions, such as protein folding, Ca2+ binding, regulation of gene expression, apoptosis and cell adhesion. Much less is known about CRTs in plants. Three Arabidopsis CRT isoforms have been identified and can be divided into two distinct CRT groups; CRT1a/1b and CRT3. To increase the understanding of plant CRTs, functional characteristics and expression profiles were examined. The regulation of cellular Ca2+ homeostasis was investigated using tobacco (Nicotiana tabacum) suspension cell lines with altered levels of maize CRT. An increased production of CRT in tobacco cell lines enabled growth in high calcium medium. Expression studies of Arabidopsis CRTs showed an overlapping expression and localization of CRT1a and 1b in floral tissues, leaves, elongating root cells, expanding cotyledons and root cap, while CRT3 was mainly expressed in cauline and wilting leaves, expanding cotyledons, elongating root cells and with no expression in pollen and root cap. For functional studies of plant CRTs, AtCRT1a and AtCRT3, respectively, were introduced into CRT-deficient (crt-/-) mouse embryonic fibroblasts. As a result of CRT-deficiency the mouse crt-/- fibroblasts have decreased levels of Ca2+ in the endoplasmic reticulum and impaired protein folding abilities. Both plant isoforms rescued these phenotypes, that is they were able to restore Ca2+-holding capacity and chaperone functions in the ER of the mouse crt-/- fibroblasts. This demonstrates that the animal sorting machinery was functional also with the plant CRTs, and that basic CRT functions are conserved across the Kingdoms. To understand the role of CRT in planta, T-DNA insertion mutants were collected for all three genes. Atcrt1a exhibited a conditional phenotype under tunicamycin stress, while disruption of AtCRT1b caused a constitutive ER stress. The double mutant Atcrt1a crt1b showed retarded growth in both normally-grown and etiolated seedlings as well as lower seed production. The Atcrt3 mutant was less sensitive to tunicamycin and CRT3 cDNA did not complement the Atcrt1b mutant, while CRT1a cDNA rescued the Atcrt1b mutant. These studies show that AtCRT1a and AtCRT1b can compensate for each other, although similar expression patterns was observed between CRT1a and 1b, cell-specific localization differences was also observed with specific antibodies. The differences observed between the CRT1 and CRT3 isoform groups suggest different physiological roles for AtCRT3 compared to AtCRT1a and 1b.