Journal of Experimental Botany, Vol. 52, No. 354, pp. 91-97,
January 2001
© 2001 Oxford University Press
Original Papers |
The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence
Department of Organismic Biology, Ecology and Evolution, University of California, Los Angeles, CA 90095–1606, USA
Received 4 August 2000; Accepted 8 August 2000
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Abstract |
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Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.
Key words: Arabidopsis, chloroplast, hypocotyl, phototropic response, zeaxanthin.
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Introduction |
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Phototropism and stomatal movements share several photobiological properties, including similar action spectra that show maxima at around 450 nm and minor peaks at around 420 and 480 nm (Quiñones et al., 1996
). Several genes have been implicated in blue light responses of Arabidopsis. The NPH1 gene product has flavin binding properties and kinase activity, and it is widely considered to be a photoreceptor for phototropic responses in higher plants (Christie et al., 1999). The nucleus-localized, flavin-binding gene products, CRY1 and CRY2 are involved in several blue light responses, including phototropism (Ahmad et al., 1998). On the other hand, mutants for the cry1, cry2 and nph1 genes have normal stomatal responses (Lascève et al., 1999), indicating that these gene products are not components of the cascade transducing blue light-dependent stomatal opening. The blue light response of guard cells is mediated by the chloroplastic carotenoid, zeaxanthin (Zeiger and Zhu, 1998). The absorption spectrum of zeaxanthin closely matches the action spectrum for blue light-stimulated stomatal opening (Quiñones et al., 1996), and stomata from the zeaxanthin-less mutant, npq1, are devoid of a specific blue light response (Frechilla et al., 1999). Biochemical and physiological studies have shown that the blue light sensitivity of guard cells is tightly coupled to their zeaxanthin content (Zeiger and Zhu, 1998).
Zeaxanthin has also been implicated as a blue light photoreceptor for phototropism in corn coleoptiles. The zeaxanthin content of coleoptile tips is linearly related to blue light-induced phototropic curvature, and inhibition of zeaxanthin formation inhibits the phototropic response (Quiñones and Zeiger, 1994). Results showing that coleoptiles of a carotenoid-less mutant of corn bend when illuminated with unilateral blue light have been interpreted as evidence that zeaxanthin is not a photoreceptor in blue light-stimulated phototropism (Palmer et al., 1996). However, phototropic bending clearly depends on several photoreceptors (Liu and Iino, 1996; Ahmad et al., 1998). Therefore, a contribution of zeaxanthin to phototropic bending could be compensated for in the carotenoid-less mutants by another photoreceptor, as observed in the normal stomatal response to white light of the zeaxanthin-less mutant, npq1 (Frechilla et al., 1999).
Blue light sensing by zeaxanthin in the guard cell chloroplast is transduced into the activation of a proton pumping ATPase at the guard cell plasma membrane (Zeiger, 2000). Several intrinsic characteristics of the guard cell chloroplast that appear to be functionally related to photosensory transduction, are also found in the coleoptile chloroplast. These features include excess PSII activity, high rates of oxygen evolution, low rates of photosynthetic carbon fixation, a xanthophyll cycle active at low light levels, reduced grana thylakoids, a high starch content, and a specific blue light response (Zhu et al., 1995; Quiñones et al., 1996).
This study investigated whether the hypocotyl chloroplast of Arabidopsis seedlings also has some of the characteristics associated with blue light sensory transduction in guard cells and coleoptile chloroplasts. Red light was used to modulate greening of dark-grown seedlings, and the phototropic response of seedlings with different extents of greening was probed with blue light. Two Arabidopsis mutants with defective greening, star (gift of Dr Judy Brasslan) and pds2 (Norris et al., 1995), were used to study the relationship between the development of hypocotyl chloroplasts and blue light-dependent phototropism. Some photosynthetic properties of the hypocotyl chloroplast of Arabidopsis were also characterized and compared with those of coleoptile and guard cell chloroplasts.
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Materials and methods |
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Plant material and growth conditions
Seeds of Arabidopsis thaliana (L.) Heynh. ecotype ‘Columbia’, the star mutant (a gift from Dr Judy Brasslan), and the pds2 albino mutant (Norris et al., 1995
) (a gift from Professor Dean DellaPenna) were cold-treated for 3 or 4 d at 4 °C, and illuminated with dim red light for 30 min to induce germination. Seeds of the pds2 mutant were harvested from plants grown from F1, heterozygous pds2 seeds. The F2 seeds segregated into wild-type and pds2 phenotypes in approximately a 3:1 ratio (Norris et al., 1995). For the phototropism experiments, wild-type and star seeds were sown on 0.8% agar supplement with MS salts (half-strength), a vitamin B6 mixture (Sigma), and 1.5% sucrose, and grown in a dark room at 25 °C. The pds2 seeds were grown on 0.8% agar with 0.5% sucrose in the dark at 25 °C. For other experiments, the seeds were sown in soil and grown in a growth chamber with a 12/12 h, dark/light cycle, 23/15 °C day/night temperature, a relative humidity of 80%, and a light fluence rate of 100 µmol m-2 s-1 at the canopy level.
Light sources
The red light used for seed pretreatment and for the induction of zeaxanthin formation was obtained from a ProDesign Network LED array (Santa Clara, CA, USA) with a peak emission at 660 nm. Blue light for the phototropism experiments was provided by a 200 W illuminator (Fiber-lite, Dolan-Jenner Industries, Inc., Lawrence, MA, USA), and two layers of a Plexiglas 2424 blue filter (Rohm and Haas, Hayward, CA, USA) with peak transmission at 470 nm. White light for the oxygen evolution and respiration measurements was provided by the 200 W illuminator described above.
Measurement of phototropic curvature
Wild-type and star seedlings of Arabidopsis were kept in complete darkness or in darkness followed by red light illumination (4 µmol m-2 s-1) of varying duration. For the phototropic stimulation, seedlings with straight hypocotyls were illuminated with unilateral blue light (0.3 µmol m-2 s-1) for 2 h. In subsequent experiments, 3-d-old, dark-grown pds2 seedlings were pretreated with 2 µmol m-2 s-1 red light from the top for 30 min, and then illuminated with unilateral blue light (0.8 µmol m-2 s-1) for 2 h. After the blue light treatment, the hypocotyl profiles were traced on paper and enlarged by photocopying. Curvatures were measured using a protractor.
Pigment analysis
Hypocotyls or cotyledons from 6-d-old, growth chamber-grown wild-type seedlings were used for pigment extraction and analysis. All solvents used were ice-cold. The tissue was homogenized in a mixture of acetone and hexane (1:4, v:v). KCl was added and the mixture was centrifuged at 3000 rpm for 3 min. The upper phase was collected and evaporated under vacuum. The dry pigments were dissolved in acetone and separated in a Beckman HPLC system equipped with an Alltech Spherisorb ODS-1µ column (Gilmore and Yamamoto, 1991; Zhu et al., 1995).
Photosynthetic oxygen evolution and CO2 fixation
Photosynthetic oxygen evolution from hypocotyls or cotyledons was measured with a Clark-type 2 electrode (Hansatech, Norfork, UK). Samples from 6-d-old, growth chamber-grown seedlings were excised, washed with distilled water, and suspended in a reaction mixture containing 0.5 mol m-3 MES-NaOH buffer (pH 6.2), 1 mol m-3 CaCl2, and 5 mol m-3 KCl. The suspension was continuously stirred in a glass chamber kept at a controlled temperature (25 °C) with a water bath. Changes in O2 concentration were calculated from chart recorder traces.
Rates of light-dependent CO2 uptake were calculated from measurements of medium alkalinization (Shimazaki and Zeiger, 1987). The experiments were conducted with the same suspension medium used for O2 evolution measurement. The medium pH was measured with a pH electrode connected to a Beckman QTM 71 pH meter. The magnitude of the pH changes was determined by back titration with 10 nmol of H+ at the end of each experiment. Rates of CO2 uptake were calculated from measured rates of alkalinization, using the Henderson-Hasselbalch equation (pKi=6.35, 25 °C) at the corresponding pH. Chlorophyll content was determined as described previously (Porra et al., 1989). Protein content was determined according to Bensadoun and Weinstein, a modified Lowry method (Bensadoun and Weinstein, 1976).
Fluorescence microscopy
Seedlings were placed on a glass slide with the direction of bending in the plane of the slide surface. Hypocotyls were viewed under a Nikon Optiphot-2 microscope, equipped with interference-contrast optics and epifluorescence and a Nikon Microflex photography attachment. Photographs were taken with Kodak Ektachrome p1600 EPH 135–36 at ISO 1600. The samples were excited with blue light, and overlapping pictures of red-fluorescing, bending hypocotyls were taken under a 10xobjective lens. Sequential overlapping pictures were pasted together to view large sections of the hypocotyl or a complete seedling.
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Results |
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Greening in wild-type Arabidopsis hypocotyls proceeds basipetally from the hook
Chlorophyll-containing chloroplasts fluoresce in the red, and this fluorescence is a useful indicator of the transition from etioplast to chloroplast (Robertson et al., 1995
). Actinic red light was used to stimulate greening in dark-grown Arabidopsis seedlings (Deng, 1994), and chlorophyll fluorescence was used to track light-induced chloroplast differentiation. Dark-grown seedlings had no detectable red fluorescence. Weakly fluorescing chloroplasts at the cotyledon and hook regions were observed after about 30 min of red light irradiation (4 µmol m-2 s-1). Greening proceeded basipetally, from the cotyledon and hook region downwards, and the extent of greening depended on the duration of the red light pretreatment.
Blue light-stimulated phototropism in wild-type Arabidopsis is altered by the extent of greening
The relationship between the extent of greening and the phototropic response of Arabidopsis seedlings to 2 h of unilateral blue light (0.3 µmol m-2 s-1) was studied in seedlings pretreated under three different conditions that altered the extent of greening: 4 d in darkness; 4 d in darkness followed by 4.5 h of red light, and 3 d in darkness followed by 24 h of red light. The red light treatment (4 µmol m-2 s-1) was applied from the top. The phototropic response of the three groups of seedlings was distinctly different (Fig. 1). Bending in dark-grown seedlings was restricted to the hook region, whereas the bending extended farther down in the red light-treated seedlings and was most pronounced in the seedlings pretreated with 24 h of red light.
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The responses to the three pretreatments were further characterized by measuring both curvature and the length of the arch of the hypocotyls (Table 1
; Firn, 1986). The dark-grown seedlings showed the smallest curvature and the shortest arch, while the seedlings pretreated with red light for 24 h had the largest curvature and the longest arch.
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The location of the phototropic arch in bending seedlings matches the distribution of mature chloroplasts
The typical distribution patterns of red-fluorescing chloroplasts in hypocotyls from Arabidopsis seedlings grown under the three conditions described above and treated with 2 h of blue light are shown in Fig. 2. The distribution of red-fluorescing chloroplasts was closely related to the length of the arch. In the dark-grown seedlings, the unilateral blue light treatment induced a limited chloroplast greening at the hook region of the hypocotyl, and the phototropic arch was restricted to that region (Fig. 2). In the red light-treated seedlings, the phototropic arches were consistently restricted to the hypocotyl regions showing mature, red-fluorescing chloroplasts.
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Seedlings of pds2 albino mutant treated with 2 h of unilateral blue light lack phototropic bending
The pds2 mutant of Arabidopsis has a defective phytoene desaturase, and is unable to desaturate phytoene, a key metabolic step in the biosynthetic pathway that generates carotenoids and xanthophylls in greening plastids (Norris et al., 1995). In addition, psd2 is completely devoid of chlorophylls, and has an albino phenotype. The pds2 mutant was used in this study to test the role of hypocotyl chloroplasts further in blue light-stimulated phototropic bending. Wild-type and psd2 seedlings were pretreated with 30 min of red light (2 µmol m-2 s-1) given from the top and illuminated with 2 h of unilateral blue light (0.8 µmol m-2 s-1). At the end of the blue light treatment, wild-type hypocotyls showed curvatures of about 60 °C (Fig. 3) and red-fluorescing chloroplasts in the phototropic arch (not shown). In contrast, the pds2 seedlings had no phototropic bending, and, as expected from their albino phenotype, no red-fluorescing chloroplasts.
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The star mutant has a delayed greening and a delayed phototropic response
The star mutant was selected by screening for delayed greening of seedlings from mutagenized Arabidopsis (J Brasslan, personal communication). Monitoring of greening by chlorophyll fluorescence showed that greening was delayed several hours in star seedlings as compared with the wild type.
The star mutant was used to test whether delayed greening was associated with a delay in the phototropic response. Four-day-old, dark-grown star seedlings were irradiated with 0.3 µmol m-2 s-1 of unilateral blue light for 2 h, and their phototropic curvature was measured. As described above, dark-grown wild-type seedlings treated with 0.3 µmol m-2 s-1 of unilateral blue light bend at the hook region (Figs 1
, 4) and show red-fluorescing chloroplasts only in that region (Fig. 2). In contrast, the star seedlings failed to bend in response to the blue light treatment (Fig. 4), and lacked any detectable red fluorescence at the end of the 2 h blue light treatment (not shown). On the other hand, star seedlings pretreated with 4 µmol m-2 s-1 red light applied from the top for 4 h, prior to the blue light treatment, bent towards the unilateral blue light at the hook region, and showed red-fluorescing chloroplasts at the phototropic arch (not shown). In star seedlings pretreated with red light for 48 h, unilateral irradiation with blue light stimulated bending comparable to that of wild-type seedlings treated with red light for 24 h (Fig. 1), and a similar distribution of red-fluorescing chloroplasts (Fig. 2). The phototropic insensitivity of dark-grown star seedlings, and the ability of red light pretreatments to restore blue light-dependent phototropic responses, provide additional evidence for a functional relationship between phototropic bending of Arabidopsis hypocotyls and the presence of mature chloroplasts within the phototropic arch.
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Unusual photosynthetic and respiratory properties of the Arabidopsis hypocotyl
Rates of photosynthetic O2 evolution and dark respiration of 6-d-old, light-grown Arabidopsis hypocotyls were measured and compared with the responses of cotyledon tissue from the same seedlings (Table 2). Net photosynthetic O2 evolution of hypocotyl tissue illuminated with 100 µmol m-2 s-1 white light was 51.6 µmol O2 mg-1 Chl h-1, about 2-fold higher than that of cotyledons. On the other hand, net photosynthetic carbon fixation of hypocotyl tissue was 23.6 µmol CO2 mg-1 Chl h-1, about the same as that of the cotyledon tissue. Thus, the ratio of O2 evolution to carbon fixation in hypocotyl chloroplasts is higher than that in cotyledon chloroplasts.
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The dark respiration rate of hypocotyl tissue was 81.2 µmol O2 mg-1 Chl h-1, about 3.5 times higher than that of cotyledon tissue (Table 2
). High rates of O2 evolution, resulting from enrichment in PSII activity, and of dark respiration are typical of guard cells and coleoptiles (Zhu et al., 1995). Thus Arabidopsis hypocotyls, guard cells and coleoptiles share unusual photosynthetic and respiratory properties.
Light dependence of zeaxanthin formation in hypocotyl and mesophyll chloroplasts of Arabidopsis seedlings
Another characteristic of guard cell and coleoptile chloroplasts is their capacity to produce the carotenoid zeaxanthin at very low irradiances. In contrast, zeaxanthin formation in mesophyll chloroplasts usually begins at light levels in which photosynthetic electron transport has been saturated (Björkman and Demmig-Adams, 1994; Hurry et al., 1997).
The light dependency of zeaxanthin formation was measured in hypocotyls and cotyledons from 6-d-old, light-grown Arabidopsis seedlings. Tissue samples were dark-adapted for 5 h and illuminated with increasing fluence rates of red light for 30 min. Pigment analysis showed that hypocotyl chloroplasts from light-grown Arabidopsis operated a functional xanthophyll cycle. Dark-adapted hypocotyls had very high levels of violaxanthin and no detectable zeaxanthin (Fig. 5). Upon illumination with 25 µmol m-2 s-1 red light, their zeaxanthin concentration increased to about 10% of the total xanthophyll cycle pool. Zeaxanthin formation in hypocotyl chloroplasts was saturated at about 50–100 µmol m-2 s-1 red light. On the other hand, zeaxanthin was undetectable in cotyledon tissue at 50 µmol m-2 s-1 red light, and reached its maximum at about 200 µmol m-2 s-1 red light (Fig. 5). These results indicate that hypocotyl chloroplasts also share with guard cell and coleoptile chloroplasts a capacity to form zeaxanthin at very low irradiances (Srivastava and Zeiger, 1995; Zhu et al., 1995).
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Discussion |
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This study shows that some of the properties typical of guard cell and coleoptile chloroplasts are also found in the hypocotyl chloroplast. These include high rates of oxygen evolution, a high ratio of oxygen evolution rate to photosynthetic carbon fixation rate (Table 2
), and zeaxanthin formation at low irradiances (Fig. 5). The absence of these characteristics in mesophyll chloroplasts, and their conservation in cells and organs exhibiting key blue light responses argue for a functional role of these properties in photosensory transduction (Zhu et al., 1995; Zeiger and Zhu, 1998).
The results showing that both phototropic curvature and arch length stimulated by unilateral blue light in Arabidopsis seedlings are closely related to chloroplast greening and the distribution of mature chloroplasts in the bending hypocotyl support the concept of a role of the hypocotyl chloroplast in phototropism. Furthermore, the fact that blue light-stimulated curvatures and arch lengths can be altered by manipulating the extent of greening of hypocotyl chloroplasts with red light pretreatments of varying durations (Fig. 2) strongly suggests that mature chloroplasts are required for phototropic bending. The phototropic insensitivity of the pds2 albino mutant (Fig. 3) and the observed temporal coupling between the delayed greening and the appearance of phototropic sensitivity in the star mutant (Fig. 4) provide genetic evidence for a requirement for mature chloroplasts in the phototropic process.
A relationship between the duration of red light pretreatments and arch length in sesame seedlings virtually identical to that shown here for Arabidopsis (Fig. 1) has been reported (Woitzik and Mohr, 1988). As shown for other species (Liu and Iino, 1996), phytochrome was clearly involved in the phototropic response of sesame hypocotyls, but whether phytochrome mediated the red light-dependent changes in arch length remained unclear. Dark-grown, etiolated seedlings synthesize large amounts of phytochrome (Parks and Quail, 1991), so greening should not be required for the supply of phytochrome in the hypocotyl. Thus, the need for mature chloroplasts appears to be different from the phytochrome requirement in both sesame and Arabidopsis. Interestingly, phytochrome regulates greening (Ken-Droor and Horwitz, 1990), so one of the roles of phytochrome in phototropism could be related to the greening process and the availability of mature chloroplasts.
Two mechanisms involving mature chloroplasts in the phototropic response can be envisioned. One pertains to recent findings showing that chloroplasts store significant amounts of IAA (Sitbon et al., 1993; Jia et al., 1997). Light-modulated fluxes of abscisic acid (ABA) across the chloroplast envelope are important in the regulation of intracellular concentrations of ABA in water stress responses (Hartung et al., 1988). A similar role of the chloroplast in the regulation of intracellular auxin concentrations mediating asymmetrical growth in the bending hypocotyl could explain the observed requirement for mature chloroplasts in the phototropic response.
A second mechanism would involve the chloroplastic carotenoid zeaxanthin. If zeaxanthin plays a role in hypocotyl phototropism, a need for a functional xanthophyll cycle in mature chloroplasts of bending hypocotyls would provide another explanation for the observed relationship between the distribution of mature chloroplasts and phototropic bending. In contrast with the detailed knowledge on the sensory transducing cascade mediating blue light perception in guard cells (Zeiger, 2000), the cascade transducing the blue light-dependent phototropic response in the hypocotyl is not well understood (Cashmore et al., 1999; Christie et al., 1999; Sakai et al., 2000). The zeaxanthin-less mutant, npq1, showed a large curvature after a 6 h exposure to unilateral blue light (Lascève et al., 1999). However, as mentioned earlier for phototropism in carotenoid-less corn (Palmer et al., 1996), a seemingly normal response of a mutant phenotype could result from compensatory responses of other photoreceptors in the system (Frechilla et al., 1999). Chloroplasts could readily sense the optical gradient between the shaded and illuminated sides of the hypocotyl or the coleoptile, and zeaxanthin excitation within the antenna bed of the chloroplast could initiate a sensory transducing cascade that might modulate auxin fluxes from the chloroplast, and bending. Interactions with nph1-mediated phosphorylation events (Christie et al., 1999) could be an integral part of this hypothetical mechanism. Although speculative, this working hypothesis illustrates the point that the body of evidence supporting a role of zeaxanthin and the chloroplast in coleoptile and hypocotyl phototropism are sufficiently compelling to warrant further examination.
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Acknowledgments |
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This research was supported by the National Science Foundation. We thank Dr Judy Brasslan for providing the star mutant and Professor Dean DellaPenna for the pds2 albino mutant.
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Notes |
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1 To whom correspondence should be addressed. Fax: +1 310 825 9433. E-mail: zeiger{at}biology.ucla.edu
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