P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Abstract

Arabidopsis thaliana has eight genes encoding members of the type 1(B) heavy metal-transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.

Figure 1.

Mutant Alleles of HMA2, HMA3, and HMA4. (A) A schematic illustration of a composite HMA gene. Boxes indicate exon coding sequences and connecting lines indicate introns. Exon and intron sizes are approximately the same for all three genes except that the first two introns and last exon vary considerably in size as indicated by a jagged line. The single base pair deletion polymorphism in HMA3 between Ws and Col and the relative position of each T-DNA insertion are shown. Arrows indicate T-DNA orientation, with the arrowhead corresponding to the left border. Details of the left border flanking sequences are shown in Table 1. (B) RT-PCR products obtained using HMA2, HMA3, HMA4, and actin (ACT2) gene-specific primers and total RNA isolated from hma2-2 (2), hma3-1 (3), and hma4-1 (4) plants. W indicates a gene-specific fragment amplified from Ws genomic DNA using the same primers. Fragment sizes (bp) are indicated.

Figure 2.

Phenotype of hma2 hma4 Double Mutant Plants. (A) Twenty-eight-day-old plants from a population (left) homozygous hma4-1 and segregating for hma2-2 with a homozygous hma2-2 hma4-1 individual shown in detail (right). (B) Fifty-four-day-old hma2-2 hma4-1 individual showing multiple aborted bolts. (C) Inflorescences of wild-type (left) and hma2-2 hma4-1 mutant (right) plants. (D) Pistils and anthers of wild-type (left) and hma2-2 hma4-1 mutant (right) flowers with insets showing dehiscing anthers.

Figure 3.

hma2 hma4 Double Mutant Phenotype Can Be Rescued by Zn. (A) Twenty-eight-day-old homozygous hma2-2 hma4-1 plants grown in soil watered with tap water (right, −Zn) or 1 mM ZnSO₄ (left, +Zn). (B) Twenty-one-day-old wild-type and hma2-2 hma4-1 plants grown on agar mineral salts medium from which Zn was omitted (top, −Zn) or containing 10 μM Zn (bottom, +Zn).

Figure 4.

Zn Accumulation in Mutant and Wild-Type Plants. (A) and (B) Zn (A) and Cu (B) levels in shoots of 21-d-old wild-type, hma2-2, hma4-1, and hma2-2 hma4-1 plants grown on agar mineral salts medium from which Zn was omitted (left) or containing 10 μM Zn (right). Values are the mean of 6 ± se. Significant differences from the wild type as determined by Student's t test are indicated by one asterisk (P < 0.05) and two asterisks (P < 0.001). (C) Zn levels in rosettes of 26-d-old wild-type and hma2-2 hma4-1 plants grown in soil irrigated with water or 1 mM ZnSO₄. (D) Zn levels in stems and inflorescences of 32-d-old wild-type and hma2-2 hma4-1 plants grown in soil irrigated with 1 mM ZnSO₄. (E) Zn levels in roots and shoots of 29-d-old wild-type and hma2-2 hma4-1 plants grown in hydroponic medium from which Zn had been omitted. Values are the mean ± se of four samples of tissue pooled from four plants per sample.

Figure 5.

HMA2p-GUS and HMA4p-GUS Expression in Transgenic Plants. (A), (C), (E), (G), (I), (K), (L), (M), (O), (Q), (S), (U), (W), (X), and (Y) HMA2p-GUS transgenic plants. (B), (D), (F), (H), (J), (L), (N), (P), (R), (T), (V), and (Z) HMA4p-GUS transgenic plants. GUS activity is indicated by blue ([A] to [V]) or red ([W] to [Z]). (A) to (D) Fourteen-day-old seedling ([A] and [B]) (bars = 5 mm) with detail of roots ([C] and [D]) (bars = 0.5 mm). (E) to (N) and (Q) to (V) Leaf ([E] and [F]) (bars = 5 mm), stem ([G] and [H]) (bars = 1 mm), and inflorescence ([I] and [J]) (bars = 0.5 mm) with developing siliques ([K] to [N]) and flowers ([Q] to [V]) of decreasing age from 5-week-old plants. Bars = 1 mm in (K) to (R) and 0.5 mm in (S) to (V). (O) and (P) Mature silique of 7-week-old plant. Bars = 1 mm. (W) Section of stem of 4-week-old plant. Bar = 10 μm. (X) Section of unopened flower. Bar = 10 μm. (Y) and (Z) Section through anther of unopened flower. Bars = 10 μm.

Figure 6.

HMA2 and HMA4 Expression in Anthers Measured by RT-PCR. PCR products were amplified using primers specific for HMA2, HMA4, and actin (ACT2) as a control using genomic DNA from Ws plants (+) or cDNA reverse transcribed from RNA isolated from immature (stage 12) anthers (IA), mature anthers (MA), and mature filaments (MF). Intensity of the cDNA band relative to ACT2 in the same sample is indicated. Fragment sizes for genomic DNA and cDNA products are indicated.

Figure 7.

Membrane Localization of HMA2 by Protein Gel Blot Analysis. (A) Protein gel blot analysis of microsomal fractions from wild type (Wt) Ws, and mutant hma2-3 plants. Equal amounts of protein (10 μg) were loaded in each lane. Sizes and positions of standards are indicated. (B) Protein gel blot analysis of plant membrane fractions from two-phase partitioning fractionation probed with antibodies that recognize HMA2, HMA2-GFP, a plasma membrane (PM)-located H⁺-ATPase, AHA2, an ER-located Ca²⁺-ATPase, ACA2, and tonoplast-located γTIP. M, total membrane proteins; U, plasma membrane–enriched upper phase proteins; L, endomembrane-enriched lower phase proteins. Equal amounts of protein were loaded in each lane. All membrane fractions were from wild-type Ws plants, except for samples probed with the anti-GFP antibody, which were from a plant line overexpressing an HMA2-GFP fusion (Figure 8). Equivalent results were obtained in two independent experiments. Approximate sizes are indicated.

Figure 8.

Membrane Localization of HMA2-GFP Fusion. (A) Confocal fluorescence image of a root tip expressing an HMA2-GFP fusion protein. (B) Detail of box shown in (A). (C) Confocal fluorescence image of a root tip expressing a GFP control. (D) Confocal fluorescence image of a root tip of a wild-type control under the same exposure settings used in (A).

Figure 9.

Cd Sensitivity of hma cad1-3 PC-Deficient Mutants. (A) Plants were grown in the presence or absence of added Cd and shoot fresh weight determined after 14 d. Values are the mean of 10 plants ± se. Significant differences from the wild type as determined by Student's t test are indicated by two asterisks (P < 0.01). (B) Plants growing in the presence of 0.06 or 0.15 μM Cd.