Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize

Abstract

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated from a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca2+ storage protein located mainly in the lumen of the endoplasmic reticulum but also in the nucleus and/or cytoplasm of some cells. A differential screening between cDNA libraries originating from 104 in vitro zygotes (18 h after in vitro fertilization) and 128 unfertilized egg cells was performed to isolate newly expressed genes or genes expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly correlated to cell division. Sequence comparison to a shorter maize calreticulin cDNA isolated from a PCR based cDNA library construction from a few plant cells [12]. It is further shown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pI 4.17) of 48 kDa sharing 77-92% and 50-54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represents to our knowledge the first cDNA clone isolated from a RT/PCR cDNA library originating from only a few plant cells and is the first gene isolated from zygotes of higher plants.

Integrated Calcium Signaling in Plants

“…Plant CRT shares the same structural domain features and basic functions identified for animal CRTs; therefore, the plant CRT might also be involved in regulation of Ca 2+ homeostasis and Ca 2+ -dependent signal pathways (Wyatt et al 2002) . Plant CRT is highly expressed during mitosis in tobacco (Denecke et al 1995) , embryogenesis of barley (Chen et al 1994) , Nicotiana plumbaginifolia (Borisjuk et al 1998) , and maize (Dresselhaus et al 1996) , and in flower tissues, including pollen tubes as well as anthers (Nardi et al 2006) . Increasing evidence also indicates that this protein is involved in the plant response to a variety of stress-mediated stimuli.…”

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

“…In animals, the protein has demonstrated many regulatory functions, such as protein processing, inner cellular Ca 2+ balance regulation, cell adhesion and control of expression of steroid-sensitive genes and apoptosis. Calreticulin genes were initially isolated from barley (Chen et al, 1994) and spinach (Menegazzi et al, 1993), additional calreticulin cDNA sequences were subsequently cloned from tobacco (Denecke et al, 1993), corn (Kwiatkowski et al, 1995, Dresselhaus et al, 1996, Chinese cabbage (Beassica pekinensis) (Lim et al, 1996), Arabidopsis (Nelson et al, 1997), rice (Li and Komatsu, 2000), pea (Pisum sativum) and Ginkgo biloba.…”

BiP and Calreticulin Form an Abundant Complex That Is Independent of Endoplasmic Reticulum Stress

“…Several reticuloplasmins have been identified in plants, including the binding protein BiP (Denecke et al, 1991;Fontes et al, 1991;Anderson et al, 1994), calreticulin (Chen et al, 1994;Dresselhaus et al, 1996), protein disulfide isomerase (PDI; Shorrosh and Dixon, 1991), and endoplasmin (Walther-Larsen et al, 1993;Denecke et al, 1993. Endoplasmin has homology with the cytosolic heat shock protein Hsp90 family, but no definitive function has been proposed.…”

“…Several reticuloplasmins are now recognized as molecular chaperones (Gething and Sambrook, 1992;Hartl, 1996), which prevent the aggregation of unfolded and partially folded polypeptides, thus increasing the yield but not the rate of correct folding and assembly. Knowledge of the binding specificity of chaperones is rapidly expanding; however, mechanistic details of how the binding of the chaperone results in the unfolding of the protein followed by refolding and release from the chaperone remain to be established.Several reticuloplasmins have been identified in plants, including the binding protein BiP (Denecke et al, 1991;Fontes et al, 1991;Anderson et al, 1994), calreticulin (Chen et al, 1994;Dresselhaus et al, 1996), protein disulfide isomerase (PDI; Shorrosh and Dixon, 1991), and endoplasmin (Walther-Larsen et al, 1993;Denecke et al, 1993. Endoplasmin has homology with the cytosolic heat shock protein Hsp90 family, but no definitive function has been proposed.…”