- © 1999 American Society of Plant Physiologists
Abstract
Calcium (Ca2+) efflux from the cytosol modulates Ca2+ concentrations in the cytosol, loads Ca2+ into intracellular compartments, and supplies Ca2+ to organelles to support biochemical functions. The Ca2+/H+ antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca2+. Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca2+ deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca2+ in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca2+ accumulation and altered activity of the tonoplast-enriched Ca2+/H+ antiporter. These results emphasize that regulated expression of Ca2+/H+ antiport activity is critical for normal growth and adaptation to certain stresses.
INTRODUCTION
Transient increases in cytosolic free calcium (Ca2+) concentrations are essential for the conversion of signals, such as red light, touch, cold shock, and pathogen infection, into adapted biological responses (Knight et al., 1991, 1996; Poovaiah and Reddy, 1993; Bowler et al., 1994; Sanders et al., 1999). Stress conditions, such as salinity, also induce cytosolic Ca2+ accumulation. During these biological responses, Ca2+ may be mobilized from the plant vacuole, an important storage compartment for Ca2+, to act as an intra-cellular signaling ion (Alexandre et al., 1990; Allen and Sanders, 1995; Barkla and Pantoja, 1996; Marty, 1999). However, little is known about the contribution of individual Ca2+ transporters in the tonoplast (vacuolar membrane) in establishing Ca2+ homeostasis during and after signal transduction events and stress responses.
Physiological data suggest an elaborate interplay between concentrations of Ca2+ and those of numerous ions within the plant. Ca2+ deficiency in plants may cause death and blackening of the growing points, but this is rarely the result of an absolute lack of soil Ca2+ (Marschner, 1995). More often, the cause is low Ca2+ concentrations in relation to other ions present in the soil. Supplemental Ca 2+ is known to mitigate the adverse effects of salinity on plant growth (LaHaye and Epstein, 1969, 1971; Epstein, 1998; Liu and Zhu, 1998). In addition, Ca2+ may be necessary for maintaining adequate K+ transport (Sussman et al., 1994). Thus, precise modulation of cytosolic to vacuolar Ca2+ ratios may be important for plants to adapt to numerous ion imbalances.
A concentration gradient of Ca2+ is established across the tonoplast by high-capacity Ca2+/H+ exchange activity (Schumaker and Sze, 1985; Blumwald and Poole, 1986). Previously, an Arabidopsis gene, CAX1 (for CALCIUM EXCHANGER 1), was identified by its ability to suppress mutants of yeast defective in vacuolar Ca2+ transport (Hirschi et al., 1996). In addition, CAX1 biochemical activities in yeast vacuoles correlate well with those described for the vacuolar Ca2+/H+ antiport activities from plant sources (Hirschi et al., 1996). A homolog of CAX1 from mung bean specifically localizes to the plant vacuole (Ueoka-Nakanishi et al., 1999). When cytosolic Ca2+ concentrations are low, the yeast vacuolar Ca2+/H+ antiporter appears to be responsible for maintaining Ca2+ homeostasis and may attenuate the propagation of Ca2+ signals (Cunningham and Fink, 1996; Miseta et al., 1999). However, the role of CAX1 in plant growth, ion homeostasis, and signal transduction is unknown.
In yeast, expression of either CAX1, the lower affinity Ca2+/H+ transporter, or CAX2 can compensate for the absence of the endogenous vacuolar Ca2+/H+ antiporter (Hirschi et al., 1996). This functional redundancy of CAX1 and CAX2 suggests that loss-of-function mutations of CAX1 in plants may not reveal a perceived phenotype because of the presence of other genes compensating for its absence. In classic genetics, the alternative to loss-of-function mutations is the creation of dominant gain-of-function mutations. Evidence suggests, however, that heterologous expression of genes from related organisms might be a more efficient and fruitful approach to the production of dominant-like gene activities. In fungi, heterologous expression of signaling molecules led to stimulus-independent, constitutively active phenotypes (Milne and Weaver, 1993; Singh et al., 1994). Similar dominant-like interactions have been documented for highly related plant orthologous myc transcription factors when overexpressed in tobacco (Payne et al., 1999), which suggests that plant-plant heterologous expression of CAX1 might be a useful and efficient way to generate constitutively active phenotypes.
Here, CAX1 gene expression in response to various stimuli is characterized. In the presence of excess Ca2+, Arabidopsis CAX1 RNA is highly expressed. Details of the growth characteristics, ion sensitivities, and chilling response of transgenic tobacco plants expressing CAX1 are also reported. The phenotypes obtained are reversible if Ca2+ is added to the plant growth media, thereby demonstrating involvement of CAX1 in Ca2+ homeostasis.
RESULTS
CAX1 Expression in Arabidopsis
Examination of the environmental stimuli that induce CAX1 expression should increase our understanding of the role this transporter plays in general ion homeostasis and stress responses. Previous reports establishing that several putative P-type Ca2+-ATPases from plants are induced by sodium (Na+) stress (Perez-Prat et al., 1992; Wimmers et al., 1992) have provided further insight into the role of Ca2+ in salt adaptation. Thus, RNA gel blot analyses were performed to determine how ion imbalances and various stresses affect CAX1 RNA accumulation. As shown in Figure 1, CAX1 RNA was moderately induced by Na+, nickel (Ni+), and osmotic stress but appeared to be highly induced (>12-fold) by Ca2+ in the media. CAX1 RNA accumulated in response to Ca2+ in a dose-dependent manner, with maximal response at the highest concentration tested (150 mM; data not shown). Given that none of the initial ion imbalances presented the same osmotic effect as 80 mM Ca2+, plants also were treated with a range of Mg2+ concentrations (10 to 150 mM) to mimic the osmotic effect of Ca2+. In these experiments, no dose-dependent induction of CAX1 RNA was observed (data not shown). The plant hormones abscisic acid, auxin, and gibberellin, at concentrations of 0.1 μM, also did not induce CAX1 expression after a 16-hr incubation (data not shown).
CAX1 RNA Expression in Response to Ion Imbalances.
RNA was isolated from whole Arabidopsis plants 16 hr after watering with various solutions (W, water; MS, nutrient media; Ca2+, 80 mM CaCl2; Cu2+, 0.1 mM CuCl2; K+, 80 mM KCl; Mg2+, 50 mM MgCl2; Na+, 80 mM NaCl; Ni2+, 0.1 mM NiCl; PEG, 10% polyethylene glycol 3350 solution (118 mOsm/kg); Zn2+, 5 mM ZnCl2). The blot was hybridized with the CAX1 cDNA. Ethidium bromide-stained rRNA before transfer is shown at bottom.
Expression of CAX1 in Transgenic Plants
Attempts to exacerbate normal calcium homeostasis in plants were made by constitutive expression of CAX1 in Arabidopsis. Transgenic plants expressing CAX1 were created by using a conventional cauliflower mosaic virus 35S promoter vector construct. CAX1 overexpression in Arabidopsis plants was expected either to attenuate endogenous CAX1 transcripts by a gene-silencing phenomenon or to exaggerate CAX1 expression. In fact, CAX1 overexpression in Arabidopsis was found by RNA gel blot analysis to augment the basal amounts of CAX1 expression. Despite the increased amounts of CAX1 RNA in these transgenic lines, no change in plant growth or development was detected (K.D. Hirschi, unpublished data). Lack of a CAX1-specific antibody, however, prevented actual quantification of alterations of CAX1 protein amounts.
Alternatively, the Arabidopsis CAX1 gene was heterologously expressed in tobacco (cultivar KY160). Transgenic lines of tobacco were generated with a CAX1 open reading frame (ORF) expressed in either the sense or antisense orientation in front of the 35S promoter. As a control, a transgenic line harboring only the expression vector (designated V) was used. For sense expression, four independent transgenic lines (S-65, S-49, S-32, and S-5) were used.
Expression of CAX1 RNA was measured in T2 transgenic lines by RNA gel blot analysis. As shown in Figure 2, CAX1 RNA accumulated in all 35S::CAX1 transgenic lines. CAX1-specific RNA was also detected in all antisense lines tested (data not shown). The inability to detect an endogenous transcript of the tobacco CAX1 homolog in the vector transgenic lines attests to the high-stringency hybridization conditions used.
Expression of CAX1 in Transgenic Tobacco Plants.
Ten micrograms of total RNA extracted from fully expanded leaves of 6-week-old T2 plants was analyzed by RNA gel blotting. The blot was hybridized with the CAX1 cDNA probe. Transgenic lines expressing the vector (V) alone do not express CAX1 RNA. Sense (S-5, S-32, S-49, and S-65) lines denote 5′;-3′; expression of the CAX1 ORF by using the 35S promoter (35S::CAX1). Ethidium bromidestained rRNA before transfer is shown at bottom.
CAX1 Expression in Tobacco Disturbs Normal Vigor
Preliminary examination suggested that heterologous CAX1 expression in tobacco had disrupted normal Ca2+ metabolism. Figure 3 demonstrates that 48 of the 60 primary transformants expressing the sense-oriented CAX1 formed necrotic lesions after 3 weeks under sterile conditions (Figure 3A). After transfer to soil, the same CAX1-transformed plants appeared to recover. Several weeks later, however, the leaves of all of the 48 primary transformants that initially displayed necrotic lesions began to redevelop altered leaf morphology. After several more weeks, the leaves were chlorotic and twisted (Figure 3B), the severity of these symptoms varying between the lines. Twenty-five of the 48 lines that displayed leaf necrosis also developed terminal buds that turned black and died (Figures 3C and 3D). Further examination of the root area among these stunted plants revealed not only reduced root mass but also, in ∼50% of the stunted plants (i.e., 12 of the original 60 primary transformants), almost no root formation (Figure 3E). In contrast, the 50 transgenic plants expressing antisense-oriented CAX1 displayed growth phenotypes indistinguishable from the 10 vector-control transgenic plants.
The 35S::CAX1 lines were selected for further study on the basis of their T1 phenotype and their ability to make seeds. Only seven of the original 48 transgenic lines that displayed altered morphology also maintained adequate fertility. In contrast, >90% of the antisense-oriented CAX1 and vector-containing transgenic lines maintained complete fertility. The same necrosis of the original transformants revisited the T2 generation of CAX1 transformants. When grown from seed in tissue culture, the T2 plants appeared normal and unperturbed for the first 5 weeks, after which their leaves began to display visible lesions. T2 CAX1-transformed plants that were sown and grown in the greenhouse also displayed necrotic lesions on their leaves as well as their apical meristems after 6 weeks of growth. Once again, the T2 CAX1-expressing plants displayed a visible reduction in root mass as well as reduced fertility.
Phenotypes of Tobacco Plants Expressing CAX1 Genes.
Sense lines denote expression of the CAX1 ORF. Antisense lines contain the ORF in the opposite orientation.
(A) Leaf phenotype of CAX1-expressing lines obtained by using a 35S promoter. Leaves are from plants grown in tissue culture.
(B) Leaf phenotype of CAX1-expressing lines after several weeks in the greenhouse.
(C) Size differences in CAX1-expressing plants. The 35S::CAX1 sense plant (in front) is stunted. The plant in the background is expressing CAX1 in the antisense confirmation. This plant is the same size as vector control plants (not shown).
(D) Magnified view of a 35S::CAX1 plant similar to (C).
(E) Root phenotype of CAX1-expressing plants. The sense roots are significantly stunted.
(F) Leaf of 10-week-old vector control plant grown for 2 weeks without Ca2+.
(G) Leaf of 10-week-old CAX1-expressing plant given Ca2+ supplementation.
Ca2+ Suppresses CAX1-Induced Symptoms
The symptoms of the CAX1-expressing plants, particularly the altered leaf morphology, could be phenocopied in both the vector control and antisense-oriented CAX1-expressing lines by growth in the absence of Ca2+ (Figure 3F). Therefore, we tested whether daily watering of plants with 2 mM CaCl2 (a sevenfold greater concentration of Ca2+ than in the normal nutrient solution) could ameliorate an apparent depletion of Ca2+ in tobacco attributable to heterologous expression of Arabidopsis CAX1. Primary transformants and T2 plants displayed altered leaf morphology and necrotic lesions on the leaves and apical meristems, regardless of the media used; however, adding Ca2+ to the medium delayed the onset and severity of these symptoms, the extent of the suppression varying among the individual lines. In some cases, this supplementation allowed CAX1-transformed plants to develop almost wild-type leaf morphology (Figure 3G).
CAX1 Expression Induces Ion Sensitivity
Ca2+ homeostasis is an important component of stress responses (Bressan et al., 1998; Sanders et al., 1999). As observed here, CAX1 expression was induced when Arabidopsis plants were watered with high concentrations of Ca2+; perturbations of other ions, however, caused no substantial alterations in CAX1 expression (Figure 1). This suggests that plants regulate CAX1 expression in response to various stimuli. Conceivably, constitutive CAX1 expression may alter the ion sensitivity of transgenic plants. Transgenic seeds germinated on standard media were transferred to various media when the seedlings were similar in size and vigor to the control plants (Figure 4A). If allowed to grow in standard media, the CAX1-transformed plants were only slightly smaller than vector controls (Figure 4B). More than 200 T2 seeds were analyzed from multiple 35S::CAX1 lines; >85% of the plants were hypersensitive to Mg2+ and K+ salts at concentrations that failed to disturb the growth of the control plants (Figures 4C and 4D). CAX1-expressing plants were also sensitive to excess Na+ in the media; however, the effects on phenotype were not as dramatic as those seen with Mg2+ (Figure 4E) and were comparable with those seen with K+ (data not shown). The growth of CAX1-expressing plants was not enhanced by above-normal concentrations of Ca2+, even though the plants were hypersensitive to Ca 2+-depleted media (Figures 4F and 4G). In none of the tests for ion sensitivity did the 100 transgenic plants expressing antisense-oriented CAX1 display a condition similar to the CAX1 sense-oriented transgenic plants or different from the vector control transgenic plants (data not shown).
Ion Sensitivity of CAX1-Expressing Plants.
Two vector control plants are shown at left and two CAX1-expressing plants (35S::CAX1) at right.
(A) Plants grown in standard media immediately after transfer to various media (pretreatment).
(B) Plants transferred to standard media and grown for 10 days.
(C) Plants transferred to standard media supplemented with 50 mM MgCl2 and grown for 10 days.
(D) Plants transferred to standard media supplemented with 100 mM KCl and grown for 10 days.
(E) Plants transferred to standard media supplemented with 50 mM NaCl and grown for 10 days.
(F) Plants transferred to standard media supplemented with 100 mM CaCl2 and grown for 10 days.
(G) Plants transferred to standard media without Ca2+ and grown for 10 days.
(H) Plants transferred to standard media supplemented with 50 mM MgCl2 and 2 mM CaCl2 and grown for 10 days.
(I) Plants transferred to standard media supplemented with 100 mM KCl and 2 mM CaCl2 and grown for 10 days.
Ca2+ Restores Ion Homeostasis
In light of the apparent Ca2+ deficiency accompanying heterologous expression of CAX1 in tobacco, we tested whether or not the ion sensitivities imposed by CAX1 expression could be attributed to such a deficiency. The transgenic plants expressing CAX1 and the vector controls were germinated on standard media and then transferred to standard media plus 100 mM KCl or 50 mM MgCl2, as described above, but were also supplemented with 2 mM CaCl2. As shown in Figures 4H and 4I, adding Ca2+ greatly reduced the ion sensitivities of the CAX1-expressing plants. Analyzing >200 T2 seeds from multiple 35S::CAX1 lines showed that under these growth conditions <20% of the plants showed any perturbations in growth.
CAX1 Expression Induces Increased Cold-Shock Sensitivity
A brief increase in cytosolic Ca2+ is one of several transient responses that occur when plants are chilled (Minorsky, 1989). Acclimation to subsequent freezing is partly the result of a modified calcium response to this stress condition (Knight et al., 1996). Cold damage in plants has been speculated to result from a lack of Ca2+ homeostasis (Minorsky, 1985). To determine whether CAX1 expression causes phenotypic alterations in plant responses, we examined the sensitivity of CAX1-expressing lines to chilling. As shown in Figure 5A, 3-week-old CAX1-expressing plants, although slightly smaller than the controls, did not display any visible growth defect. Both 3-week-old vector control and CAX1-expressing plants were subjected to a mild cold treatment. Six days after the cold shock, ∼50% of 200 CAX1-expressing plants (50 plants from each of the T2 plants analyzed in Figure 2) that were chilled developed necrotic lesions that were not found on any of the 200 vector or antisense transgenic plants analyzed (Figures 5B and 5C). CAX1-expressing plants also appeared to grow slightly more slowly after the cold treatment (data not shown). Addition of exogenous Ca2+ reduced the chilling sensitivity of these plants. When 2 mM CaCl2 was added to the standard media, only ∼15% of the 200 CAX1-expressing plants developed necrotic lesions when cold-shocked (Figure 5D).
Cold-Shock Sensitivity of CAX1-Expressing Plants.
Vector control plants are shown at left and CAX1-expressing plants (35S::CAX1) at right.
(A) Plants grown in standard media for 21 days (before cold treatment).
(B) Twenty-seven-day-old plants 6 days after cold treatment.
(C) Leaves of plants at 21 days after cold treatment.
(D) Twenty-seven-day-old plants grown in standard media supplemented with 2 mM CaCl2, seen 6 days after cold treatment.
Ca2+ Accumulation in CAX1-Expressing Plants
To ascertain whether CAX1 expression altered total Ca2+, presumably through sequestration into the vacuole, we measured the total accumulation of Ca2+ in the roots and leaves of transgenic plants. As shown in Figure 6, CAX1-expressing plants contained almost twice as much total Ca2+ in root tissue as did the vector control plants. Leaves of CAX1-expressing plants contained 30% more total Ca2+ than did plants expressing the vector alone.
Ca2+/H+ Antiport in CAX1-Expressing Plants
Because expression of CAX1 in yeast restored vacuolar Ca2+/H+ antiport activity to yeast strains deficient in this transporter (Hirschi et al., 1996), Ca2+/H+ antiport activity was measured in tonoplast-enriched vesicles from transgenic tobacco roots to determine whether CAX1 expression caused altered Ca2+/H+ transport. Proton gradients (acidic inside the vesicles) were formed artificially by adding nigericin to K+-loaded vesicles (Salt and Wagner, 1993). As shown in Figure 7A, treatment with nigericin caused a rate of proton accumulation (monitored with 14C-methylamine) similar to those reported in oat and observed in tobacco (Schumaker and Sze, 1985; V. Korenkov and G.J. Wagner, unpublished results). Addition of CaCl2 (10 to 100 μM) caused efflux of methylamine, an indication that Ca2+ was exchanged (antiported) with H+ (Schumaker and Sze, 1985).
Tonoplast-enriched vesicles were prepared from plants watered with either nutrient solution or Ca2+. In the absence of Ca2+, the endogenous Ca2+/H+ antiporter may be weakly expressed (Figure 1); regardless of the environmental conditions, however, the 35S::CAX1 transgene should produce constitutive amounts of CAX1. As shown in Figure 7B, CAX1-expressing transgenic plants watered with nutrient solution were capable of accumulating Ca2+ to steady state concentrations of ∼14 nmol/mg protein, compared with ∼7 nmol/mg protein for the vector controls. When tonoplast-enriched vesicles were prepared from transgenic plants watered with Ca2+, the CAX1-expressing plants were capable of accumulating steady state concentrations of Ca2+ to ∼16 nmol/mg protein; the comparable value for the vector controls was ∼14 nmol/mg protein (Figure 7C). Under both growth conditions, the amounts of Ca2+/H+ antiport activity varied in the CAX1-expressing plants, being greater in S-65 plants than in S-49 or S-32 plants (data not shown); this coincided with the greater abundance of CAX1 transcripts in S-65 plants (Figure 2). Despite some variation among lines, the CAX1-expressing plants always expressed at least 30% more Ca2+/H+ antiport activity than did control plants watered with nutrient solution.
Calcium Concentrations in Roots and Leaves of Transgenic Plants.
Ca2+ content of vector controls (open bars) and CAX1-expressing plants (hatched bars) grown in standard media was determined by atomic absorption spectrophotometry. Data represent the means (±sd) of three independent assays. wt, weight.
In our attempts to measure the kinetic properties of Ca2+/H+ transport activity as a function of Ca2+ concentration, variations between experiments made it difficult to obtain precise measurements of apparent Km and Vmax. However, a dependence on Ca2+ concentration and a saturation in Ca2+ uptake were observed. Preliminary experiments indicated an apparent Km for Ca2+ of ∼15 ± 5 μM and a Vmax of 13 ± 5 nmol of Ca2+ mg–1 protein min–1, values in general agreement with those reported for Ca2+/H+ in plants (Schumaker and Sze, 1985). In addition, the CAX1 sense- and antisense-expressing lines did not appear to differ from the vector controls in these kinetic measurements (data not shown).
DISCUSSION
Exogenous Ca2+ Induces CAX1 RNA
The induction of CAX1 expression in plants with exogenous Ca2+ is consistent with the CAX1 protein playing a role in loading the vacuole with Ca2+ (Figure 1). The plant vacuole contains millimolar concentrations of Ca2+, whereas Ca2+ in the cytosol is submicromolar under resting conditions. In plants and fungi, this asymmetric distribution is achieved by P-type Ca2+-ATPases and Ca2+/H+ antiport systems (Cunningham and Fink, 1996; Ferrol and Bennett, 1996; Malmström et al., 1997; Ueoka-Nakanishi et al., 1999). The vacuolar Ca2+-ATPase has a high affinity for Ca2+ compared with the antiporter (Ueoka-Nakanishi et al., 1999). In response to a Ca2+ load, the vacuolar antiporter may lower cytosolic Ca2+ content to a few micromolar, and the Ca2+-ATPase then may act to further lower the cytosolic Ca2+ pools. Although the Ca2+/H+ antiporter may have some role in controlling the cytosolic Ca2+ concentrations during signal transduction events (see below), the Ca2+-specific induction of CAX1 RNA is too slow to account for any portion of this rapid redistribution of Ca2+.
The data presented here demonstrate that CAX1 also could be weakly induced by Na+, osmotic shock, and Ni+, indicating a need for future studies to address the specificity of CAX1 transport and mechanisms of CAX1 induction. Potentially, CAX1 may have a role in Ni+ transport; however, oats do not appear to possess a tonoplast high-affinity Ni+/H+ transporter (Gries and Wagner, 1998).
CAX1 Phenotypes: Importance of Regulated Gene Expression
The results presented here show that constitutive expression of CAX1, a putative vacuolar Ca2+/H+ antiport, can alter growth, increase the sensitivity of the plant to various ions, and alter the plant response to chilling. Expression of CAX2, an antiporter with different ion affinities, did not produce these phenotypes (K.D. Hirschi, unpublished data). Furthermore, the antisense CAX1 constructs produced no visible or biochemical differences from the vector controls in any of our studies. The lack of sequence similarity between the Arabidopsis CAX1 gene and the tobacco homolog (Figure 2) may explain these results.
The phenotypes described here may reflect the inability to modulate Ca2+/H+ antiport activity in certain cells and during certain events. In fact, given the expression pattern of CAX1 in Arabidopsis (Figure 1), the CAX1-expressing tobacco plants were expressing amounts of Ca2+/H+ antiport activity that may be appropriate only in the presence of high concentrations of Ca2+ in the soil. In CAX1-expressing plants, the tonoplast-enriched Ca2+/H+ activity was independent of the watering conditions (Figures 7B and 7C). In contrast, the vector control plants, when watered with Ca2+, demonstrated tonoplast-enriched Ca2+/H+ antiport activity nearly equal to that of the CAX1-expressing plants. Thus, the CAX1-expressing plants appeared to possess constitutively high amounts of Ca2+/H+ antiport activity.
Constitutive Ca2+/H+ antiport activity in transgenic tobacco plants appears to cause stress sensitivities. Organisms usually are rendered sensitive to various ions through defects in transporters and regulatory proteins. In yeast, a defect in a transport protein causes increased metal sensitivity (Li and Kaplan, 1998). In plants, a defect in a putative regulatory Ca2+ sensor causes hypersensitivity to salt (Liu and Zhu, 1998).
K+/Nigericin-Dependent Methylamine and Ca2+ Uptake Activity in Tonoplast-Enriched Vesicles from Tobacco Roots.
(A) K+/nigericin was used to generate a ΔpH that was monitored as the accumulation of 14C-methylamine (filled diamonds). After ∼10 min, Ca2+ was added to a final concentration of 10 μM (open diamonds) or 100 μM (open triangles). Methylamine equilibration was monitored by direct filtration (Schumaker and Sze, 1985). The data shown are representative of the >10 experiments that were performed with tonoplast-enriched vesicles from various transgenic plants. Data are presented as nanomoles of CH3NH2 mg–1 of protein, after subtraction of the control values (obtained in the absence of nigericin).
(B) and (C) Ca2+ uptake activity in tonoplast-enriched vesicles from vector control tobacco roots (open squares) and CAX1-expressing roots (open circles) watered with nutrient solution (B) or CaSO4 (C). Ca2+ uptake was monitored by using the direct filtration method (Schumaker and Sze, 1985). After 14 min, Triton X-100 was added to a final concentration of 0.03% to dissipate the accumulation of Ca2+. Results are from assays of two tonoplast preparations with the vector control and three tonoplast preparations from transgenic line S-65 (35S::CAX1) with both the nutrient water solution and CaCl2 watering treatments. Data represent the means (±sd) of these assays. Although the Ca2+ uptake varied with each line tested, similar results were obtained with S-49 and S-32 lines assayed in the same manner (data not shown). These results are presented as nanomoles of Ca2+ mg–1 of protein, after subtraction of the control values (absence of nigericin).
Ca2+ partitioning may be altered by expression of CAX1 in the transgenic plants. This model is supported by the constitutive amounts of tonoplast-enriched Ca2+/H+ activity and the increased total Ca2+ accumulation (Figures 6 and 7). Future experiments will have to specifically address localized fluctuations in Ca2+ and the possibility that CAX1 functions at other membranes in the plant cell to cause these changes in Ca2+ homeostasis.
The phenotypes of CAX1-expressing plants are consistent with Ca2+ deficiencies. In crop plants, Ca2+-deficient symptoms include necrosis of young leaves, often followed by necrosis of the apical meristem (Scaife and Turner, 1984); roots may appear brownish and short. These phenotypes are very similar to those seen in the CAX1-expressing plants (Figure 3). Furthermore, Ca2+-deficient crop plants often are diagnosed by their increased sensitivity to other cations (Scaife and Turner, 1984). The CAX1-expressing transgenic tobacco plants studied here were hypersensitive to K+ and Mg2+ (Figures 4C and 4D). The high concentrations of these ions may compete with Ca2+ for uptake and may induce Ca2+ deficiency-like symptoms (Figure 3G). The ability to suppress the growth defects and ion sensitivities of CAX1-expressing plants with exogenous Ca2+ strongly suggests that these plants are deficient in Ca2+ (Figures 3G, 4H, 4I, and 5D).
Chilling Sensitivity: Ca2+ Deficiency or Altered Ca2+ Signaling?
The cold-shock sensitivity data presented here suggest that even in optimal growth media, CAX1-expressing plants have altered Ca2+ homeostasis (Figure 5). These growth phenotypes may be caused by alterations in the availability of Ca2+ at the plasma membrane. Displacement of membrane-associated Ca2+ can impair membrane stability (Läuchli, 1990). In fact, Ca2+ may protect membranes from the effects of high salt and cold shock (LaHaye and Epstein, 1969; Lynch and Steponkus, 1987).
Alternatively, the chilling sensitivity may be attributable to a dampening in the cytosolic burst after this environmental stimulus. In plants, Ca2+ is mobilized from the vacuole (Alexandre et al., 1990; Gilroy et al., 1993; Allen and Sanders, 1995; Trewavas and Malhó, 1997). In fact, coldinduced vacuolar release of calcium has been demonstrated in tobacco and Arabidopsis (Knight et al., 1996). Thermodynamic considerations argue that the vacuolar Ca2+-ATPase is necessary to account for the restoration of resting concentrations of cytosolic Ca2+ (Malmström et al., 1997); however, localized increases in cytosolic Ca2+ can reach micromolar concentrations (McAinsh and Hetherington, 1998). Thus, a major function of the vacuolar Ca2+/H+ antiporter may be to reduce cytosolic Ca2+ concentrations after they have been increased as a result of external stimuli (Ueoka-Nakanishi et al., 1999). Conceivably, constitutive expression of CAX1 in transgenic plants may perturb these transient concentrations of cytosolic Ca2+ during adapted biological responses.
In summary, a critical finding in this study is the ability to significantly perturb the growth parameters of a plant through the constitutive expression of a single transport protein. The CAX1-expressing plants displayed altered phenotypes and increased stress sensitivities, emphasizing that modulated expression of a Ca2+/H+ antiporter is important for normal growth and several biological responses.
METHODS
Plant Materials
Arabidopsis thaliana ecotype Columbia was used in this study. For stress treatment, surface-sterilized seeds were grown on one-half strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 2% (w/v) sucrose and 1% (w/v) agar, pH 5.7 (standard media), for 3 weeks and then transferred to a water bath containing the appropriate stressor. Osmolality of the 10% polyethylene glycol 3350 (PEG) solution was measured with an osmometer (Wescor, Logan, UT). Tobacco cultivar KY160 was also used in this study. Tobacco plants were grown in a greenhouse as previously described (Grusak and Pomper, 1999). For optimal growth, plants in the greenhouse were watered three times a day, with soil-saturating volumes of a nutrient solution containing 3 mM KNO3, 0.275 mM CaCl2, 0.25 mM MgSO4, and 0.25 mM KH2PO4. For studies of calcium deficiency, the CaCl2 was omitted from this mixture. For Ca2+ supplementation and measurement of total Ca2+ content, a soil-saturating volume of 2 mM CaCl2 was added daily.
Surface-sterilized tobacco seeds were plated on standard media and maintained in a temperature-controlled room at 25°C with continuous cool-fluorescent illumination. To produce a cold shock, we placed the plants germinated on standard media at 4°C for 24 hr at 21 days after plating, then returned the plants to 25°C for 6 or 21 days. For ion sensitivity analysis, surface-sterilized seeds were germinated in standard media; 14 days after plating, they were transferred to standard media supplemented with the appropriate ion. To make media deficient in Ca2+, we deleted the CaCl2 from the nutrient solution and added sucrose and agar, as given for the standard media. Standard media plus 2 mM CaCl2 was used to suppress the CAX1-conferred phenotypes.
Most experiments were performed with segregating T2 populations of tobacco lines S-65 and S-49. Phenotypes did not drastically differ among the cauliflower mosaic virus 35S::CAX1-expressing plants. Antisense line AS-46 was used in most experiments; however, the phenotypes displayed by antisense and vector control lines were indistinguishable in all experiments performed.
Plant Transformations
Standard techniques of DNA cloning were performed as described by Ausubel et al. (1998). The coding region of CAX1 was cloned into pBin19 (Clontech, Palo Alto, CA), which contained the cauliflower mosaic virus 35S promoter fragment. The recombinant plasmids, or vector controls, were introduced into Agrobacterium tumefaciens LB4404 (Life Technologies, Grand Island, NY). Tobacco leaf disc transformation was conducted according to published procedures by using tobacco strain KY160 (Tarczynski et al., 1992). Transformants were selected on standard media containing 100 μg/mL kanamycin. Approximately 60 primary transformants harboring the 35S::CAX1 construct were placed in soil.
RNA Extraction and RNA Gel Blot Analysis
RNA was isolated from Arabidopsis plants (leaves, stems, and roots) and tobacco leaves, according to previously published procedures (Niyogi and Fink, 1992). After electrophoresis on a 1% agarose gel in formaldehyde, total RNA was blotted onto Hybond N+ membrane (Amersham), according to the manufacturer's recommendations. The full-length CAX1 cDNA was radiolabeled with 32P-dCTP by using a random-primed labeling kit (Amersham). Blots were hybridized at 65°C according to the method of Church and Gilbert (1984). Blots were washed three times (15 min each time) in 0.1 × SSC (1 × SSC is 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at 65°C, and hybridization was visualized by autoradiography. Results were quantified with a PhosphorImager SI and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Calcium Analysis
Fifty-day-old tobacco plants were grown in the greenhouse as described in Plant Materials. The young leaves were harvested, and the roots were rinsed in water and harvested. The leaves and roots were dried at 70°C for 3 days. At least 0.3 g (dry weight) of roots was analyzed individually; roots weighing less were pooled to yield the required weight. The tissues then were wet digested, and the digestate was resuspended as described in Grusak (1994). Total Ca2+ content per gram of dry mass was determined by atomic absorption spectrophotometry (model 2100; Perkin-Elmer, Norwalk, CT).
Isolation of Sealed Tonoplast-Enriched Vesicles
To determine tonoplast transport properties of individual transgenic plant types in studies such as this one, we also determined transport activity in vesicles prepared from soil-grown tobacco plants. Tobacco roots (without the tap root) teased from soil by gentle washing with water yielded vesicles with good transport activity (V. Korenkov, K.D. Hirschi, and G.J. Wagner, unpublished results). After root isolation, the procedure of Salt and Wagner (1993) was used with some modifications (Gonzales et al., 1999). All steps were conducted at 4°C, and when used, DTT and phenylmethylsulfonyl fluoride (PMSF) were added just before the buffer was used. Fifty-day-old tobacco plants were watered with 0.5 × MS medium or 10 mM CaSO4 for the last 7 days of growth. Roots were harvested (40 g) and soaked for 10 min in homogenization buffer (3 mL/mg roots) containing 250 mM mannitol, 20 mM Hepes adjusted with Bis-Tris propane (BTP) to pH 7.4, 6 mM EGTA, 0.5% polyvinylpyrrolidone-40, 0.1% BSA, 0.1 mM PMSF, and 1 mM DTT. Half of the root samples (i.e., ∼20 g) were transferred to a cold mortar holding 40 mL of homogenization buffer, cut into 2-cm pieces, and homogenized with a mortar and pestle. The remaining root samples were divided and homogenized the same way, and the root suspension from all portions was filtered through four layers of cheesecloth. The supernatants were decanted and centrifuged for 5 min at 1000g, followed immediately by 10 min at 12,000g. The homogenates were decanted and centrifuged for 30 min at 60,000g. The supernatants were discarded, and the pellet was resuspended in 250 mM mannitol, 2.5 mM Hepes-BTP, pH 7.2, 0.1% BSA, 0.1 mM PMSF, and 0.1 mM DTT. The resuspended pellet then was layered onto a 10-mL solution containing 6% (w/v) dextran, 250 mM mannitol, and 2.5 mM Hepes-BTP, pH 7.2. The resulting gradient was centrifuged for 2 hr at 70,000g. The visible band at the mannitol-dextran interface (vesicles) was collected.
Loading of Vesicles with Potassium
Vesicles from the dextran solution were recovered and diluted to 36 mL with K+-loading buffer containing 50 mM mannitol, 2.5 mM Hepes-BTP, pH 7.2, 150 mM KCl, 0.1% BSA, 0.1 mM PMSF, and 0.1 mM DTT. After incubation at 10°C for 1 hr, vesicles were sedimented at 90,000g for 30 min. The pellet was resuspended in 100 to 200 μL K+-loading buffer to a final concentration of 1 to 2 mg protein/mL. Protein analysis was performed as previously described (Salt and Wagner, 1993).
Ca2+ Transport Assay
45Ca2+ uptake was measured by a filtration assay as previously described (Schumaker and Sze, 1985). The loaded vesicle preparations were pelleted and resuspended in buffer without KCl just before use, and an aliquot was diluted into a reaction mixture containing 250 mM mannitol, 25 mM Hepes-BTP, pH 7.0, with or without 5 μM nigericin. After incubation with 10 μM 45Ca2+ (American Radiolabeled Chemicals, St. Louis, MO) at 4°C for the indicated period of time, the samples were filtered through prewetted HATF Millipore filters (0.45 μm pore size; Millipore Corp., Bedford, MA) and then washed in resuspension buffer containing 0.1 mM CaCl2. The filters were dried, and the radioactivity was determined by liquid scintillation counting. Results are presented as nanomoles of Ca2+ taken up per milligram of protein. Each transgenic line was tested for Ca2+ transport at least twice. Absolute rates of Ca2+ transport varied with each preparation; however, the difference between CAX1-expressing lines and vector controls was always >30% when plants were watered with nutrient solution.
Methylamine Accumulation
ΔpH generation in vesicles (acid inside) was determined by 14C-methylamine (American Radiolabeled Chemicals) accumulation by using the direct filtration method previously described (Churchill and Sze, 1983). All vesicles appeared to be well sealed, as evidenced by maintenance of the steady state gradient in the control. The rate and magnitude of proton dissipation in response to Ca2+ were concentration dependent. Absolute rates of methylamine accumulation varied with each preparation.
Acknowledgments
I thank Maricar Miranda, Nathaniel Wilganowski, and Jean Sunega for technical support, and George Wagner and Victor Korenkov for assistance establishing the Ca2+ transport assays. I am also grateful to Bonnie Bartel, Andrew Diener, Toshiro Shigaki, and George Wagner for critical reading of the manuscript. This work was funded in part by the National Institutes of Health (Grant Nos. CHRC 5 P30 and 1R01 GM 57427). This work is a publication of the U.S. Department of Agriculture, Agricultural Research Service (USDA/ARS), and the Children's Nutrition Research Center (Department of Pediatrics, Baylor College of Medicine, Houston, TX); it has been partially funded by the USDA/ARS under Cooperative Agreement No. 58-6250-6001. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
- Received June 29, 1999.
- Accepted August 31, 1999.
- Published November 1, 1999.