Phytochrome Modulation of Blue Light-Induced Chloroplast Movements in Arabidopsis1 Stacy L. DeBlasio, Jack L. Mullen, Darron R. Luesse, and Roger P. Hangarter* Department of Biology, Indiana University, Bloomington, Indiana 47405
Photometric analysis of chloroplast movements in various phytochrome (phy) mutants of Arabidopsis showed that phyA, B, and D are not required for chloroplast movements because blue light (BL)-dependent chloroplast migration still occurs in these mutants. However, mutants lacking phyA or phyB showed an enhanced response at fluence rates of BL above 10 mol m⫺2 s⫺1. Overexpression of phyA or phyB resulted in an enhancement of the low-light response. Analysis of chloroplast movements within the range of BL intensities in which the transition between the low- and high-light responses occur (1.5–15 mol m⫺2 s⫺1) revealed a transient increase in light transmittance through leaves, indicative of the high-light response, followed by a decrease in transmittance to a value below that measured before the BL treatment, indicative of the low-light response. A biphasic response was not observed for phyABD leaves exposed to the same fluence rate of BL, suggesting that phys play a role in modulating the transition between the low- and high-light chloroplast movement responses of Arabidopsis.
Plants have evolved a number of developmental and physiological mechanisms that allow them to adapt to changes in their environment. Many of these pathways are modulated in response to various environmental stimuli such as light, gravity, temperature, and nutrient availability (Hangarter, 1997). In the case of light, plants possess a variety of photoreceptor molecules that are sensitive to the quality, quantity, and direction of light within the environment. The known photoreceptors of Arabidopsis include the phytochromes (phys A, B, C, D, and E), which mainly function to detect red light (RL) and far-red light (FRL) and two distinct classes of blue light (BL) photoreceptors, the “photolyase-like” cryptochromes (cry1, cry2, and cry3) and the phototropins (phot1 and phot2; Somers et al., 1991; Ahmad and Cashmore, 1993; Dehesh et al., 1993; Reed et al., 1993, 1994; Clack et al., 1994; Liscum and Briggs, 1995; Guo et al., 1998: Kleine et al., 2003). The crys regulate inhibition of stem elongation, photomorphogenesis, entrainment of the circadian clock, leaf expansion, and anthocyanin production (Ahmad et al., 1995; Somers et al., 1998; Toth et al., 2001; Wang et al., 2001; Mockler et al., 2003). The phots mediate such BL responses as early phase inhibition of hypocotyl elongation (phot1), phototropism, stomatal regulation, and chloroplast movements (Liscum and Briggs, 1995; Christie et al., 1998; Lasceve et al., 1999; Folta 1
This work was supported by the National Science Foundation (grant no. IBN– 0080783) and by the U.S. Department of Agriculture (National Needs Fellowship no. 98 –38420 –584). * Corresponding author; e-mail
[email protected]; fax 812– 855– 6082. Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.029116.
and Spalding, 2001a; Kagawa et al., 2001; Kinoshita et al., 2001; Sakai et al., 2001). Analysis of mutants lacking one or more of these photoreceptors has led to a better understanding of how plants respond to changes in their light environment. It has become increasingly clear that these photoreceptors often act redundantly, synergistically, and/or antagonistically in several different light-mediated pathways (Casal and Boccalandro, 1995; Ahmad and Cashmore, 1997; Ahmad et al., 1998; Mockler et al., 1999; Mazzella and Casal, 2001). For example, the co-action of phys and crys has been well documented in such responses as anthocyanin accumulation, inhibition of hypocotyl elongation, and flowering time (Ahmad et al., 1998; Casal and Mazzella, 1998; Neff and Chory, 1998; Casal, 2000; Devlin and Kay, 2000; Folta and Spalding, 2001b; Mazzella et al., 2001; Nagy and Schafer, 2002). Similarly, phot1-dependent BL-induced phototropism is subjected to modulation by both phyA and phyB, indicating that a functional connection exists between phy and phot (Hangarter, 1997; Janoudi et al., 1997). The nature of this relationship and the involvement of phy in other phot-mediated pathways are unclear. In this paper, we investigate whether phy could be involved in modifying phot-dependent chloroplast movements. Chloroplast movements are light-directed responses that occur in a number of diverse plant groups including algae, moss, ferns, and angiosperms (Zurzycki, 1961; Inoue and Shibata, 1973; Lechowski, 1974; Brugnoli and Bjo¨rkman, 1992; Dong et al., 1996; Park et al., 1996; Trojan and Gabrys, 1996; Augustynowicz and Gabrys, 1999; Gorton et al., 1999; Kagawa and Wada, 1999; Kadota et al., 2000). In species that contain multiple chloroplasts per cell, exposure to dim light causes chloroplasts to accumu-
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late along cell walls oriented perpendicular to the incident light. When the fluence rate of light is high, chloroplasts migrate to the anticlinal walls, parallel to the incident light. In the dark, the ratio of chloroplasts along the anticlinal walls compared with the periclinal walls has been shown to depend upon the light condition in which the plant is grown (Trojan and Gabrys, 1996). Because lensing effects, light reflectance, and other properties of plant cells result in fluence rates being highest along the periclinal walls and lowest along the anticlinal walls (Seitz, 1972; Vogelmann et al., 1996a, 1996b), it has been hypothesized that light-induced chloroplast movements provide an adaptive function allowing for more efficient photon absorption when light is scarce and protection against photodamage via self-shading when light intensities are too high (Zurzycki, 1955; 1972; Haupt and Scheuerlein, 1990; Haupt and Hader, 1993; Terashima and Hikosaka, 1995; Park et al., 1996; Trojan and Gabrys, 1996). Mutants that are not able to move their chloroplasts in response to light were shown to be more sensitive to photodamage than wild-type (WT) plants (Kasahara et al., 2003). Chloroplasts migrating to the anticlinal walls in the upper cell layer of the leaf may also be contributing to the photosynthetic efficiency of the plant by allowing more light to penetrate into the deeper cell layers and/or to leaves located below (Brugnoli and Bjo¨ rkman, 1992; Terashima and Hikosaka, 1995; Gorton et al., 1999). In nonflowering plant species like algae, moss, and ferns, chloroplast relocation can be induced by either RL or BL with the signals acting synergistically when the plants are exposed to both (Kraml and Herrmann, 1991; Kagawa and Wada, 1996; Augustynowicz and Gabrys, 1999; Kadota et al., 2000; Sato et al., 2001). In those plants exhibiting RL-induced chloroplast movement, the response has been shown to be FR reversible, indicating mediation by phy (Kadota et al., 1989, 2000; Kagawa and Wada, 1996; Augustynowicz and Gabrys, 1999). Recent studies have shown that in the fern Adiantum capillus-veneris, RLinduced chloroplast photorelocation is regulated by phy3 (Kawai et al., 2003). In angiosperms like Arabidopsis, light-induced chloroplast movement is BL dependent (Inoue and Shibata, 1973; Trojan and Gabrys, 1996; Kagawa and Wada, 2000). A short pulse of BL is sufficient to initiate directed movement, whereas continuous irradiation will sustain the movement (Trojan and Gabrys, 1996; Kagawa and Wada, 2000). It has been determined recently that BL-induced chloroplast movement in angiosperms is mediated by the phots, with phot2 inducing the high-light response and phot1 and phot2 acting redundantly to mediate the low-light response (Sakai et al., 2001). To determine if phy could be involved in these photmediated pathways, we analyzed BL-induced chloroplast movements in various phy-deficient mutants. 1472
Our results indicate that although phyA, B, and D are not required for induction of chloroplast movements in Arabidopsis, deficiencies in phyA or phyB result in an enhancement of the high-light response. The effects were most pronounced when leaves were exposed to an intermediate fluence rate of BL, suggesting that phy may act to modulate the transition and/or competition between the phot1- and phot2dependent signals for the low- and high-light responses, respectively. RESULTS Chloroplast Movements in Phy-Deficient Mutants
Previous studies have established that BL-induced chloroplast movements can be analyzed by measuring the change in RL transmittance through leaves (Inoue and Shibata, 1973; Trojan and Gabrys, 1996; Jarillo et al., 2001). These light-induced changes in leaf transmittance are due to rearrangements of chloroplasts (Zurzycki, 1961; Trojan and Gabrys, 1996). Chloroplast migration to the periclinal cell walls in response to low-fluence rates of light results in a decrease in light transmittance through leaves due to greater surface area coverage by chloroplasts, whereas under high-light conditions, chloroplasts migrate to the anticlinal walls, and light transmittance increases as the amount of surface area covered by chloroplasts decreases. This technique measures movement in all cell layers simultaneously. Figure 1 shows a time course of the change in RL transmittance in WT Arabidopsis leaves in response to sequential 1-h treatments of low-, intermediate-, and high-intensity BL (450 ⫾ 25 nm; 0.3, 20, and 60 mol m⫺2 s⫺1, respectively). Because sustained exposure to RL alone does not induce chloroplast movement in Arabidopsis (Trojan and Gabrys, 1996), RL transmittance through each leaf was monitored for 1.5 h before exposure to BL to establish the baseline from which to gauge the responses (Fig. 1). Upon exposure to 0.3 mol m⫺2 s⫺1 BL, RL transmittance through WT leaves decreased approximately 0.9% as chloroplasts migrated to the periclinal walls. Irradiation with 20 mol m⫺2 s⫺1 BL increased RL transmittance through WT leaves by about 0.3% compared with the RL-only phase. After increasing the BL to 60 mol m⫺2 s⫺1, RL transmittance through leaves increased by another 1.3%. Thus, the total change in RL transmittance in WT leaves over the sequential BL regime was about 2.5%. During the intermediate and high BL treatments, the change in RL transmittance reached a steady state after 30 min of BL exposure, whereas RL transmittance was still slightly decreasing even after 1 h of the low-fluence rate treatment, indicating that the low-light response occurs slower than the high-light response. Similar differences in the kinetics of the low- and high-light responses also have been observed in other species (Inoue and Shibata, 1973). Plant Physiol. Vol. 133, 2003
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D at the intermediate-fluence rates where the enhancement was the largest to determine which specific phy or combination of phys were involved in the enhanced response (Fig. 2). Dark-acclimated leaves of phy single, double, and triple mutants, except for phyD, showed marked enhancement compared with WT leaves after exposure to 20 mol m⫺2 s⫺1 BL. In addition to the increase in magnitude of the highlight response, the movement reached completion as quickly as WT (average half times were 11.7 ⫾ 3.8 min for WT and 10.1 ⫾ 2.8 min for the phyA and B mutant combinations combined). Thus, removal of the phys resulted in both a greater magnitude and increased rate of change in light transmittance through the leaf. Fluence rate response curves were generated for WT and phyABD to determine if phys impact the light intensity dependencies required for the low- or high-light responses. Dark-treated leaves were exposed to various fluence rates of broad-band BL (480 ⫾ 50 nm) for 1 h. Figure 3 shows the overall change in percentage RL transmittance from the ini-
Figure 1. BL-induced chloroplast movements in WT and phydeficient mutants. The plots show the average change (⫾SE) in the percentage of RL transmittance of leaves relative to the average transmittance measured for the leaves before the first BL treatment. RL transmittance was measured in dark-acclimated leaves for 90 min before sequential treatments of 0.3, 20, and 60 mol m⫺2 s⫺1 BL (450 ⫾ 25 nm) indicated by the arrowheads at 90, 150, and 210 min. Number of leaves (n) is shown for each treatment.
To determine if phy is required for co-activation of BL-induced chloroplast movements, we monitored changes in RL transmittance in leaves from phyABD triple-null mutant leaves (Fig. 1A) and the phy chromophore-deficient mutant hy1-100 (Fig. 1B). BLinduced chloroplast movement responses occurred in both of the phy-deficient mutants, indicating that phys A, B, and D are not required for BL induction of chloroplast movements in Arabidopsis. The involvement of phyC and E could not be completely ruled out by these results because the hy1-100 defect allows some functional phy chromophore to be produced (Davis et al., 1999; Muramoto et al., 1999). Although the phys were not required for BL-induced chloroplast movements, phy deficiency resulted in enhanced responses to intermediate- and high-fluence rates of BL, with the enhanced behavior in both mutants being greatest at the intermediate fluence rate (P ⬍ 0.05 measured at the end of the light treatment; Fig. 1, A and B). We conducted a time course analysis of triple, double, and single mutants of phyA, B, and Plant Physiol. Vol. 133, 2003
Figure 2. Chloroplast movement in response to intermediate BL in phy mutants. Results for phyABD triple mutant and WT (Landsburg erecta [Ler]) are included in each graph for reference. RL transmittance was measured in dark-acclimated leaves for 45 min before exposure with 20 mol m⫺2 s⫺1 BL (450 ⫾ 25 nm). Results are presented as the average change ⫾ SE in the percent of RL transmittance of leaves relative to the average value measured before turning on the BL. Number of leaves (n) is shown for each treatment. 1473
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Figure 3. BL fluence rate dependence in WT and the phyABD mutant. The change in RL transmittance through leaves was measured after dark-acclimated leaves were exposed to 1 h of the indicated fluence rates of broadband BL. The graph shows the average change (⫾SE) in the percentage of RL transmittance of leaves relative to the percent transmittance measured before illumination (n ⫽ 8 and 32 for phyABD and WT, respectively). The curves were obtained by fitting the data to the sum of two competing exponential responses.
tial dark measurement after 1 h. At low-fluence rates of BL, light transmittance decreased in WT leaves with the maximum low-light response occurring at 1.5 mol m⫺2 s⫺1 BL. As the fluence rate of BL was increased from 1.5 to 15 mol m⫺2 s⫺1, the decrease in RL transmittance observed diminished and eventually returned to the level seen in dark-acclimated leaves. Fluence rates of 20 mol m⫺2 s⫺1 or higher were required to cause an increase in RL transmittance above that in dark-acclimated leaves with the magnitude of change in RL transmittance increasing in proportion to the fluence rates of BL over the range we were able to test (up to 60 mol m⫺2 s⫺1 BL). Although exposing WT and mutant leaves to broadband BL (480 ⫾ 50 nm) resulted in slightly less of an overall change in magnitude of percentage RL transmittance compared with the change induced by using a narrower band of BL (450 ⫾ 25 nm; Figs. 1 and 2), the response of the phyABD triple mutant is still altered compared with WT beginning in the transition zone (1.5–15 mol m⫺2 s⫺1) and at higher fluence rates (P ⬍ 0.05 for fluence rates 4–60 mol m⫺2 s⫺1; Fig. 3). The light-dependent chloroplast movements in the pallisade cells of phot mutants (phot1-5 and phot2-1) suggested that phot1 and phot2 have partly redundant roles in mediating the low-light response when the fluence rate of BL is between 2 and 16 mol m⫺2 s⫺1 (Sakai et al., 2001). It is within this range of BL intensities that we observed what appeared to be a transitional low-light response. Specifically, the kinetics of transmittance changes in response to fluence rates of BL within this transition range showed biphasic behavior (Fig. 4). For example, upon exposure to 10 mol m⫺2 s⫺1 continuous BL (450 ⫾ 25 nm), transmittance increased for about 20 min before decreasing to a value lower than that measured before 1474
the BL treatment (Fig. 4). Although the final change in transmittance is consistent with an overall lowlight response, it appears that a high-light response occurred initially. Similar biphasic changes in light transmittance have been observed in foxtail (Setaria viridis; Inoue and Shibata, 1973). Interestingly, phyABD mutant leaves of Arabidopsis failed to develop a biphasic response under these same conditions (Fig. 4). In the phyABD leaves, 10 mol m⫺2 s⫺1 continuous BL caused an increase in leaf transmittance for 30 min, after which it remained constant, indicative of only a high-light response occurring. These results demonstrate that the effect of phy is not limited to BL conditions in which phot2 alone mediates the high-light response but extends to BL conditions in which phot1 and phot2 function have been shown to overlap. Chloroplast Movements in Plants Overexpressing PhyA and PhyB
Because the absence of phyA or phyB resulted in an enhancement of the high-light response, we examined plants overexpressing phyA or phyB (Wagner et al., 1991; Bagnall et al., 1995) to determine if these photoreceptors directly function to attenuate BLinduced chloroplast movements or if the enhancement phenotype is a result of physiological differences between WT and the phy mutants. Following the same protocol used in Figure 1, we monitored chloroplast movements in leaves from plants overexpressing either phyA or phyB (Fig. 5). The low-light response was enhanced at the lower fluence rate of BL (0.3 mol m⫺2 s⫺1) in both overexpressing lines relative to WT with the enhanced low-light response more pronounced in the phyB-overexpressing leaves
Figure 4. Chloroplast movement response in WT and phyABD leaves induced by a “transitional” fluence rate of BL. RL transmittance was measured in dark-acclimated leaves for 60 min before exposure to 10 mol m⫺2 s⫺1 BL (450 ⫾ 25 nm). Results are presented as the average change ⫾ SE in the percentage of RL transmittance of leaves relative to the value measured before turning on the BL. Number of leaves (n) is shown for each treatment. Plant Physiol. Vol. 133, 2003
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than phyA-overexpressing leaves (P ⫽ 0.014 and 0.050 1 h after exposure to 0.3 mol m⫺2 s⫺1, respectively). At the higher BL intensity (60 mol m⫺2 s⫺1), there was a slight enhancement of the high-light response in leaves overexpressing phyB. However, statistical analysis revealed this enhancement to be insignificant (P ⬎ 0.1 at all time points). The overexpressing lines displayed no statistically significant affect on the magnitude of movement after irradiation with 20 mol m⫺2 s⫺1 compared with WT (P ⬎ 0.2 at all time points). Effect of RL and FRL on BL-Induced Chloroplast Movements
To determine if the effect of phy on BL-induced chloroplast movements was dependant upon RL activation in WT plants, we needed to use a region of the spectrum outside the red that could be used to measure the movement responses. Transmittance spectra of an Arabidopsis leaf after dark acclimation and exposure to a high-fluence rate of white light revealed that the regions of greatest change in transmittance were those associated with chlorophyll a, chlorophyll b, and carotenoids with the largest trans-
Figure 5. Time course analysis of chloroplast movements in leaves overexpressing PhyA or PhyB. RL transmittance was measured in dark-acclimated leaves as described in Figure 1. Number of leaves (n) is shown for each treatment. Plant Physiol. Vol. 133, 2003
Figure 6. The effect of RL and FRL on BL-induced chloroplast movements in WT leaves (Ler). The time course was generated by exposing dark-treated leaves to 1 h of 25 mol m⫺2 s⫺1 continuous BL (450 ⫾ 25 nm). For the RL ⫹ BL and BL ⫹ FRL time courses, leaves were also exposed to 25 mol m⫺2 s⫺1 continuous RL or 45 mol m⫺2 s⫺1 continuous FRL during the BL treatments. Chloroplast movements were monitored by measuring the change in BL transmittance. Number of leaves (n) is shown for each treatment.
mittance change occurring in the blue wavelengths (data not shown) similar to that observed in foxtail (Inoue and Shibata, 1973). Thus, by measuring changes in BL transmittance, it is possible to monitor chloroplast movement without exposing samples to RL. We exposed dark-treated WT leaves to either 25 mol m⫺2 s⫺1 BL (450 ⫾ 25 nm), 25 mol m⫺2 s⫺1 RL (660 ⫾ 40 nm) plus BL, or 25 mol m⫺2 s⫺1 BL plus 45 mol m⫺2 s⫺1 FRL (750 ⫾ 50 nm), and measured the change in percentage BL transmittance every 5 min for 1 h (Fig. 6). Because BL is absorbed more strongly by chlorophyll and carotenoids, the change in transmittance through WT leaves when measured with BL is considerably larger than what was observed when RL was used to monitor movement. For example, continuous irradiation with 25 mol m⫺2 s⫺1 BL resulted in an average increase of about 8% above the initial transmittance of BL (Fig. 6) compared with values of less than 1% when the effect of 20 mol m⫺2 s⫺1 BL was measured with RL (Figs. 1–3). At higher fluence rates of BL, we often observed changes in BL transmittance of 12% to 15%, whereas the changes in RL transmittance are typically less than 3% (data not shown). Although the measurements with BL and RL produced different magnitudes of changes in transmittance, our measurements of percentage BL transmittance in WT leaves showed that simultaneous treatments with RL or FRL failed to produce a significant change in chloroplast movements compared with treatment with BL alone (P ⬎ 0.1 for all time points; Fig. 6). DISCUSSION
Light-induced chloroplast movements may be ubiquitous in angiosperms (Zurzycki, 1961; Inoue 1475
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and Shibata, 1973; Brugnoli and Bjo¨ rkman, 1992; Dong et al., 1996; Park et al., 1996; Trojan and Gabrys, 1996; Gorton et al., 1999) and also have been observed in algae, moss, and ferns (Kadota et al., 1989, 2000; Augustynowicz and Gabrys, 1999; Kagawa and Wada, 1999). In nonflowering plant species, chloroplast relocation can be induced by BL or RL. These signals can act additively, but only the RL-induced movement response is FR reversible, indicating roles for both phy and BL receptor systems (Kadota et al., 1989, 2000; Kagawa and Wada, 1996; Augustynowicz and Gabrys, 1999; Kawai et al., 2003). In contrast, induction of chloroplast movement in angiosperms has been proposed to be strictly BL dependent (Inoue and Shibata, 1973; Trojan and Gabrys, 1996) and is dependent on the phot family of photoreceptors (Kagawa et al., 2001; Sakai et al., 2001). According to Sakai et al. (2001), phot1 acts primarily to induce the low-light response at fluence rates of BL less than 2 mol m⫺2 s⫺1. Between 2 and 16 mol m⫺2 s⫺1, phot1 and phot2 function overlap in mediating the low-light response, although it is unclear if the two photoreceptors contribute equally within this range. Above 16 mol m⫺2 s⫺1 BL, phot2 drives the highlight response. Although RL alone does not induce chloroplast movement in Arabidopsis, it has been suggested that simultaneous irradiation with RL is required for full induction of the responses by BL (Kagawa and Wada, 2000). Similarly, phot-mediated phototropism has been shown to be enhanced by RL activation of phys A and B, indicating a functional synergy between the two classes of photoreceptors. To test whether or not phy could be involved in BL-induced chloroplast movements, we measured chloroplast movements in WT and phy-deficient mutants using a photometric procedure based on measuring changes in light transmittance through leaves induced as a result of chloroplast migration (Inoue and Shibata, 1973; Walczak and Gabrys, 1980). These changes in light transmittance reflect light-dependent chloroplast movements as verified by microscopic data that demonstrate correspondence between chloroplasts relocating to the periclinal and anticlinal cell walls and light transmittance of leaves (Zurzycki, 1955, 1972; Inoue and Shibata, 1973; Lechowski, 1974; Trojan and Gabrys, 1996; Augustynowicz and Gabrys, 1999; Gorton et al., 1999). This technique provides a measure of chloroplast movement in all cell layers and works well in Arabidopsis (Walczak and Gabrys, 1980; Trojan and Gabrys, 1996). The most significant finding in this work is that phys A and B can suppress chloroplast movements at fluence rates of BL above 5 mol m⫺2 s⫺1 as shown by a striking enhancement in the change in RL transmittance in phy-deficient mutants (Figs. 1–4). This apparent enhancement of the high-light response was much more substantial for intermediate fluence rates (20 mol m⫺2 s⫺1; Fig. 2). However, the differ1476
ence between mutants and WT was still significant at higher fluence rates of BL and in the range of fluence rates in which the transition from the low- to highlight responses occurred in WT leaves (between 2–15 mol m⫺2 s⫺1). In the transition range, there was a general decrease in RL transmittance consistent with a low-light response, yet the magnitude of change was less than that observed when leaves were exposed to lower fluence rates of BL (Fig. 3). The effect of phy is also apparent in the transition between the low- and high-fluence responses as suggested by the kinetic response of the change in RL transmittance we observed when leaves were irradiated with a transitional fluence rate of BL (Fig. 4). In WT leaves, RL transmittance increased above the dark level for about 20 min before decreasing to a level lower than that measured before treatment with BL. This biphasic response was not observed when the phyABD leaves were irradiated with the same “transitional” fluence rate of BL as WT. In the phyABD mutant, RL transmittance increased and remained high for over 2 h (Fig. 4). Phys A and B may modulate this response by either directly inhibiting the high-light signal and/or indirectly by enhancing the low-light response signal. The results with the phy overexpressors tend to support the latter hypothesis because in the presence of excess phyA or phyB, the low-light response was enhanced (Fig. 5). Although the final effect with the transition fluence rate is indicative of an overall low-light response, initially it appears that there is a transient high-light response. Inoue and Shibata (1973) also reported a biphasic response in leaves of foxtail at some intensities of BL. It is possible that the kinetics reflect differences in the responses between mesophyll layers. For example, the palisade cells may first undergo a high-light response followed by the spongy mesophyll initiating the low-light response. The slower kinetics of the low-light response relative to the highlight response coupled with mesophyll layer-specific responses could lead to biphasic changes in light transmittance through a leaf. However, Kagawa and Wada (1999) found that when low-light-acclimated fern prothallial cells were irradiated with a microbeam of 5 W m⫺2 (approximately 20 mol m⫺2 s⫺1) of BL continuously, chloroplasts located within the irradiated area initially moved out of the light but changed direction and migrated back into the light after 10 min of exposure. Because the fern response was observed in a single cell, the biphasic response may represent an antagonistic relationship between the phot2-induced signal for high light and the lowlight response signal generated by activation of both phot1 and phot2 by these transition fluence rates. Thus, differences in longevity and kinetics of the low- and high-light responses may result in a faster generation of the high-light signal by phot2 that eventually gets overtaken by a slower generation of the low-light response signal. Plant Physiol. Vol. 133, 2003
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We were not able to observe a significant effect of RL or FRL on chloroplast migration in WT Arabidopsis when chloroplast movements are measured using the change in percentage transmittance of BL (Fig. 6). Exposure of WT leaves to simultaneous RL and an intermediate fluence rate of BL (25 mol m⫺2 s⫺1) did not result in a significant change in response comparable with leaves exposed to BL only. To try to inactivate phyB, we also exposed WT leaves to FRL during the BL treatments, but we were not able to observe an effect of FRL (Fig. 6). Based on the results with the phy-deficient mutants, FRL might be expected to enhance the high-light response because FR reversibility is used as an indicator of phy action in low-fluence rate responses (Casal et al., 1998). The lack of effect of simultaneous RL and FRL treatments may result from phy activation by BL (Quail et al., 1995) or from the necessity of using continuous light treatments instead of the short pulses of light typically used to demonstrate R/FR reversibility of phy action (Casal et al., 1998). However, the results confirm that RL is not required for substantial chloroplast movement in Arabidopsis as it is in algae, moss, and ferns (Kadota et al., 1989, 2000; Kagawa and Wada, 1996; Augustynowicz and Gabrys, 1999; Kawai et al., 2003). Because chloroplast movements are fluence rate dependent and leaves of some phy mutants develop differently than WT in ways that may increase light conditions within the cell layers (e.g. smaller chloroplasts, smaller cells, and lower chlorophyll content; Chory et al., 1989; Neff and Chory, 1998), it seemed possible that the enhancement phenotype may be related to leaf cells in the phy mutants being more “sensitive” to lower intensities of BL. However, the differences in the fluence rate response that we observed in the phyABD line compared with WT indicate that the enhancement in the phyABD mutant is probably not the result of a change in light penetration because the enhancement is not apparent until the fluence rates reach the transition between the low- and high-light responses (Fig. 3). An alternative hypothesis is that phot2 may be more sensitive to small changes in light intensity if the low-light response is primarily mediated by phot1. Given this scenario, increased light penetration in the phy mutants could lead to an enhancement of the high-light response without shifting the range of fluence rates that induce a low-light movement response. Yet, in the phy overexpressors, we did not observe an attenuation of the high-light response that would be indicative of less light penetration due to increased levels of chlorophyll (Fig. 5). Overall, this study suggests that phyA and phyB may contribute to BL-dependent chloroplast movements by modulating the transition between the high- and low-light responses meditated by phot1 and phot2. However, at this time, it is not possible to determine if phyA and phyB are acting directly to Plant Physiol. Vol. 133, 2003
enhance the low-light response signal, which in turn inhibits the signal for the high-light response, or if phyA and phyB have different effects, with phyA primarily enhancing the low-light response and phyB inhibiting the high-light response. Analysis of various phy and phot double mutants should help differentiate between these models or suggest an alternative mechanism. Regardless of the exact mechanism, this work suggests that the phys may play a role in the fine-tuning of light transmittance properties of leaves for maintenance of maximal photosynthetic productivity during periods when light levels are transitioning close to the photosynthetic light compensation point. MATERIALS AND METHODS Growth Conditions Arabidopsis phy-deficient mutant seeds were obtained from Bob Sharrock (Montana State University, Bozeman; single, double, and triple mutants) and Joanne Chory (The Salk Institute, La Jolla, CA; hy1-100). PhyA and phyB overexpressor seeds were obtained from Peter Quail (University of California, Berkeley). All phy null mutants were in the Ler background. Overexpressors were in the RLD background. Seeds were sown in watersoaked Scott’s Plug mix (Scotts-Sierra, Marysville, OH) and incubated at 4°C in darkness for 3 to 4 d. Plants were then grown at 23°C in a Percival growth chamber (Percival Scientific, Perry, IA) with a 12-h photoperiod using white light (80–100 mol m⫺2 s⫺1) provided by a mixture of cool-white fluorescent and incandescent bulbs. After 2 weeks of growth, seedlings were fertilized with K-grow All-Purpose Plant Food (Kmart, Troy, MI) and every 2 weeks thereafter.
Transmittance Measurements Leaves from 4- to 6-week-old WT (Columbia, Ler, or RLD), phy-deficient, and phy-overexpressing plants were excised and incubated in a dark humid chamber for 9 to 15 h. Individual leaves were then placed with the leaf blade gently sandwiched between two glass slides, and the petiole, which extended beyond the slides, was wrapped with a water-soaked paper towel to keep the leaves hydrated. The glass slides were placed so that the leaf blade covered a 5-mm hole cut in black electrical tape that covered a red Plexiglas base (Rohm and Haas no. 2423, Dayton Plastics, Columbus, OH). The sensor from a LI-COR 1800 spectroradiometer (LI-COR Inc., Lincoln, NE) was fastened directly under the 5-mm area. A 660-nm RL-emitting diode (LED, Radio Shack, Fort Worth, TX) mounted 2 cm above the 5-mm aperture of the stage provided the RL source (20–25 mol m⫺2 s⫺1) for transmittance measurements. RL transmittance of dark-acclimated leaves was measured for at least 45 min before initiation of the indicated BL treatments. BL was provided by filtering light from a halogen fiber optic light microscope illuminator (Cole Palmer, Chicago) with a blue interference filter (450 ⫾ 25 nm, Melles Griot, 03FIB304, Mellis Griot, Rochester, NY). The fiber optic light guide for the BL was positioned at an angle of 60° relative to the surface of the leaf. The fluence rates of BL were achieved with neutral density filters placed in the light path. RL transmittance though leaves was measured with the LI-COR 1800 spectroradiometer every 2 nm and integrated between 650 and 670 nm at the indicated time intervals. For each leaf, change in percentage RL transmittance was calculated as: ⌬% RL transmittance ⫽ ((It/Io) ⫻ 100)/IA), where Io and It are the incident and transmitted RL fluence rate, respectively, and IA is the average percentage RL transmittance value measured before the BL treatments. Results are presented as the average change in percentage RL transmittance for the indicated number of leaves. For the experiments that measured chloroplast movements using the change in transmittance of BL, excised WT (Ler) leaves were handled as described above except they were dark acclimated for 18 h and mounted on a clear 1-mm-thick glass stage. The dark-acclimated leaves were then exposed to 25 mol m⫺2 s⫺1 BL (450 ⫾ 25 nm, Melles Griot, 03FIB304) or in
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some treatments simultaneously with 25 mol m⫺2 s⫺1 RL or 45 mol m⫺2 s⫺1 FRL (750 ⫾ 50 nm) generated by a 660-nm-emitting LED and an RL-/FRL-emitting diode array (Qream-2200, Barnevled, WI). The BL source was mounted perpendicular to the leaf surface, whereas the RL and FRL sources were positioned at angles of 60° and 10° relative to the surface of the leaf, respectively. BL transmittance was measured every 5 min using the LI-COR 1800 spectroradiometer and change in percentage BL transmittance was calculated as: ⌬% BL transmittance ⫽ ((It/Io) ⫻ 100)/IA), where Io and It are the incident and transmitted BL fluence rate, respectively, and IA is the percentage BL transmittance value measured at time 0 min. Results are presented as the average change in percentage BL transmittance for the indicated number of leaves.
Fluence Rate Response WT (ecotype Ler) and phyABD triple mutant leaves were excised from 6-week-old plants and dark acclimated for 17 to 24 h sandwiched between an inverted petri dish bottom and lid. Eight leaves were arranged in each petri dish so that their petioles pointed toward the center and were positioned to be on moistened Whatman filter paper (42.5-mm diameter, Whatman, Clifton, NJ). RL transmittance was measured through each leaf using a custom device that consisted of a clear Plexiglas turntable. At one position, an LI-190SA Quantum sensor (connected to a LI-COR LI-189 Quantum Radiometer Photometer) was mounted below the turntable with red LED’s positioned directly above the quantum sensor. The turntable was built to hold each inverted petri dish in a specified orientation so when the turntable was rotated to eight precise positions, each of the eight leaves could be located in turn between the red LEDs and the quantum sensor for light transmittance measurements. The device design ensured that each transmittance measurement was made through the same 5-mm-diameter area of each leaf, even after a petri dish was removed and later returned to the turntable. RL transmittance was measured in the dark-acclimated leaves before and after exposure to 1 h of the different fluence rates of broadband BL (480 ⫾ 50 nm) given from above. For each leaf, the change in percentage RL transmittance was calculated as previously described. The data presented are the average change in percentage RL transmittance for a total of eight or 32 phyABD and WT leaves, respectively, for each fluence rate of BL. Fluence rates of BL from 0.3 to 10 mol m⫺2 s⫺1 were provided by filtering light from cool-white fluorescent light bulbs through blue Plexiglas. To obtain fluence rates between 15 and 60 mol m⫺2 s⫺1, light from halogen flood lamps (150-W Quartzline, General Electric, Fairfield, CT) was filtered through 7 cm of 1.5% (w/v) CuSO4.7H2O (Sigma, St. Louis) and blue Plexiglas. Both BL sources provided similar spectral outputs peaking near 480 nm with approximately 100-nm half bandwidth (measured with a LI-COR 1800 spectroradiometer). Received June 24, 2003; returned for revision August 1, 2003; accepted September 18, 2003.
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