The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast

Abstract

Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress. We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase. Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles. The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616. ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain. An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect.

Figure 1

Sequence alignment of deduced amino acid sequences of Arabidopsis Ca²⁺-ATPase isoform ACA4 with ACA2 and cauliflower BCA1. Sequences were aligned by the Jotun-Hein method (PAM250 comparison) using the MEGALIGN program (DNAstar, Inc., Madison, WI). Identical residues are shaded; gaps introduced to maximize alignment are denoted by hyphens. The 10 putative transmembrane domains (M1–M10) predicted using the SOAP programs are overlined. Double lines indicate a putative CaM-binding domain (CBD). PKC indicates a sequence recognition motif for protein kinase C. The ACA4 cDNA sequence reported here has the GenBank accession number AF200739 and is identical to the extracted sequence of the genomic clone (AC002510).

Figure 2

The N-terminal domain of ACA4 binds CaM. A, The purified fusion protein ACA4N27 (covering K₁₃–P₁₃₉ of the ACA4 N terminus; lane 1) or the GST protein (lane 2) was blotted onto nitrocellulose and incubated with ¹²⁵I-CaM in the presence of 500 μm CaCl₂ (+Ca) or 2 mm EGTA (−Ca). After washing, blots were exposed to a phosphor-imager. B, Forty nanomolar dansyl-CaM was titrated with equimolar amounts of fusion protein (N_t) in the presence of 333 μm CaCl₂ (+Ca) or 2.5 mm EGTA (−Ca). Fluorescence intensity was measured by a 400- to 550-nm scan upon excitation at 340 nm in a fluorescence spectrophotometer.

Figure 3

ACA4 is localized to internal membranes. A, To determine the intracellular localization of ACA4, microsomal fractions obtained from aqueous two-phase partitioning of root microsomes were probed with anti-ACA4N27. Efficient partitioning of the microsomes (M) to the lower (L) or to the upper (U) phase was ascertained by western blots using marker antisera against subunit B of the vacuolar V-ATPase (Manolson et al., 1988) and the plasma membrane-bound H⁺-pump isoform AHA3 (Pardo and Serrano, 1989). B, Arabidopsis root microsomes subjected to Suc density gradient fractionation. Each fraction was immunoprobed with anti-ACA4N27, anti-V-ATPase (vacuolar membrane marker; Ward et al., 1992), BIP (ER marker), and anti-AHA3 (plasma membrane marker, see above). In addition, chlorophyll and Suc in the fractions were quantified.

Figure 4

ACA4-GFP fluorescence is detected in small vacuoles in transiently transformed protoplasts. A, ACA4-targeted GFP fluorescence detected under blue light excitation is found in small vacuoles (sV) of transformed Arabidopsis protoplasts. B, The same sample of protoplast detected under bright field. C, GFP fluorescence of protoplasts expressing Δ47-ACA4-GFP is found in vacuoles of similar size and distribution. D, Fluorescence of transformed protoplasts immunoprobed with antisera directed against radish (Raphanus sativus) γ-TIP microscopy labeling the membrane of the large vacuole (V) and of small vacuoles (sV). E, In a minor fraction of protoplasts, imaging of ACA4-GFP revealed ER-like structures. F, Confocal fluorescence image of protoplast expressing GFP alone. Arabidopsis protoplasts were transformed with plasmids p35S-ACA4-GFP, p35S-Δ47-ACA4-GFP, and p35S-GFP-JH2 encoding for full length and an N-terminally truncated ACA4 fused to a C-terminal GFP, and for a GFP-only control, respectively. Confocal laser scanning images were recorded after 24 h and imaged for GFP (A–C, E–F) and lissamine rhodamine fluorescence-conjugated secondary antibody (D). The stored images were colored as green (GFP) or red (lissamine rhodamine) images using Adobe PhotoShop 5.5 (Adobe Systems, Mountain View, CA). Images were evaluated from hundreds of cells obtained from three independent experiments showing similar results. V, Large (central) vacuole; sV, small vacuoles; N, nucleus. Scale bars are 10 μm.

Figure 5

ACA4 expression is throughout the plant and is induced by salt stress. A, ACA4-specific transcripts (ACA4) were monitored in total RNA from different tissues by RT-PCR amplification of a 561-bp cDNA fragment. To circumvent problems with genomic DNA contamination, primers for ACA4 were chosen to cover an intron-spanning region (intron 3). As an internal control, RT-PCR on the actin Aac1 gene was employed. Control RT-PCR amplification yielded essentially constant signals (<10% deviation). B, Changes in ACA4-specific mRNA levels upon salt treatment were detected by semiquantitative RT-PCR. Arabidopsis seedlings were treated with different amounts of NaCl for 24 h, and amplified cDNA sequences were separated and quantitated. Relative intensities of the PCR bands are given in percentages as compared to control plants (no NaCl).

Figure 6

An N-terminally modified form of ACA4 complements a yeast strain devoid of Ca²⁺-ATPases. Triple mutant K616 (Pmr1 Pmc1 Cnb1) strain was transformed with control vectors pYES2 (vector control), pYES2-ACA4 (ACA4), or pYES2-Δ44ACA4 (Δ44-ACA4), respectively. Single colonies were streaked out on SC-Ura/Gal or SC-Ura/Glu plates containing 10 mm Ca²⁺ (+ calcium) or 10 mm EGTA, pH 5.5 (− calcium) and incubated for 3 d at 30°C. In the presence of Gal only the N-terminal truncated ACA4 was able to provide growth of strain K616 on EGTA that was comparable to wild-type strain K601, whereas the full-length enzyme was not.

Figure 7

Effect of salinity on growth of yeast strain K616 complemented by ACA4. Triple mutant K616 (Pmr1 Pmc1 Cnb1) strain was transformed with control vectors pYES2, pYES2-PMC1 (PMC1), pYES2-ACA4 (ACA4), and pYES2-Δ44ACA4 (Δ44-ACA4), respectively. For drop tests, mid-log precultures in SC-Ura plus 10 mm CaCl₂ were pelleted, washed twice, and an optical density at 600 nm of 1.0 in water was obtained by dilution. Cells were diluted 10-fold and each 5 μL was spotted on SC-Ura/Gal plates supplemented with different EGTA, KCl, NaCl, or mannitol concentrations. Growth was recorded after 4 d of incubation at 30°C.