Results

BL Transiently Increases [Ca²⁺]_c in Arabidopsis Rosette Leaves. To analyze BL-dependent changes in [Ca²⁺]_c in Arabidopsis leaves, we made transgenic A. thaliana that expressed aequorin in WT plants, phot1 and phot2 mutants, and phot1 phot2 double mutants. Because the phot1 mutant has a Ler background and phot2 has a WS background, we used WT plants of both ecotypes as controls.

When rosette leaves from 3-wk-old WT plants were irradiated with 100 μmol·m^-2·s^-1 of BL for 10 s, each leaf exhibited a transient increase in aequorin luminescence lasting 50–70 s (n > 100). Because 95–98% of transgenic aequorin is located in the cytoplasm (28), the [Ca²⁺]_c can be estimated from the aequorin luminescence according to Baum et al. (18). Fig. 1 shows the averaged BL-induced transient increase in [Ca²⁺]_c of leaves from Ler (n = 10) and WS (n = 10). The resting level of [Ca²⁺]_c in the dark was estimated to lie between 25 and 50 nM, with a mean of 34.5 ± 2.5 nM. It took ≈10 s to reach the peak level of [Ca²⁺]_c after the BL source was shut off, then another 40–60 s for the [Ca²⁺]_c to return to the resting level. The kinetics of BL-induced Ca²⁺ increase is similar to results obtained with deetiolated seedlings (18). The peak [Ca²⁺]_c induced by BL was 214.3 ± 14.2 nM in WS and 154.0 ± 9.3 nM in Ler leaves (Fig. 1). It is not clear whether the difference is due to genetic differences between the ecotypes without further investigation. The kinetics of increase in [Ca²⁺]_c during and immediately after BL irradiation could not be determined in these experiments for the same reasons as in the previous study by Baum et al. (18).

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Fig. 1.

BL-induced changes in [Ca²⁺]_c. The averaged increases in [Ca²⁺]_c were obtained from 10 independent experiments in leaves from Ler (circles) and WS (squares) plants. Leaves were illuminated for 10 s (gray zone) with 100 μmol·m^-2·s^-1 of BL. The vertical bars indicate the standard error. Dots represent dark control ([Ca²⁺]_c in the same plants before illumination in Ler plants).

Next, we examined the BL-induced elevation of [Ca²⁺]_c (Δ[Ca²⁺]_c) in the leaves of phot1 and phot2 mutants, phot1 phot2 double mutants, and WT plants at various fluence rates. We examined two lines of transformants, which exhibited high aequorin luminescence, in each mutant and WT plant. Because the results were similar between the two lines, the results of only one are presented. Δ[Ca²⁺]_c was calculated as the difference between [Ca²⁺]_c in the dark before BL irradiation and the BL-induced peak in [Ca²⁺]_c. In leaves of WT plants, BL-induced Δ[Ca²⁺]_c increased in a fluence-rate-dependent manner from 0.1 to 10 μmol·m^-2·s^-1 and remained constant up to 250 μmol·m^-2·s^-1 (Fig. 2 A and B, filled bars).

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Fig. 2.

BL-induced increase in [Ca²⁺]_c (Δ[Ca²⁺]_c, the difference between [Ca²⁺]_c in the dark before illumination and the BL-induced peak in [Ca²⁺]_c)in leaves of WT plants, phot1 and phot2 mutants, and phot1 phot2 double mutants and relative contributions of phot1 and phot2 to the BL responses. (A and B) Δ[Ca²⁺]_c in leaves of Ler plants and a phot1 mutant (A) and those in leaves of WS plants and phot2 and phot1 phot2 mutants (B) at various fluence rates of BL. (C and D) Relative contributions of phot1 (C) and phot2 (D) to the BL-induced Δ[Ca²⁺]_c. The contributions of phot1 in the presence and absence of phot2 were calculated as the percent difference in Δ[Ca²⁺]_c between phot1 and Ler (i.e., the results in A) and between phot1 phot2 and phot2 (results in B), respectively. The contribution of phot2 was calculated as the percent difference in Δ[Ca²⁺]_c between phot2 and WS. The vertical bars indicate the standard error of 5–20 independent experiments. * and **, significantly different from the data in the WT plant and the phot2 mutant, respectively (P < 0.05).

In leaves of phot1 mutants (Fig. 2 A, open bars), the BL-induced Δ[Ca²⁺]_c was very small at 0.1 μmol·m^-2·s^-1, remained lower than in the WT at fluence rates between 1 and 50 μmol·m^-2·s^-1, and reached the same level as in the WT only at fluence rates higher than 100 μmol·m^-2·s^-1 (Fig. 2 A). The relative contribution of phot1 to the response to BL was calculated from the percent difference between the BL-induced Δ[Ca²⁺]_c values from phot1 and Ler (Fig. 2C, gray bars) and showed that phot1 functions at fluence rates between 0.1 and 100 μmol·m^-2·s^-1. At 100 μmol·m^-2·s^-1, however, the Δ[Ca²⁺]_c values in the phot1 mutant and in Ler did not differ significantly (P > 0.05; Fig. 2 A). We thus conclude that phot1 functions at fluence rates between 0.1 and 50 μmol·m^-2·s^-1.

In the leaves of phot2 mutants (Fig. 2B, gray bars), Δ[Ca²⁺]_c was the same as in the WT at a fluence rate of 0.1 μmol·m^-2·s^-1; however, between 1 and 250 μmol·m^-2·s^-1 of BL, Δ[Ca²⁺]_c was less in the phot2 mutant than in the WT. The relative contribution of phot2 (Fig. 2D) showed that phot2 functions between 1 and 250 μmol·m^-2·s^-1 of BL. These results suggest that, despite their structural similarity, phot1 and phot2 have different photosensitivities with respect to inducing increases in [Ca²⁺]_c, as was also reported for BL-dependent induction of chloroplast movements and phototropism (6, 23).

In leaves of phot1 phot2 double mutants (Fig. 2B, open bars), Δ[Ca²⁺]_c was barely detectable at BL fluence rates of 0.1 and 1 μmol·m^-2·s^-1, was lower than in the phot2 mutant at fluence rates of 10 μmol·m^-2·s^-1, and was the same as in the phot2 mutant at 100 and 250 μmol·m^-2·s^-1 (Fig. 2B), suggesting photoreceptor(s) other than phototropins may induce transient increases in [Ca²⁺]_c at fluence rates between 10 and 250 μmol·m^-2·s^-1. The relative contribution of phot1 in the absence of phot2, calculated from the differences in Δ[Ca²⁺]_c between the phot2 mutant and the phot1 phot2 double mutant (Fig. 2C, open bars), is similar to its relative contribution in the presence of phot2 (Fig. 2C, gray bars), suggesting that phot1 and phot2 additively contribute to the BL-induced increase in [Ca²⁺]_c.

phot1 and phot2 Mediate Increase in [Ca²⁺]_c in Different Manners. To determine the source of Ca²⁺ that appeared in the cytoplasm after BL irradiation, we tested the effects of several Ca²⁺ channel blockers, Ca²⁺ chelators, and inhibitors of PLC on the BL-induced increase in [Ca²⁺]_c. Because both phot1 and phot2 contribute to the transient increase in [Ca²⁺]_c at 1 μmol·m^-2·s^-1 (Fig. 2) and no other photoreceptors were functioning (Fig. 2B), we used this fluence rate for the inhibitor tests.

After leaves were treated with the Ca²⁺ channel blocker CoCl₂ (5 mM) for 2 h, the BL-induced increase in [Ca²⁺]_c in WT plants and in phot1 and phot2 mutants was significantly reduced to ≈15–35% of control levels (Fig. 3A and Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org). Pretreatment with LaCl₃ at 0.5 mM (data not shown) for 2 h or a dihydropyridine-type Ca²⁺ channel blocker, nifedipine, at 100 μM for 4 h (Fig. 3B), similarly inhibited the response in leaves from WT plants and from phot1 and phot2 mutants to 17–30% of control levels. After treatment with Ca²⁺ chelating agents, EGTA (1 mM) or O,O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (5 mM), for 30 min, the dark level of [Ca²⁺]_c increased by 10–20 nM compared with the control, and the increase in [Ca²⁺]_c was reduced to 1–10% of control leaves (data not shown). These results suggest that both phot1- and phot2-induced increases in [Ca²⁺]_c require extracellular Ca²⁺ and that phot1 and phot2 modulate Ca²⁺ influx through a PM Ca²⁺ channel.

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Fig. 3.

Effects of inhibitors on BL-induced Δ[Ca²⁺]_c in the WT plants and phot1 and phot2 mutants. A leaf, treated with each inhibitor in the dark, was irradiated with 1 μmol·m^-2·s^-1 of BL. Effects of 5 mM CoCl₂ (A), 100 μM nifedipine (B; 0.4% DMSO was used as the control), 10 μM neomycin (C), and 10 μM U-73122 (D; 10 μM U-73343, an inactive analogue, was used as the control) are shown. The vertical bars indicate the standard error of 3–18 independent experiments.

When leaves were treated with an inhibitor of PLC, neomycin (10 μM) or U-73122 (10 μM), for 30 min before BL irradiation, a different pattern of results was obtained. The dark level of [Ca²⁺]_c increased by 5–10 nM compared with the control (Fig. 7, which is published as supporting information on the PNAS web site), and the BL-induced increase in [Ca²⁺]_c was partly suppressed in the Ler and WS plants and in the phot1 mutant (Fig. 3 C and D). However, both inhibitors did not significantly inhibit the BL-induced increase in [Ca²⁺]_c in the phot2 mutant (Fig. 3 C and D). The results suggest that PLC-mediated phosphoinositide signaling is involved in the phot2-mediated increase in [Ca²⁺]_c. Both neomycin and U-73122 have been described as inhibiting PLC activity, thus reducing the amount of inositol 1,4,5-triphosphate (InsP₃) released and blocking InsP₃-sensitive Ca²⁺ release from internal stores in plant cells (29–31). It is thus strongly suggested that phot2 induces Ca²⁺ release from internal stores, such as vacuoles and ER, depending on PLC-mediated InsP₃ production. Because the phot2-mediated increase in [Ca²⁺]_c was suppressed in the presence of either Ca²⁺ chelating agents or Ca²⁺ channel blockers (Fig. 3 A and B and Fig. 6), Ca²⁺ release from an internal store occurred only when [Ca²⁺]_c was increased via PM Ca²⁺ channels. Ca²⁺-induced Ca²⁺ release (CICR) was likely involved in the phot2-mediated transient increase in [Ca²⁺]_c.

phot1 and phot2 Are Located on the PM. The location of phot1 on the PM (9) is consistent with the idea that phot1 regulates a Ca²⁺ channel in the PM. Although the precise location of phot2 remains unknown, autophosphorylation activity of phot2 was detected in a CM fraction (8). To determine whether phot2 locates on the PM and/or on membranes of internal Ca²⁺ stores, we made transgenic plants expressing HA-epitope-tagged PHOT1 and PHOT2 in phot1 and phot2 mutants, respectively. These transgenic phot1 and phot2 mutants exhibited BL-induced phototropism and chloroplast relocation indistinguishably from WT plants (data not shown), so we refer to them as HA-PHOT1 and -PHOT2 transformants, respectively. We attempted to prepare a PM-rich and an inner membrane-rich fraction by an aqueous two-phase partitioning method.

Assays of marker enzymes showed that the upper and lower phases from the two-phase partitioning system were enriched in PM and inner membranes, respectively (Table 1). The activity of vanadate-sensitive ATPase, which is a marker for PM, was about three times more concentrated in the PM-rich fraction than in the CM fraction, whereas its activity in the inner membrane-rich fraction was only half that in the CM fraction (Table 1). Activities of Triton-stimulated UDPase, antimycin A-resistant NADH–cytochrome c reductase, and antimycin A-sensitive NADH–cytochrome c reductase, which are markers for Golgi, ER, and mitochondrial membranes, respectively, were detected in the inner membrane-rich fractions, with relative activities roughly the same as in the CM fraction. They were not detected or were barely detectable in the PM-rich fraction (Table 1). A marker for vacuole (nitrate-sensitive ATPase) was detected at equal levels in both PM- and inner membrane-rich fractions. These results suggest that our PM-rich fraction is enriched in the PM but contains an amount of vacuolar membranes similar to the inner membrane fraction. The inner membrane fraction contains all kinds of membranes, but the content of the PM was diminished to one-sixth of that in the PM-rich fraction.

We analyzed both membrane fractions by immunoblotting with an anti-HA antibody. In the HA-PHOT1 transformant, a 137-kDa band was detected in the PM-rich fraction but was barely detected in the inner membrane fraction (Fig. 4). This result is consistent with those of Sakamoto and Briggs (9), indicating that phot1 is localized mainly to the PM. In the HA-PHOT2 transformants, a 129-kDa band was detected in the PM-rich fraction but was only faintly detected in the inner membrane-rich fraction (Fig. 4). These results indicate that both phot2 and phot1 are localized mainly to the PM rather than the inner membranes.

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Fig. 4.

Immunodetection of HA-PHOT1 and -PHOT2 from inner membrane-rich (IM) and PM-rich (PM) fractions isolated from leaves of HA-PHOT1 and -PHOT2 transformants, respectively. The proteins (3 μg in each lane) were separated by SDS/PAGE. Epitope-tagged PHOT1 and PHOT2 were detected as slightly larger bands than predicted molecular masses of phot1 (112 kDa) and phot2 (102 kDa), respectively.

Discussion

In this study, we demonstrated that (i) phot2 as well as phot1 is involved in the BL-induced elevation of cytosolic Ca²⁺;(ii) phot1 and phot2 share partly redundant roles for the Ca²⁺ elevation (namely, phot1 is responsible for the lower fluence rate of BL and phot2 for the higher fluence rate); (iii) phot1 and phot2 could induce the Ca²⁺ influx from the apoplast through the Ca²⁺ channel in the PM; and (iv) phot2 could trigger CICR from internal Ca²⁺ stores such as the ER or vacuoles, depending on PLC-mediated InsP₃ production (Fig. 5). In animal cells, CICR through an InsP₃-sensitive Ca²⁺-release channel in the ER and sarcoplasmic reticulum has been reported (32–34). Similarly, phot2 may activate the CICR via an InsP₃-sensitive Ca²⁺-release channel in membranes of internal Ca²⁺ stores.

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Fig. 5.

A schematic model of the signal pathway of phot1- and phot2-dependent increase in [Ca²⁺]_c in leaves of A. thaliana. phot1 and phot2 in the PM could mediate Ca²⁺ influx through a Ca²⁺ channel in the PM. phot2 alone induces CICR from internal Ca²⁺ stores through PLC-mediated signaling. In WT leaves, phot1 possibly increases the phot2-dependent PLC pathway and suppresses phot2-dependent Ca²⁺ influx from the apoplast. We describe the possibility in Discussion. See text for details.

We noticed that the inhibitory effects of PLC inhibitors on the BL response in WT leaves were larger than those of the phot1 single mutant (Fig. 3 C and D). Although phot1 was not involved in the PLC pathway in the phot2 mutant (Fig. 3 C and D), phot1 possibly has some relationship to the PLC pathway in WT leaves. Because the PLC pathway mediates the BL-induced elevation of Ca²⁺ only in the presence of phot2 (Fig. 3 C and D), we propose a model that phot1 increases the phot2-dependent PLC pathway in the WT. Although we should investigate further whether phot1 actually increases the phot2-dependent PLC pathway in WT leaves, this model indicates the possibility that there are some functional interactions between phot1 and phot2 to induce the elevation of cytosolic Ca²⁺. Because there are no reports that phot1 and phot2 interact in mediating the BL-induced phototropism, chloroplast movement, and stomatal opening, it is important for understanding the phototropin-dependent signaling pathways to clarify whether each phototropin functionally interacts with another phototropin to induce the BL-induced elevation of Ca²⁺.

We indicated that phot2-dependent Ca²⁺ influx from the apoplast mediates the BL response in the phot1 mutant (Fig. 3). Babourina et al. (19), on the other hand, indicated that phot2 did not mediate the BL-induced Ca²⁺ influx from the apoplast in etiolated hypocotyls of A. thaliana by using a noninvasive ion-selective electrode under continuous BL illumination. In this study, we also found that phot2-dependent Ca²⁺ influx from the apoplast would be extremely low in the WT because PLC inhibitors reduced the BL-induced increase in [Ca²⁺]_c to a level similar to that in the phot2 mutant [Fig. 3 C and D; the increases in Ca²⁺ in the presence of each drug in WS plants and the phot2 mutant were not significantly different (P > 0.05)]. In the WT, the phot2-dependent PLC pathway increased by phot1 might cover all of the phot2-dependent elevation of [Ca²⁺]_c, and phot2-dependent Ca²⁺ influx from the apoplast would be equivocally eliminated. It is suggested that phot2 alternatively mediates the Ca²⁺ influx from the apoplast according to different circumstances. There are differences between our experimental conditions and those of Babourina et al. (19): (i) tissues examined, leaves or hypocotyls; (ii) plants, deetiolated or etiolated; (iii) fluence rates of BL, 1 μmol·m^-2·s^-1 or 25 μmol·m^-2·s^-1; and (iv) [Ca²⁺]_c measured after a 10-s pulse of BL irradiation or Ca²⁺ influx measured during continuous BL irradiation. Further investigation would be required to clarify whether phot2 affects the Ca²⁺ influx according to the conditions.

PLC-mediated phosphoinositide signaling plays pivotal roles in both animal and plant cells (35, 36). LaBelle et al. (37) revealed that a PLC isoform, PLCβ2, was associated with the PM. In the present study, immunoblot analysis revealed not only phot1 but also phot2 mainly in the PM-rich fraction (Fig. 4). This result is consistent with the idea that the phototropin in the PM regulates PLC activity associated with the same membrane. In mammalian systems, a PLC isoform, PLCβ, is regulated by heterotrimeric G proteins (36). In plant guard cells, abscisic acid activates PLC and can elevate [Ca²⁺]_c via InsP₃ (30), and heterotrimeric G protein regulates abscisic acid signaling (38). G protein might be involved in the phototropin-mediated signaling pathway, as was also found for the phytochrome A-dependent signaling pathway (39).

At fluence rates of 10–250 μmol·m^-2·s^-1, BL induced an elevation in [Ca²⁺]_c in the leaves of the phot1 phot2 double mutants, indicating that some BL photoreceptor(s) other than phototropins are involved in BL-induced changes in [Ca²⁺]_c. In deetiolated seedlings of A. thaliana, it is reported that neither cryptochrome1 nor cryptochrome2 is involved in BL-induced increases in [Ca²⁺]_c (18). However, we observed that red light induced a transient increase in [Ca²⁺]_c in leaves from both WT plants and phot1 phot2 double mutants (data not shown). A phytochrome-mediated elevation of [Ca²⁺]_c was detected in wheat cell protoplasts (40). The possible involvement of phytochrome(s) in BL-induced increases in [Ca²⁺]_c should be examined by using aequorin transformants of these mutants.

This study indicates that the two phototropins are responsible for the BL-dependent increase in cytosolic Ca²⁺ differently. It should be noted that the two phototropins induce the Ca²⁺ increase with different photosensitivities, as was also the case in inducing phototropism and chloroplast movements (6, 23); namely, phot1 works at a lower fluence rate of BL, whereas phot2 works at a higher fluence rate of BL. Although it is not clear whether the phototropin-related responses are mediated by the same intracellular signaling pathways, the BL-induced increase in [Ca²⁺]_c could be one of the intermediate signals for the BL-dependent responses. This study shows that the increase in Ca²⁺ is a common role of phototropins. Furthermore, phot1 and phot2 might increase Ca²⁺ concentration adjacent to the PM and to the inner compartments, respectively, and these differences in localized changes in [Ca²⁺]_c might function in transferring messages to separate signal transduction pathways. Further examination is required to clarify whether and how phototropin-dependent Ca²⁺ signaling mediates BL-induced movements in plants, such as chloroplast movement, phototropism, and stomatal opening; how the two phototropin molecules respond to BL of different fluence rates; how the phototropins mediate the light perception signal to each signal-transduction pathway; what the components of the pathways are; and how the signaling system acts on the Ca²⁺ channels.