Article Figures & Data
Figures
- Fig. 1.
ECA1 behaves like a transporter with multi-cation specificity. A through C, Ca2+, Mn2+, and Zn2+ stimulated phosphoprotein formation in membranes of ECA1 transformants. [32P]ATP (300 nm) was added to a 2-mL reaction mixture containing 25 mmHEPES-1,3-bis(tris[hydroxymethyl]methylamino) propane (BTP; pH 7.0), 100 mm KCl, and 80 μg of vesicle with 0.5 mm EGTA alone (C, ▪ and ■) or in the presence of 5 μm divalent cations. The final free concentration of the cations was estimated from total cation added in the presence of 0.5 mm EGTA using the Maxchelator program. Aliquots were sampled, and unlabeled ATP was added at 120 s (arrow) to a final concentration of 1 mm. Black and white symbols represent [32P] phosphoprotein level before and after addition of ATP, respectively. The data are from the average of two experiments. A, Ca2+, ●; Mn2+, ▴; Cd2+, ▪. B, Zn, ●. C, Ni2+, ▴. D,45Ca transport into membrane vesicles of ECA1 transformants is blocked by Mn. ATP-dependent45Ca (approximately 0.6 μm at 0.3 μci mL−1) uptake at 5 min was measured without EGTA in the presence of Mn2+ as indicated. Pump activity shown is the difference in uptake by vesicles of mutants transformed with ECA1 and that of vector alone. Average of two experiments.
- Fig. 2.
ECA1, but not ACA2, expression restored growth of K616 mutant on Mn2+-supplemented medium. Wild-type (W303) or K616 (pmr1 pmc1 cnb1) yeast cells were transformed with control vector (p426 Gal1) alone. K616 cells were transformed with pECA1 or pACA2-2. Each transformant was diluted with complete synthetic medium (SC)-uracil (URA)/Gal medium to a density atA 600 of 1.0, 0.1, 0.01, or 0.001. Then, 10 μL of each dilution was dotted on SC-URA/Gal media (control), or with 1 mm MnCl2 or 3 mm ZnCl2, and incubated for 3 d at 30°C.
- Fig. 3.
Position of the T-DNA insert in eca1-1mutant. A, T-DNA inserts in the 10th transmembrane (TM) domain of ECA1 protein. Position 1,025 marks the last amino acid of ECA1 ineca1-1 mutant corresponding to wild-type ECA1. B, PCR analysis showing the absence of a wild-type ECA1 gene in a homozygouseca1-1 mutant plant. Genomic DNA samples from a wild-type plant (Wt) or eca1-1 homozygous plant (Mutant) were used as templates in a PCR reaction using a primer pair that can amplify the entire ECA1 coding sequence (5′ + 3′), or a primer pair that amplifies the sequence between the T-DNA left border and the 5′ end ofECA1 (5′ + TL). A picture of a UV-illuminated ethidium bromide-stained gel is shown.
- Fig. 4.
Growth of the eca1-1 mutant is inhibited by high Mn2+ and is rescued by an ECA1 transgene. Seeds of wild-type plant (ECA1), mutant (eca1-1), and mutants expressing wild-type ECA1 (35S-#6 and 35S-#7) were germinated on a 0.8% (w/v) agarose plate with 0.5× Murashige and Skoog. Five-day-old seedlings were transferred to 0.5× Murashige and Skoog medium alone (control, −Mn) or supplemented with 0.5 mm Mn2+ (+Mn). A, Plant morphology and size 12 d after transfer. B, Fresh weight of 25 plants 10 d after transfer (±se,n = 4). C, Root hairs of plant 12 d after transfer. D, Chlorophyll content from seedlings 10 d after transfer. Average (±se) from four extractions. Bar = 1 mm.
- Fig. 5.
eca1-1 (−/−) (homozygous) mutant grew poorly on low Ca2+. Seeds were germinated on a 0.8% (w/v) agarose plate with 0.5× Murashige and Skoog salts at pH 5.8. Seedlings (5 d old) of similar size were transferred to medium containing reduced Ca2+ (0.2 mm) and grown for 10 d at 21°C. Plants are representative of mutant (eca1-1) and wild type (ECA1) from three independent experiments.
- Fig. 6.
Reduction of ECA1 protein and Ca2+ pumping in the eca1-1 (−/−) mutant. A, ECA1-like protein is found in all organs of wild-type plants. Total protein extracts (10-μg samples) from roots (R), leaves (L), flowers (F), and siliques (S) were subjected to SDS-PAGE (8% [w/v] gel), transferred to nitrocellulose, and probed with pre-immune or anti-ECA1(C) serum (1:1,000 [v/v]). Secondary antibody (1:5,000 [v/v]) from donkey anti-rabbit IgG was conjugated with horseradish peroxidase, and activity was detected using enhanced chemiluminescence (Amersham-Pharmacia Biotech, Uppsala). The arrow marks the expected position of ECA1 (116 kD). B, ECA1 protein content in mutant and transgenic plants. Microsome (25 μg of protein) from 1-week-old seedlings of wild-type (ECA1), mutant (eca1-1), and transgenic mutants expressing ECA1 (35S#6 and 35S#7) were separated by SDS-PAGE (10% [w/v] acrylamide), transferred, and probed with rabbit anti-ECA1 (M; 1:1,000 [v/v]). Secondary antibodies (1:5,000 [v/v]) were linked to alkaline phosphatase. Arrow marks expected size (116 kD) of ECA1. C, Ca2+ transport. Assay mixture consisted of 1 mm ATP and 10 μm Ca in the absence of EGTA, and membrane from 1-week-old seedlings of wild type (ECA1) or mutant (eca1-1). Pump activity shown is the difference between uptake at 30 min with and without Mg2+. When added, CPA was 100 nmol mg−1 protein. ΔCPA, Pump activity sensitive to CPA. Total, Activity without CPA. Average of two experiments.
Tables
Medium Plant Ca Mn Mg Fe Zn μg/g dry wt−1 −Mn Wild 7,796 338 2,952 323 247 eca1-1 6,913 295 2,644 253 200 +Mn Wild 7,352 2,045 2,617 272 303 eca1-1 6,170 1,830 2,203 275 202 Five-day seedlings of wild-type plant (ECA1) and mutant (eca1-1) were grown in liquid medium with 0.5× Murashige and Skoog alone (−Mn) or supplemented with 0.5 mM Mn2+(+Mn) for 2 weeks. Ion content of whole plants was analyzed by ICP emission spectrometry. Average of two to three experiments.
