1. Top of page
  2. Abstract
  3. Introduction
  4. Phototropin-dependent calcium mobilization in A. Thaliana
  5. Relationships between blue light-dependent responses and calcium
  6. Future perspectives
  7. References

Plants have several kinds of photoreceptors, which regulate growth and development. Recent investigations using Arabidopsis thaliana revealed that the newly found blue light receptor phototropins mediate phototropism, chloroplast relocation, stomatal opening, rapid inhibition of hypocotyl elongation and leaf expansion (1,2). Several physiological studies suggest that one of the intermediates in phototropin signaling is cytosolic Ca2+. Studies using phototropin mutants have demonstrated that phototropins induce an increase in cytosolic Ca2+ concentration. However, the function of Ca2+ in the phototropin-mediated signaling process remains largely unknown. This review presents findings about phototropin-mediated calcium mobilization and the involvement of calcium in blue light-dependent plant responses.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Phototropin-dependent calcium mobilization in A. Thaliana
  5. Relationships between blue light-dependent responses and calcium
  6. Future perspectives
  7. References

Because of their sessile features, plants use light not only as an energy source but also as signals for their growth and development to adapt to various environmental conditions. In particular, the UV-A (320–390 nm) and blue (390–500 nm) regions of light act upon and regulate different plant responses. Recent genetic studies using mutants of Arabidopsis thaliana revealed that at least two distinct types of blue light receptors are responsible for the blue light-induced responses. Cryptochromes (cry1 and cry2) participate in photomorphogenic processes, which require transcriptional activity (reviewed in 3,4), whereas phototropins (phot1 and phot2, formerly known as NPH1 and NPL1) are involved in rapid and reversible movement responses such as phototropism, chloroplast movement and stomatal opening, in addition to photomorphogenic responses, such as leaf expansion and rapid inhibition of hypocotyl elongation (reviewed in 1,2,5). These phototropin-mediated responses serve to optimize photosynthesis, which results in promoting plant growth in low-light environments (6).

The photoreceptors phot1 and phot2 have a serine/threonine protein kinase domain in the C-terminus and two repeated motifs designated LOV1 and LOV2 (light, oxygen, or voltage-sensing domain) in the N-terminus (7,8) (Fig. 1a). The phototropins undergo autophosphorylation upon absorption of blue light by flavin mononucleotide (FMN) in the LOV domains (9,10). Both phot1 and phot2 localize on the plasma membrane while some phot2 receptors become localized on the Golgi apparatus in response to blue light (11–13). Phototropins are distributed in other green organisms (reviewed in 14), and phototropin genes have been isolated from green algae (Chlamydomonas reinhardtii CrPHOT; 15), ferns (Adiantum capillus-veneris AcPHOT1 and AcPHOT2; 16,17), and moss (Physcomitrella patens PHOTA1, PHOTA2, PHOTB1, and PHOTB2; 18) as well as seed plants.


Figure 1.  Structures, functions and photosensitivity of phot1 and phot2 of A. thaliana. (a) Schematic drawings of protein structures of Arabidopsis phot1 and phot2 (996 and 915 amino acids, respectively). Both phototropins have two FMN molecules associated with the LOV (light, oxygen and voltage) domains LOV1 and LOV2 on the N-terminus region and serine/threonine kinase in the C-terminus region. (b) Functions of phot1 and phot2 in plant movement and growth. (c) The photosensitivities of phot1 and phot2 in the induction of phototropism, chloroplast movement, stomatal opening*, and cytosolic Ca2+ increases. Arrows in chloroplast movement represent the chloroplast accumulation response and the broken arrow shows the chloroplast avoidance response. 1. Sakai et al. (10), 2. Kagawa et al. (8), 3. Jarillo et al. (19), 4. Folta and Spalding (21), 5. Folta et al. (22), 6. Kinoshita et al. (20), 7. Sakamoto and Briggs (11), 8. Takemiya et al. (6), 9. Harada et al. (12). *We observed the increase in stomatal conductance in response to very low fluence rate of blue light (less than 0.1 μmol m−2 s−1) in intact leaves by gas exchange method (Doi, M. and Harada, A., unpublished data), but we did not include the data in this figure.

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In A. thaliana, phot1 and phot2 have different sensitivities to blue light (Fig. 1c). phot1 functions over a wide range of fluence rates of blue light, mediating phototropism from 0.01 to 100 μmol m−2 s−1 and chloroplast accumulation from 0.4 to 100 μmol m−2 s−1 (8,10) (Fig. 1c). phot2 requires higher fluence rates, mediating phototropism from 1 to 100 μmol m−2 s−1 and chloroplast accumulation from 2 to 16 μmol m−2 s−1 (8,10) (Fig. 1c). phot2 alone mediates the chloroplast avoidance response under strong light in mesophyll cells between 32 and 100 μmol m−2 s−1 (8,10,19) (Fig. 1c). In guard cells, phot1 and phot2 contribute redundantly to blue light-induced stomatal opening, and phot1 has a higher light sensitivity than phot2 (20) (Fig. 1c). Both phot1 and phot2 are involved in leaf expansion, and phot1 has been shown to be more sensitive to blue light than phot2 (6,11) (Fig. 1c). Folta and Spalding (21) and Folta et al. (22) discovered that phot1 elicited rapid inhibition of hypocotyl elongation by blue light, but phot2 did not (Fig. 1b).

As most of the responses via phototropins are rapid, and phototropins play minor roles in transcriptional regulation in A. thaliana (23), signal intermediates, which act immediately after blue light perception, have been pursued extensively. Recent genetic analysis revealed that NPH3 and RPT2 function as regulators of the phot1-dependent pathway in some responses (24–27). The endogenous substrates for phototropins have not yet been identified, although a phototropin kinase phosphorylated the artificial substrate in vitro when the kinase was irradiated with blue light (28).

A candidate for the signal intermediate is cytosolic Ca2+. Ca2+ is the most versatile intracellular messenger, able to couple a wide range of extracellular signals to specific responses in plants (29–31). Ca2+ comes from the extracellular space and/or from inner Ca2+ stores such as the endoplasmic reticulum (ER) and vacuoles (29,32). Different sources of Ca2+ produce different patterns of cytosolic Ca2+ increase. Signals arising from the amplitude, rate and spatiotemporal patterning of increases in cytosolic Ca2+, called “Ca2+ signatures” (reviewed in 32, 33, Fig. 2), are delivered to particular effectors such as calmodulin, Ca2+-dependent protein kinases (CDPKs), calcineulin B-like protein (CBL)-CBL interacting protein kinase (CIPK) complexes (CBL-CIPKs) and other Ca2+-binding proteins to initiate cellular responses (Fig. 2, reviewed in 34–36).


Figure 2.  Schematic model indicating how Ca2+ signatures are decoded to various responses. This cartoon represents how signals arising from various pattern of Ca2+ increase are delivered to particular effectors such as calmodulins, CDPKs (calcium-dependent protein kinases) and CBL–CIPKs (calcineurin B-like protein-CBL interacting protein kinase complexes) to initiate various cellular responses.

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In this article, we first introduce recent studies on the phototropin-dependent Ca2+ mobilization using Arabidopsis mutants. We next describe physiological studies on the involvement of Ca2+ in phototropism, chloroplast movement, rapid inhibition of hypocotyl growth, stomatal opening and leaf movement in response to blue light, and discuss the relationships between the results from mutant studies and the physiological evidence.

Phototropin-dependent calcium mobilization in A. Thaliana

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phototropin-dependent calcium mobilization in A. Thaliana
  5. Relationships between blue light-dependent responses and calcium
  6. Future perspectives
  7. References

Recent reports have demonstrated that phototropins mediate the mobilization of Ca2+ in response to blue light using Arabidopsis phototropin mutants (Table 1 and Fig. 3). Baum et al. (37) discovered the phot1-dependent elevation of cytosolic Ca2+ in de-etiolated whole seedlings of A. thaliana that had been transformed with cytosol- or organelle-targeted aequorin, a calcium-sensitive luminescent protein. Ten seconds of blue-light illumination induced a transient increase in cytosolic Ca2+ within a minute, but not in the chloroplasts and nucleus. The blue light-dependent elevation of Ca2+ was not affected in either cry1 or cry2 mutants, whereas the phot1 (nph1) mutant exhibited only half the elevation of the wild type (37), indicating that the Ca2+ increase was partly mediated by phot1. The increase in cytosolic Ca2+ was inhibited by Ca2+ channel blockers (La3+ and nifedipine), and a Ca2+ chelator (O,O′-bis(2-aminophenyl)ethyleneglycol-N, N, N’, N′-tetraacetic acid, BAPTA), suggesting that phot1 mobilizes Ca2+ from extracellular space through Ca2+ channels in the plasma membrane (37).

Table 1.   Studies indicating blue light-dependent Ca2+ mobilization through phototropins in Arabidopsis thaliana.
OrganGrowth conditionIlluminationFluence rate of blue light (μmol m−2 s−1)Contribution of phot1Contribution of phot2Reference
  1. Organs, illumination time, fluence rate of blue light and contribution of phot1 and phot2 in blue light-induced Ca2+ mobilization are summarized. ND, not determined.

10–16 d seedlingsDe-etiolated10 s of illumination∼600 Partly contributed to cytosolic Ca2+ elevationNDBaum et al. (37)
Seedlings (7–10 mm long)De-etiolatedContinuous illumination25Contributed to Ca2+ influxDid not contribute to Ca2+ influxBabourina et al. (38)
Mesophyll protoplasts from 3–10 wk-old leavesDe-etiolatedContinuous illumination275Contributed to Ca2+ currentDid not contribute to Ca2+ currentStoelzle et al. (39)
Rosette leaves from 3–4 wk-old plantsDe-etiolated10 s of illumination0.1–250 Contributed to Ca2+ influx (0.1 ∼ 50 μmol m−2 s−1 of blue light)Contributed to Ca2+ influx and PLC-dependent pathway (1∼250 μmol m−2 s−1)Harada et al. (12)
Hypocotyls (2–3 mm long and with tightly closed hooks)Etiolated10 s of illumination100NDNDFolta and Spalding (22)

Figure 3.  Schematic models of phot1 and phot2-depedent Ca2+ mobilization under weak (a), medium (b), and strong blue light (c) summarized from recent studies. Blue arrow heads above phot1 and/or phot2 indicate the activated state of phototropins. Dark-, medium- and light-blue color of arrow heads indicate strong, medium, and weak blue light, respectively. (a) Under low fluence rate of blue light, phot1 in the plasma membrane (11,12) solely mediated Ca2+ influx through the Ca2+ channels in the plasma membrane. (b) Under medium blue light, phot1 mediated Ca2+ influx through the Ca2+ channels in the plasma membrane. Under the same conditions, phot2 in the plasma membrane (12,13) might alone induce CICR from internal Ca2+ stores through PLC-mediated signaling and also induces Ca2+ influx through Ca2+ channels. Since phot2 induced Ca2+ influx from the apoplast in the leaf cells under medium light (12) but not in de-etiolated seedlings (38), the arrow from phot2 to the plasma membrane Ca2+ channel is colored in gray. In leaves, there is a possibility that phot1 increases the phot2-dependent PLC pathway and suppresses phot2-dependent Ca2+ influx from the apoplasts (green dotted lines; see discussion in 12 for details). (c) Under high fluence rate of blue light, phot2 in the plasma membrane (12,13) might induce CICR through PLC-mediated signaling and Ca2+ influx through plasma membrane Ca2+ channels (12). phot2 was associated with Golgi apparatus at 20–48 μmol m−2 s−1 or higher (13). phot2 in Golgi apparatus may therefore participate in Ca2+ release from internal Ca2+ store. Possible involvement of phot2, likely localized on the chloroplasts (Wada, M., personal communication), in induction of Ca2+ release from the chloroplasts through hypothetical Ca2+ transporters under strong light is also drawn in this figure. As phot1 did not induce Ca2+ influx from the apoplast in the leaf cells under strong light (12) but in de-etiolated seedlings (37) and mesophyll cells (39), we drew the gray arrow from phot1 to the plasma membrane Ca2+ channel. 1. Harada et al. (12), 2. Babourina et al. (38), 3. Baum et al. (37), 4. Stoelzle et al. (39).

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Babourina et al. (38) measured net Ca2+ fluxes across the plasma membrane and cell wall by measuring the concentration gradient of Ca2+ in the surface of Arabidopsis de-etiolated seedlings using vibrating microelectrodes. In both wild-type plants and phot2 mutants, blue light induced immediate Ca2+ influx within 2–3 min and showed a peak at 3–5 min after the illumination, whereas in phot1 mutants and phot1 phot2 double mutants, blue light did not elicit a net Ca2+ influx (38). These results indicate that phot1 solely induces Ca2+ uptake into the cytosol from the apoplast. Stoelzle et al. (39) found that blue light activated the voltage-dependent and calcium-permeable channels in the plasma membrane of mesophyll cells of A. thaliana mature leaves. The Ca2+ current was nearly eliminated in phot1 mutants and completely eliminated in phot1 phot2 double mutants. In contrast, essentially the same Ca2+ current was found in the cry1 cry2 double mutant as in the wild type. These results indicate that phot1 predominantly mediates the blue light-dependent activation of voltage-dependent Ca2+ channels in the plasma membrane of mesophyll cells.

Harada et al. (12) indicated that phot2 as well as phot1 mediated cytosolic Ca2+ increases using rosette leaves of aequorin-transformed phot1 and phot2 single mutants, and those of phot1 phot2 double mutants. phot1 mediated the Ca2+ increase at lower fluence rates, from 0.1 to 50 μmol m−2 s−1 ; phot2 mediated it at higher fluence rates, from 1 to 250 μmol m−2 s−1 (Fig. 1c and Table 1). Despite their structural similarity, phot1 and phot2 seem to have different photosensitivities with respect to inducing increases in cytosolic Ca2+, as was also reported for blue light-induced chloroplast movement and phototropism (8,10) (Fig. 1c). As phot2 elicited the Ca2+ increase in a similar magnitude irrespective of the presence or absence of phot1, phot1 and phot2 additively contributed to the blue light-induced increase in cytosolic Ca2+ (12).

Pharmacological tools indicated that phot1 and phot2 mediated Ca2+ increases with distinct mechanisms in leaf cells (12) (Fig. 3). Ca2+ channel blockers (Co2+, La3+, and nifedipine) and Ca2+ chelating agents (BAPTA and ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N′-tetraacetic acid; EGTA) inhibited Ca2+ increases in the wild-type and phot1 and phot2 mutants, suggesting that both phot1 and phot2 are responsible for the Ca2+ influx through Ca2+ channels in the plasma membrane in leaf cells. In contrast, the inhibitors of phospholipase C (U-73122 and neomycin) inhibited the Ca2+ increase in the wild type and the phot1 mutant, but did not affect the phot2 mutant (12), although the inhibition was partial. As both U-73122 and neomycin inhibit phospholipase C (PLC) activity and thus reduce the amount of inositol 1,4,5-triphosphate (InsP3), which induces Ca2+ release from internal stores (40–42), phot2 likely induces Ca2+ release from vacuoles or ER in plant cells. As the phot2-mediated Ca2+ increase was largely suppressed by both Ca2+ chelating agents and Ca2+ channel blockers, the Ca2+ release might occur only when Ca2+ in the cytosol was increased via plasma membrane Ca2+ channels. Ca2+-induced calcium release (CICR) is likely involved in the phot2-mediated transient increase in cytosolic Ca2+ (Fig. 3b,c) in leaf cells.

It is possible that phot1 inhibits the phot2-mediated activity of Ca2+ influx across the plasma membrane because the magnitude of PLC inhibitions was larger in the presence of phot1 than its absence (see Fig. 3b and discussion in 12 for details). As there is no report that phot1 and phot2 functionally interact in phototropin-induced responses, it is important to clarify whether phototropins interact with each other in increasing cytosolic Ca2+.

Babourina et al. (38) indicated that phot2 did not mediate the Ca2+ influx from the apoplasts in etiolated hypocotyls in the absence of phot1. Harada et al. (12), on the other hand, indicated that phot2 induced Ca2+ influx from the apoplast in the absence of phot1 in leaf cells. Stoelzle et al. (39) showed that blue light induced the small Ca2+ current in the phot1 mutant, whereas the Ca2+ current was absent in mesophyll cells of phot1 phot2 double mutants, indicating that phot2 induces Ca2+ influx. It is suggested that phot2 might alternatively induce the Ca2+ influx according to the tissues and light conditions (Fig. 3b, the gray arrow from phot2 to plasma membrane Ca2+ channel).

These studies indicate that Ca2+ is responsible for the phototropin-dependent signaling pathway and that phot1 and phot2 mediate the elevation of cytosolic Ca2+ differently; phot1 increased cytosolic Ca2+ from extracellular spaces through the plasma membrane Ca2+ channels, whereas phot2 might induce Ca2+ uptake from both internal calcium stores and the extracellular spaces (Fig. 3). The different sources of Ca2+ for phot1 and phot2 may produce different “Ca2+ signatures” and result in different signaling processes.

Relationships between blue light-dependent responses and calcium

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phototropin-dependent calcium mobilization in A. Thaliana
  5. Relationships between blue light-dependent responses and calcium
  6. Future perspectives
  7. References


Unilateral light induces differential growth between the shaded and illuminated sides, and results in phototropic bending. It has been reported that calcium plays a role in the process of differential growth.

Baum et al. (37) found blue light-induced increases in cytosolic Ca2+ under similar conditions as those that induce phototropism in de-etiolated cells of seedlings of A. thaliana and Nicotiana plumbaginifolia. The Ca2+ increase started within a few seconds. Pretreatment of the seedlings with red light had a strong inhibitory effect on the blue light-dependent increase in cytosolic Ca2+ in tobacco seedlings, whereas this treatment had no effect on the Ca2+ increase in Arabidopsis seedlings (37). As phototropic bending was substantially inhibited by prior illumination by red light in many plant species except Arabidopsis (43–47), the different effects of red light on the Ca2+ increase between Arabidopsis and tobacco were in accordance with the phototropic responses of the two plant species. Furthermore, blue light desensitized the responses to a second blue light (37), with sensitivity recovering within 2–3 h after the first blue light illumination. The desensitization and recovery time courses were in good accordance with phototropic responses reported in other plant species (45–47). The results suggest that the calcium signal is involved in the phototropic response.

In contrast, Folta et al. (22) suggested that Ca2+ influx is not responsible for phototropism. BAPTA, which inhibited Ca2+ increase in cells of etiolated seedlings of A. thaliana, did not suppress the phototropism. The experimental conditions between measuring the Ca2+ increase and determination of phototropism, however, were different; Ca2+ increase was observed after a 10 s pulse of blue light irradiation, but phototropism was observed after a continuous blue light irradiation. It is unclear whether the BAPTA treatment completely inhibited the Ca2+ increase during the continuous illumination. Further investigation is required.

As Arabidopsis seedlings are too small to detect differences in the calcium response between shaded and illuminated sides, Ma and Sun (48) suggested the involvement of Ca2+ in cells of shaded and illuminated sides of sunflower hypocotyls. Application of EGTA to the shaded side solely suppressed phototropic bending, whereas application of EGTA, a Ca2+ channel blocker verapamil, and calmodulin antagonists (W-7 and trifluoperazine) to the illuminated side slightly increased the phototropic bending (48).

In monocotyledon maize coleoptiles, Ca2+ differences between illuminated and shaded sides could be found because of their larger size. Gehring et al. (49) revealed that unilateral light induces increases in cytosolic Ca2+ concentration in shaded-side maize coleoptile cells using the calcium-fluorescent reagent Fluo-3. The increase started within 5 min and showed a peak 15 min after illumination (49). Babourina et al. (50) determined net Ca2+ efflux across plasma membranes and cell walls only on the shaded side of maize coleoptiles. The Ca2+ efflux was observed 10–15 min after the start of illumination. Goswani and Audus (51) supplied 45Ca2+ to maize coleoptiles and investigated 45Ca2+ movement in response to unilateral white light. 45Ca2+ began to move to the illuminated side from the shaded side 15 min after the illumination. The measured Ca2+, however, was likely to be derived from both intracellular and extracellular spaces. As the amount of apoplastic Ca2+ is much larger than that of intracellular Ca2+, the differential distribution might represent apoplastic Ca2+. As Ca2+ reduces the cell wall extensibility (52), the increased apoplastic Ca2+ suppresses growth in the illuminated side (please refer to the extensive discussion of the light-induced distribution of apoplastic Ca2+ in phototropism (53)).

We noticed that the mobilization of Ca2+ in monocotyledons occurred slower (within a few minutes; 49,50) than in dicotyledons (within a few seconds; 37). Although the differences are partly because of the different experimental conditions of light, the signaling processes of phototropism between dicotyledons and monocotyledons might be intrinsically different. In the rice genome, there are three copies of phototropin genes (54). Retrotransposon-based or T-DNA-insertional mutants of rice are available (55–57). As rice is large enough to distinguish the responses between shaded and illuminated sides, phototropin-deficient rice expressing Ca2+-sensitive proteins will be useful in examining the phototropin-dependent differential distribution of Ca2+. Differences in the mechanism of phototropism between monocots and dicots should be elucidated.

What is the role of calcium increase during phototropism? During the event of phototropic bending, auxin is indicated to move from the illuminated side to shaded side and becomes asymmetrically distributed (58–61). Blakeslee et al. (62) reported the phot1-dependent relocalization of the auxin efflux carrier PIN1, which is suggested to be involved in auxin redistribution during phototropism. Calcium likely plays a role in PIN1 relocation. Recently, Friml et al. (63) demonstrated that the PINOID kinase influenced the localization of the auxin efflux carrier PIN1 and determined the direction of polar auxin transport. We note here that both the calmodulin-related protein TCH3 and the novel calcium-binding protein AtPBP1 bind to the PINOID kinase (64). Benjamins et al. (65) suggested that PINOID is involved in a gravitropic response. Although it is not investigated whether PINOID kinase is also involved in phototropism, PINOID may function downstream of the phototropin-dependent increase in Ca2+ in illuminated side.

On the other hand, as auxin can induce cytosolic Ca2+ increases (66), it is possible that the increase in cytosolic Ca2+ is caused by auxin but not directly caused by phototropins. Gehring et al. (49) suggested that the Ca2+ increase on the shaded side was caused by translocated auxin, not by light. Changes in both the distribution of auxin and the localization of the auxin efflux carrier PINs have been monitored in intact Arabidopsis seedlings during gravitropism (61). We can determine whether Ca2+ change precedes auxin movement or vice versa by combining intracellular Ca2+ monitoring with the measurement of auxin and PIN distributions in A. thaliana during phototropism.

Rapid inhibition of hypocotyl elongation

The hypocotyls of etiolated seedlings elongate in darkness, whereas blue light quickly inhibits this elongation (reviewed in 5). High-resolution imaging analysis of hypocotyl growth revealed that several blue light receptors participated in the distinct steps of this process; phot1 acted in the primary phase, which started within 30 s after illumination and lasted for 30 min; cry1 and cry2 exerted a role in the second phase, which occurred after anion channel activation; and cry1 but not cry2 mediated the several-hour final phase, which is independent of anion channel activity (5,21). As phot1-induced rapid-growth inhibition did not depend on NPH3, which interacts with phot1 and mediates phototropism, the growth inhibition may have different signaling pathways from phototropism (21).

Folta et al. (22) indicated that blue light induced cytosolic Ca2+ increases in etiolated seedlings of A. thaliana and that BAPTA inhibited both the light-induced Ca2+ increase and the rapid-growth inhibition at a similar concentration dependency. From the results, the authors concluded that Ca2+ influx from apoplasts is important in mediating phot1-dependent growth inhibition. This is the only report showing that the same drug inhibited both the Ca2+ increase and the response. The result of Babourina et al. (38), which showed that phot1 but not phot2 induced Ca2+ influx from apoplasts in hypocotyls, supports the conclusion that Ca2+ influx is important in phot1-dependent rapid-growth inhibition (Table 1).

What is the target of Ca2+ in phot1-induced growth inhibition? In the phot1 mutant, cryptochrome-dependent anion-channel activation was delayed and partially inhibited (21). As the blue light-dependent anion-channel activation required Ca2+ (67), the anion channel can be a target of Ca2+. If so, it is interesting because Ca2+ may therefore mediate the functional interaction of the phototropin and cryptochromes.

Blue light-dependent chloroplast movement

In various kinds of plant cells, chloroplasts change their position in response to light (68,69). Chloroplasts move toward the irradiated area when the light is weak, and chloroplasts accumulate on the front face of the cell. This chloroplast accumulation response enhances photosynthesis by improving the light capture under low-intensity light (70). When cells are irradiated with strong light, the chloroplasts move to the anticlinal wall of the cell to avoid photodamage (71–73).

Both accumulation and avoidance movements of chloroplasts are suggested to require Ca2+, which is released from internal Ca2+ stores such as ER or vacuoles but not Ca2+ influx from apoplasts. Long-term (12 h) but not short-term incubation of Lemna trisulca leaves with EGTA or La3+ or short-term incubation with higher concentrations of nifedipin or verapamil, markedly inhibited both accumulation and avoidance movements of chloroplasts, but short-term incubation with Ca2+ ionophore A23187, EGTA and both A23187 and La3+ did not affect the movement (74,75). The authors concluded that the chloroplast movement was not dependent on Ca2+ influx from apoplasts. In accordance with this, Sato et al. (76) showed that the chloroplast accumulation was affected neither by extracelluar Ca2+ nor by Ca2+ channel blockers in protonematal cells of Adiantum capillus-veneris. In caulonematal cells of P. patens, both UV-A and blue light induced phototropin-dependent Ca2+ influx from apoplasts (77,78). However, inhibition of the Ca2+ influx by La3+ and EGTA did not suppress the chloroplast movement (78,79).

In A. thaliana, phot1 mediates chloroplast accumulation responses (8,19) (Fig. 1b,c); however, phot1-dependent Ca2+ release from internal Ca2+ stores was not found. These results conflict with those described above (see Future Perspectives).

The photoreceptor phot2 mediates both the chloroplast accumulation and the avoidance responses, at lower and higher fluence rates of blue light, respectively (8) (Fig. 1c). Under these light conditions, phot2 induced an increase in cytosolic Ca2+, probably through Ca2+ release from internal calcium stores (12) (Fig. 1c, Fig. 3b,c). The released Ca2+ may play a role in chloroplast movement. The question is how light intensity switches the direction of chloroplast movement through phot2. As the increased Ca2+ concentration through phot2 depended on blue light intensity—larger at a higher fluence rate and smaller at a lower fluence rate (12)—the differences in the concentration of cytosolic Ca2+ may generate specific signals to determine the direction of chloroplast movement. There is also the possibility that differentially localized phot2 may be activated and consequently result in making different calcium signatures in combination with the intensity of the light. phot2 localized on the plasma membrane under dark conditions, and some phot2 became localized on the Golgi apparatus under blue light (13) (Fig. 3c). The association of phot2 with the Golgi apparatus was induced effectively by blue light of 20–48 μmol m−2 s−1 or higher (13). This switching fluence rate of blue light is similar to that required to change the direction of chloroplast movement, 32 μmol m−2 s−1 (8) (Fig. 1c). Weaker blue light less than 20–48 μmol m−2 s−1 may only activate phot2 on the plasma membrane (Fig. 3b) whereas stronger blue light may activate phot2 on the Golgi apparatus as well as on the plasma membrane (Fig. 3c), inducing distinct Ca2+ signatures, which determine the direction of chloroplast movement. phot2 also localized on the chloroplasts (Wada M, personal communication). This is in good accordance in that chloroplasts show avoidance response only in the irradiated small area of the cell by a strong light (8). Because chloroplasts can store Ca2+ and possesses Ca2+ transporters in the envelope membranes (29,32,80–86), it is likely that phot2 on the chloroplast induces the release of Ca2+ to the cytosol from chloroplast themselves under strong light to induce the chloroplast avoidance response (Fig. 3c).

Chloroplast movement is regulated by cytoskeletal proteins. Actin filaments and/or microtubules provide the tracks of movement, and dynamic reorganization of actin filaments was found during chloroplast movement (87–91). Some actin-related proteins such as gelsolin/fragmin of the villin superfamily (actin filament-bundling and/or -severing proteins) and myosin (actin motor) are Ca2+ sensitive (92,93). Some microtubule-related proteins interact with the calcium-binding proteins, and microtubule polymerization is sensitive to Ca2+ (93–96). These cytoskeleton-related proteins may be receptors for Ca2+.

Recently, DeBlasio et al. (97) isolated the plastid-movement-impaired1 (pmi1) mutant exhibiting severely attenuated blue light-induced chloroplast movements. The PMI1 gene product has a domain similar to that of a rice protein, which interacts with calcium-binding proteins. Although it should be investigated whether PMI1 functions in the blue light-dependent signaling pathway or chloroplast motility itself, this finding should help us understand the mechanism of chloroplast movement to find other Ca2+-binding proteins mediating chloroplast movement.

Stomatal opening and other turgor movements of plants

Stomata regulate gas exchange between plants and the atmosphere by changing the size of the stomatal pore, which is surrounded by a pair of guard cells (98,99). Stomata open in response to blue light, and blue light increases the proton pump activity via phosphorylation of the plasma membrane H+-ATPase in guard cells (100–102). The pump activation creates an electrical potential across the plasma membrane and drives K+ uptake through voltage-gated K+ channels in the same membrane (103). The accumulated K+-salt facilitates water influx into guard cells, and the turgor-driven swelling of guard cells leads to stomatal opening.

Irving et al. (104) reported that auxin induced both cytosolic Ca2+ increase in guard cells and stomatal opening. In the signaling processes of blue light-induced stomatal opening, Shimazaki et al. (105) suggested the involvement of calcium by pharmacological tools using both calmodulin antagonists (W-7, trifluoperazine, compound 48/80, prenylamine) and inhibitors of the calmodulin-dependent myosin light chain kinase (ML-9). W-7 and ML-9 inhibited both blue light-dependent proton pumping and stomatal opening in the epidermis of Commelina benghalensis ssp. These results suggest that calmodulin-like proteins or myosin light chain kinase may play a role in the blue light signaling process in guard cells. Shimazaki et al. (106) suggested that the calcium might be released from an intracellular calcium store. Blue light-dependent proton pumping was affected by neither Ca2+ channel blockers nor extracellular Ca2+, but was inhibited by drugs that affected the internal Ca2+ stores.

Extensive investigations have demonstrated that the stomata close in response to phytohormone abscisic acid (ABA) through the oscillation of cytosolic Ca2+ concentration with a definite period in Arabidopsis guard cells (107,108). Such oscillation was required for inducing stomatal closure (108). However, the increase in cytosolic Ca2+, which is mediated by phot1, phot2 and auxin, showed no oscillatory change (12,37,104). If Ca2+ is involved in opening response of the stomata, guard cells should distinguish Ca2+ signals produced by blue light from those by ABA, resulting in the opening or closing of stomata.

Leaves bend through the blue light-induced decrease in turgor pressure in pulvinar motor cells, particularly in Leguminosae (reviewed in 109). Recently, Inoue et al. (110) suggested that phototropin is most likely a blue light receptor that regulates the leaf movement of kidney bean plants. The decrease in turgor pressure is caused by water efflux from motor cells, which is brought about by efflux of K+ and Cl (111,112). The K+-salt efflux is driven by depolarization-activated K+ channels in the plasma membrane (113–115), and the depolarization is triggered by inactivation of plasma membrane H+-ATPase in response to blue light (110,116–119). Moyen et al. (120) found the increase in cytosolic Ca2+ concentration in protoplasts isolated from pulvinar cells of Mimosa pudica in response to UV-A light. Because extracellular Ca2+, La3+, and nifedipine neither affected the UV-A-dependent Ca2+ mobilization nor inhibited blue light-induced leaf movement, Ca2+ was most likely released from internal Ca2+ stores in the motor cells (120).

Future perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Phototropin-dependent calcium mobilization in A. Thaliana
  5. Relationships between blue light-dependent responses and calcium
  6. Future perspectives
  7. References

It has become evident that phot1 and phot2 induce increases in cytosolic Ca2+ concentration using Arabidopsis mutants. These findings strongly support the hypothesis that calcium plays important roles in blue light-induced responses. Strong evidence for the involvement of Ca2+ has been obtained from phot1-dependent rapid-growth inhibition of hypocotyls (22). Other physiological studies of chloroplast movements, stomatal opening and leaf movements suggest the requirement of Ca2+ released from internal stores. phot2 might induce Ca2+ release from internal Ca2+ stores; however, no direct evidence was obtained for the induction of phot1-dependent Ca2+ release from intracellular Ca2+ stores.

In order to obtain further progress in this area, we should determine the “Ca2+ signatures” produced by phototropins. No one has observed the subcellular changes of Ca2+ under blue light as aequorin luminescence is too faint. Most Ca2+-sensitive fluorescent dyes or proteins require blue light as an excitation light and thus interfere with the measurement of cytosolic Ca2+ in response to blue light. We should improve the techniques for finding calcium changes. Such technical advances may clarify whether phot1 induces Ca2+ release from ER or vacuoles as well as clarifying detailed calcium signatures.

In order to identify the components that function downstream of Ca2+ increases, it is necessary to screen the mutants that show the defects in phototropin-mediated responses and to isolate the interactors of both phototropins and phototropin-related proteins such as NPH3/RPT2. It is possible that the obtained factors include Ca2+-regulated proteins. PMI1 and PINOID kinase are possible signaling candidates for chloroplast movement and phototropism, respectively, because these proteins might be regulated by Ca2+-binding proteins (62–64,97). Further analyses of PMI1 and PINOID would reveal how Ca2+ is involved in these blue light-dependent responses.

Finally, we should clarify how phototropins act on Ca2+ transporters such as Ca2+ channels in the plasma membrane and Ca2+ release channels in organelle membranes. It is useful to isolate mutants that have defects in blue light-dependent Ca2+ increases. Pharmacological studies have indicated that phot2-dependent Ca2+ increase is sensitive to PLC inhibitors (12). It should be elucidated whether phot2 activates the PLC-mediated signaling pathway to increase Ca2+ release.

Acknowledgements We thank our laboratory colleagues for their helpful advice. A.H. is supported by Research Fellowship of the JSPS Young Scientists.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Phototropin-dependent calcium mobilization in A. Thaliana
  5. Relationships between blue light-dependent responses and calcium
  6. Future perspectives
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