Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants.
Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.
Wyatt
Sarah E
SE
Department of Botany, North Carolina State University, Raleigh 27695-7612, USA.
Tsou
Pei-Lan
PL
Robertson
Dominique
D
D016960:Agrobacterium tumefaciens / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D001682:Biological Availability; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D037282:Calreticulin; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D010641:Phenotype; D030821:Plants, Genetically Modified; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D008164:Luminescent Proteins; D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins; D002118:Calcium