MCA1 and MCA2 that mediate Ca2+ uptake have distinct and overlapping roles in Arabidopsis

Abstract

Ca(2+) is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca(2+)-permeable channels or transporters involved in Ca(2+) uptake in roots are largely unknown. We recently identified a candidate for the Ca(2+)-permeable mechanosensitive channel in Arabidopsis (Arabidopsis thaliana), named MCA1. Here, we investigated the only paralog of MCA1 in Arabidopsis, MCA2. cDNA of MCA2 complemented a Ca(2+) uptake deficiency in yeast cells lacking a Ca(2+) channel composed of Mid1 and Cch1. Reverse transcription polymerase chain reaction analysis indicated that MCA2 was expressed in leaves, flowers, roots, siliques, and stems, and histochemical observation showed that an MCA2 promoter::GUS fusion reporter gene was universally expressed in 10-d-old seedlings with some exceptions: it was relatively highly expressed in vascular tissues and undetectable in the cap and the elongation zone of the primary root. mca2-null plants were normal in growth and morphology. In addition, the primary root of mca2-null seedlings was able to normally sense the hardness of agar medium, unlike that of mca1-null or mca1-null mca2-null seedlings, as revealed by the two-phase agar method. Ca(2+) uptake activity was lower in the roots of mca2-null plants than those of wild-type plants. Finally, growth of mca1-null mca2-null plants was more retarded at a high concentration of Mg(2+) added to medium compared with that of mca1-null and mca2-null single mutants and wild-type plants. These results suggest that the MCA2 protein has a distinct role in Ca(2+) uptake in roots and an overlapping role with MCA1 in plant growth.

Figure 1.

Function of MCA2 in yeast cells. A, MCA2 has the ability to increase Ca²⁺ accumulation even in the mid1 cch1 double mutant. MCA1 or MCA2 cDNA on a multicopy plasmid was expressed under the control of the TDH3 promoter in each yeast mutant (mid1, cch1, or mid1 cch1) as well as the parental strain (wild type). The yeast MID1 gene with its own promoter was also expressed from a multicopy plasmid, YEpMID1 (Nakagawa et al., 2007). Data are the mean ± sd for three independent experiments. *P < 0.05 versus the vector in each mutant. Note that the data for vector, MID1, and MCA1 are from our previous article (Nakagawa et al., 2007). B, MCA2 has the ability to increase viability after exposure to α -factor even in the mid1 cch1 double mutant. The yeast cells used in this experiment are the same as in A. Data are the mean ± sd for three independent experiments. *P < 0.05 versus the vector (control) in each mutant.

Figure 2.

Expression of MCA2. A, RT-PCR of the MCA2 transcript. Total RNA was isolated from leaves, flowers, roots, siliques, and stems of wild-type Arabidopsis plants (5 to 6 weeks old) and subjected to RT-PCR. β -Tubulin mRNA was used for an internal control. B, Spatial patterns of MCA1 and MCA2 transcription as revealed by GUS staining. Ten-day-old (a–e and i–m) and 22-d-old (f–h and n–p) MCA1p::GUS and MCA2p::GUS plants are shown. a and i, A whole plant (an arrow indicates the extreme end of the primary root); b and j, a leaf (not a cotyledon); c and k, the tip of a primary root; d and l, a region corresponding to the shoot apical meristem; e and m, a cross section of a primary root, showing GUS activity in the stele and endodermis; f and n, an upper view of a 22-d-old plant; g and o, magnification of the base of the inflorescence shown in f and n, respectively; h and p, a side view of a 22-d-old plant. Note that two photographs were joined to make h. C, GFP fluorescence images of MCA2-GFP expressed in root cells. The top row represents intact roots of 6-d-old seedlings and the bottom row those treated for at least 10 min with 0.8 m mannitol to induce plasmolysis. The membrane marker proteins used are as follows: plasma membrane, a GFP fusion to the plasma membrane channel protein PIP2A expressed in line Q8; endoplasmic reticulum (ER) membrane, a GFP fusion to an endoplasmic reticulum membrane protein expressed in line Q4; vacuolar membrane, a GFP fusion to the vacuolar membrane channel protein δ -TIP expressed in line Q5 (Cutler et al., 2000); and cytoplasmic GFP.

Figure 3.

Construction of the mca1-null mca2-null double mutant. A, Genomic organization of the MCA1 and MCA2 genes. Boxes represent exons, and their black areas show the open reading frame. The T-DNA is drawn to an arbitrary size. B, RT-PCR showing no detectable production of the MCA2 transcript in the mca2 and mca1-null mca2-null line. β -Tubulin mRNA is a control. RNA was purified from whole seedlings grown for 10 d.

Figure 4.

Growth phenotypes. A, Growth of wild-type, mca1-null, mca2-null, and mca1-null mca2-null plants. Plants were grown for 20 d on MS medium and for an additional 5 d in soil under a 16-h-light/8-h-dark cycle at 22°C. Bolting of the mca1-null mca2-null mutant was retarded. B, Timing of the appearance of first and second rosette leaves. Thirty-eight to 40 plants were grown for the indicated times on MS medium under a 16-h-light/8-h-dark cycle at 22°C. Percentages of plants that had developed first and second rosette leaves are indicated. A result representative of three independent experiments is shown. In all three experiments, the appearance of first and second rosette leaves was retarded most severely in the mca1-null mca2-null mutant followed by the mca1-null mutant, although the absolute timing of leafing varied from experiment to experiment. C, Timing for bolting. Thirty-eight to 40 plants were grown for 14 d on MS medium and for additional days in soil under a 16-h-light/8-h-dark cycle at 22°C. Percentages of bolted plants are indicated. A result representative of three independent experiments is shown. In all three experiments, the timing of bolting was retarded most severely in the mca1-null mca2-null mutant followed by the mca1-null mutant, although the absolute timing of bolting varied from experiment to experiment.

Figure 5.

Ca²⁺ uptake activity of the roots of wild-type, mca1-null, mca2-null, and mca1-null mca2-null plants. Transgenic and wild-type plants were grown for 21 d on MS medium and for an additional 14 d in aerated hydroponics. Shoots were removed and roots were weighed and incubated for 20 min in an assay solution (2 mm KCl, 0.1 mm CaCl₂, and 3 MBq/L ⁴⁵CaCl₂ [30 MBq/mmol]) in the absence and presence of 1 mm GdCl₃, a blocker of mechanosensitive ion channels, or 20 μm verapamil, a blocker of voltage-gated Ca²⁺ channels. The roots were washed five times with a washing solution composed of 2 mm KCl, 0.1 mm CaCl₂, and 1 mm LaCl₃, and the radioactivity retained in the roots was counted in a liquid scintillation counter. Data are the mean ± sd for five independent experiments. *, P < 0.05 versus the wild type.

Figure 6.

Primary roots of mca1-null mca2-null plants, but not of mca2-null plants, fail to enter the lower, harder agar (1.6%) medium from the upper, softer agar (0.8%) medium. Seeds of transgenic and wild-type lines were sowed on the surface of the upper medium, incubated for 9 d, and photographed to examine the ability of the primary roots to enter the lower medium. The percentage of primary roots that had not entered the lower medium was calculated. Number of seedlings examined: wild type, n = 104; mca1-null, n = 91; mca2-null, n = 52; mca1-null mca2-null, n = 52. Data are the mean ± sd. *, P < 0.01; **, P > 0.05.

Figure 7.

Effect of the concentration of agar and CaCl₂ on primary root growth. Seeds of transgenic and wild-type lines were sowed on MS medium containing 0.8% or 1.6% agar and 0.1, 0.5, 3.0, or 13.0 mm CaCl₂ and incubated for 9 d. Seedlings were picked up, laid on another MS/1.6% agar medium, and photographed. Primary root length was measured with ImageJ software. The length is presented as the mean for 15 seedlings of each line with the sd. A result representative of three independent experiments is shown. a, Wild-type seedling; b, mca1-null seedling; c, mca2-null seedling; d, mca1-null mca2-null seedling. The black bar represents null lines whose primary root lengths are significantly shorter (P < 0.05) than those of the wild-type line under each incubation condition.

Figure 8.

Severe growth defect of the mca1-null mca2-null mutant grown on medium containing high Mg²⁺. Plants were grown for 24 d on MS medium under 16-h-light/8-h-dark conditions at 22°C and photographed. Note that the color tone of each sample varies delicately from photograph to photograph; thus, a difference in the color tones presented here is not a significant factor for the interpretation of the results. Also note that the retardation of growth in the mca1-null mca2-null mutant presented in Figure 4 is not obvious in the first row of Figure 8 showing the transgenic lines grown on MS medium with no additive because upper views are shown.

Figure 9.

Ca and Mg accumulation in mca1-null mca2-null and wild-type plants. Plants were grown for 24 to 26 d under the conditions described in the legend to Figure 8, and roots and shoots were subjected to inductively coupled plasma atomic emission spectrometer analysis to determine the content of Ca (A) and Mg (B). a, Wild-type plants; b, mca1-null mca2-null plants. Data are the mean ± sd of three independent experiments. Note that there was no significant difference (P > 0.05) in the Ca and Mg contents between mca1-null mca2-null and wild-type plants, except for two cases (*P = 0.0207; **P = 0.0026).