The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants

Abstract

To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

Figure 1

CRT and BiP, an ER-localized chaperone, show similar distribution on Suc gradients. Microsomes were isolated from the wild-type cell line and layered onto a discontinuous Suc gradient (45%, 38%, and 22% [w/v] Suc). One-milliliter fractions were collected and equal amounts of protein were analyzed by 10% (w/v) SDS-PAGE (10 μg protein lane⁻¹). The bottom and top of the gradient are indicated. A, Equal amounts of protein from collected gradient-fractions of a wild-type cell line, visualized with Gelcode staining. B, Immunostaining of gradient fractions from a wild-type cell line with polyclonal antibodies against maize CRT (1:5,000) and polyclonal antibodies against BiP (1:10,000), an ER marker (Denecke et al., 1991). C, Comparison of CRT in ER-enriched fraction 2 from gradient-fraction of wild-type and CRT overproducing cell line 7 (Nt CRT:7). Gelcode-stained gel is on the left and immunostained western blot is on the right. Lane 1, Heat-shocked wild type; Lane 2, heat-shocked CRT sense line 7 (Nt CRT:7). Migrations of standards and CRT are indicated.

Figure 2

Altered CRT expression in ER-enriched vesicles from transgenic cell lines. ER-enriched vesicles from CRT, GFP transgenic, and wild-type cell lines were separated on Suc gradients. Fraction 2, containing ER-enriched membrane vesicles (Fig. 1), was analyzed by 10% (w/v) SDS-PAGE (15 μg protein lane⁻¹), blotted, and immunostained with polyclonal antibodies against maize CRT (1:5,000). A, Increased production of CRT in CRT sense lines. Lane 1, Non-heat-shocked wild type. Lane 2, Heat-shocked wild type. Lane 3, Non-heat-shocked CRT sense line (Nt CRT:7). Lane 4, Heat-shocked CRT sense line (Nt CRT:7). B, Lowered production of CRT in CRT antisense lines. Lane 1, Non-heat-shocked wild type. Lane 2, Heat-shocked wild type. Lane 3, Non-heat-shocked antisense CRT (Nt CRT-A:3). Lane 4, Heat-shocked antisense CRT (Nt CRT-A:3). C, No significant changes in CRT production in mgfp5 transgenic cell lines (Nt GFP:4). Lane 1, Non-heat-shocked wild type. Lane 2, Non-heat-shocked Nt GFP:4 transgenic line. Lane 3, Heat-shocked wild type. Lane 4, Heat-shocked Nt GFP:4 transgenic line. D, Expression of a maize CRT in the transformed line Nt CRT:7. Lane 1, Purified maize CRT. Lane 2, Heat-shocked Nt CRT:7. Lane 3, Non-heat-shocked wild type. Lane 4, Standard. E, Protein visualized with silver staining. Lane 1, Purified maize CRT. Lane 2, Heat-shocked Nt CRT:7. Lane 3, Non-heat-shocked wild type. Lane 4, Standard.

Figure 3

Increased levels of CRT increase Ca²⁺ uptake capacity in vitro. ER-enriched membrane vesicles (fraction 2, Fig. 1), were obtained from CRT transgenic, Nt GFP:4 transgenic, and wild-type cell lines as indicated in “Materials and Methods.” ATP-dependent ⁴⁵Ca²⁺ uptake was performed on the ER-enriched vesicles (see “Materials and Methods”). The ATP-dependent Ca²⁺ uptake was measured in presence and absence of 3 mm ATP, and is shown as Δ ATP. Black symbols denote heat-shocked cells and white symbols denote non-heat-shocked cells. A, ER-enriched membrane vesicles from CRT overproducing (triangles), wild-type (squares), and CRT antisense (circle) cell lines were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹ see “Materials and Methods”). The ⁴⁵Ca²⁺ recovered was 11.1 ± 0.7 nmol ⁴⁵Ca²⁺ mg protein⁻¹ at 20 min for heat-shocked wild type. B, ER-enriched membrane vesicles from GFP expressing (triangles) and wild-type (squares) cell lines were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹). Data (mean of two values ± the range) are shown from one experiment. The experiment has been repeated at least three times with consistent results. The increase in ⁴⁵Ca²⁺ uptake in the CRT sense lines compared with heat-shocked wild type was 2.0 ± 0.3-fold (mean ± sd) for three experiments.

Figure 4

CRT affects the amount of released and retained ER Ca²⁺ after treatment with ionomycin in vitro. ER-enriched membrane vesicles (fraction 2, Fig. 1), were collected from CRT transgenic, Nt GFP:4 transgenic, and wild-type cell lines. Membranes were incubated with ATP in the presence of ⁴⁵Ca²⁺ for 22 min (see “Materials and Methods”). The Ca²⁺ ionophore ionomycin (1.5 μm) was added and membrane vesicles were analyzed for ⁴⁵Ca²⁺ after 5 min. White bars, vesicles from non-heat-shocked cells; black bars, vesicles from heat-shocked cells. A, ER-enriched vesicles from CRT over- and under-producing cell lines were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹), and compared with wild type. Data are shown as amount ⁴⁵Ca²⁺ released after addition of ionomycin. The heat-shocked CRT sense lines showed a 2 ± 0.5-fold higher amount of released ⁴⁵Ca²⁺ than heat-shocked wild type (8.1 ± 1.8 nmol ⁴⁵Ca²⁺ mg protein⁻¹ for heat-shocked wild type, and 16 ± 1.6 nmol ⁴⁵Ca²⁺ mg protein⁻¹ for the CRT sense line). B, ER-enriched vesicles from the Nt GFP:4 cell line were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹), and compared with wild type. Data are shown as amount ⁴⁵Ca²⁺ released after addition of ionomycin. C, ER-enriched vesicles from CRT over- and under-producing cell lines were assayed for ATP- dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹), and compared with wild type. Data are shown as amount ⁴⁵Ca²⁺ associated with the ER vesicles after ionomycin treatment. The CRT sense lines had a 2.5- ± 1-fold higher amount of retained ⁴⁵Ca²⁺ after ionomycin treatment, compared with heat-shocked wild type (2.9 ± 1.0 nmol ⁴⁵Ca²⁺ mg protein⁻¹ for heat-shocked wild type, and 8.2 ± 1 nmol ⁴⁵Ca²⁺ mg protein⁻¹ for the CRT sense line). D, ER-enriched vesicles from Nt GFP:4 cell line were assayed for ATP-dependent ⁴⁵Ca²⁺ uptake (10 μg protein aliquot⁻¹), and compared with wild type. Data are shown as amount ⁴⁵Ca²⁺ associated with the ER vesicles after ionomycin treatment. Data are shown from one experiment. All experiments have been repeated at least three times with similar trends.

Figure 5

Increased transient expression of CRT decreases chlorosis of Arabidopsis plants transferred to calcium-depleted medium. Seeds from At CRT:3 and At CRT-A:5 were germinated on nutrient medium. Sixteen days after germination, plants were incubated at 35°C for 2 h (heat shock) and allowed to recover at 21°C overnight. The heat shock procedure was repeated for 3 consecutive d. Heat-shocked plants were either placed on fresh calcium-depleted medium on d 4 or harvested, homogenized, and analyzed by 10% (w/v) SDS-PAGE (2.5 μL homogenate lane⁻¹). Proteins were blotted and immunostained with polyclonal antibodies against CRT. A, Immunostaining of CRT in homogenized transgenic plant lines with polyclonal antibodies against maize CRT (1:5,000). Lanes 1 through 5, Non-heat-shocked plants; Lanes 6 through 10, heat-shocked plants. Lanes 1 and 6, At CRT:7; lanes 2 and 7, At CRT:3; lanes 3 and 8, At CRT-A:3; lanes 4 and 9, At CRT-A:4; lanes 5 and 10, At CRT-A:5. B, Immunostaining of CRT in homogenized At CRT:3 with polyclonal antibodies against castor bean CRT (1:10,000). Lane 1, Non-heat-shocked At CRT:3. Lane 2, At CRT:3 heat shocked 1 d. Lane 3, At CRT:3 heat-shocked 2 d. Lane 4, At CRT:3 heat-shocked 3 d. Lane 5, Standard. C, Total protein visualized with Gelcode staining. Lanes as indicated in B. D, Upper, photographs show non-heat-shocked and heat-shocked wild type (left), At CRT:3 (center), and At CRT-A:5 (right) plants 16 h after induction; lower, photographs show a non-heat-shocked and heat-shocked wild type (left), At CRT:3 (center), and At CRT-A:5 (right) plants transferred after induction to calcium-depleted medium (Arabidopsis [AT] medium containing 10 mm EGTA) for 9 d. Twelve seedlings for each transgenic line and medium treatment were germinated, one-half were induced for transgene expression, and one-half were maintained as non-induced controls. (Note: the wild-type plants shown were from a separate experiment.) The experiment has been repeated three times with similar results. In subsequent experiments, 20 to 50 seedlings for each line were assessed. In all experiments, a minimum of 80% of the plants showed the phenotypes represented.