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Keywords:

  • phytochrome A;
  • red light;
  • irradiance;
  • photoprotection;
  • hypocotyl;
  • Arabidopsis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plants perceive red (R) and far-red (FR) light signals using the phytochrome family of photoreceptors. In Arabidopsis thaliana, five phytochromes (phyA–phyE) have been identified and characterized. Unlike other family members, phyA is subject to rapid light-induced proteolytic degradation and so accumulates to relatively high levels in dark-grown seedlings. The insensitivity of phyA mutant seedlings to prolonged FR and wild-type appearance in R has led to suggestions that phyA functions predominantly as an FR sensor during the early stages of seedling establishment. The majority of published photomorphogenesis experiments have, however, used <50 µmol m−2 sec−1 of R when characterizing phytochrome functions. Here we reveal considerable phyA activity in R at higher (>160 µmol m−2 sec−1) photon irradiances. Under these conditions, plant architecture was observed to be largely regulated by the redundant actions of phytochromes A, B and D. Moreover, quadruple phyBphyCphyDphyE mutants containing only functional phyA displayed R-mediated de-etiolation and survived to flowering. The enhanced activity of phyA in continuous R (Rc) of high photon irradiance correlates with retarded degradation of the endogenous protein in wild-type plants and prolonged epifluorescence of nuclear-localized phyA:YFP in transgenic lines. Such observations suggest irradiance-dependent ‘photoprotection’ of nuclear phyA in R, providing a possible explanation for the increased activity observed. The discovery that phyA can function as an effective irradiance sensor, even in light environments that establish a high Pfr concentration, raises the possibility that phyA may contribute significantly to the regulation of growth and development in daylight-grown plants.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The ability to respond and adapt to changing environmental stimuli is essential to the growth and development of plants. Of these stimuli, light signals are of fundamental importance. Plants perceive light signals using specialized photoreceptors, the red (R) and far-red (FR) light-absorbing phytochromes and the blue/UV-A-absorbing cryptochromes and phototropins. All higher plant phytochromes are thought to exist as dimers of two identical approximately 120-kDa polypeptides. Each monomer is attached to a light- absorbing linear tetrapyrrole, phytochromobilin (Lagarias and Rapoport, 1980). Higher plants contain multiple phytochromes, the apoproteins of which are encoded by a small family of divergent genes (Sharrock and Quail, 1989). In Arabidopsis thaliana, five phytochromes (A–E) have been sequenced and characterized (Clack et al., 1994). Phytochromes are synthesized in their biologically inactive R-absorbing (Pr) form and acquire activity on photoconversion to their active FR-absorbing (Pfr) form. The absorption of light also triggers translocation of phytochrome to the nucleus, where it has been suggested to regulate gene expression (reviewed by Nagatani, 2004; Nagy et al., 2000). Processes under phytochrome control include seedling de-etiolation, plant architecture, leaf development, and the timing of transition from vegetative to reproductive development (for review see Whitelam and Devlin, 1997).

In contrast to other family members, phyA, which is relatively stable in the Pr form, displays rapid proteolytic degradation on conversion to Pfr, and thereby accumulates to relatively high levels in etiolated seedlings (Clough and Vierstra, 1997; Quail, 1994). The degradation of phyA in the light is accompanied by a decrease in PHYA transcription (Cantón and Quail, 1999; Sharrock and Quail, 1989). It has therefore been proposed that phyA functions predominantly in promoting seed germination and the early stages of seedling de-etiolation (Smith and Whitelam, 1990). Studies using Arabidopsis mutants that are deficient in individual phytochromes have revealed an exclusive role for phyA in promoting seedling de-etiolation in continuous FR (FRc) (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993). Under these conditions, where phyA is relatively stable, phyA mutants displayed long hypocotyls and closed, unexpanded cotyledons, thus resembling wild-type (WT) seedlings that have been grown in darkness. Such observations provided clear and unambiguous evidence of the role of phyA as an effective FR sensor. In contrast, Arabidopsis mutants deficient in phytochromes B–E displayed short hypocotyls and fully opened cotyledons when grown in FRc (Aukerman et al., 1997; Devlin et al., 1998; Franklin et al., 2003b; Reed et al., 1994). In FR-rich environments (such as perceived or actual vegetational shading), the photoconversion of phytochromes B, D and E to their inactive Pr form relieves the inhibition of axis elongation, resulting in a suite of responses termed the shade-avoidance syndrome (Smith and Whitelam, 1990, 1997). Under these FR-enriched conditions, phyA action leads to an inhibition of extension growth, thus antagonizing shade-avoidance responses and preventing excessive elongation, which could ultimately prove lethal. Observations showing phyA mutant seedlings to display longer hypocotyls than WT seedlings when grown in continuous low R:FR-ratio light support this notion (Johnson et al., 1994). The conditional lethality of the phyA mutation in natural light environments was demonstrated by Yanovsky et al. (1995), who showed phyA mutants grown in the field under dense vegetational shade to display extreme hypocotyl elongation and die.

In addition to its role in seedling establishment, phyA performs a multitude of functions throughout Arabidopsis development. These include daylength perception (Johnson et al., 1994; Lin, 2000) and the redundant suppression of internode elongation with phytochromes B and E, leading to a compact rosette phenotype (Devlin et al., 1998). Despite the WT appearance of phyA monogenic mutants grown in white light (Whitelam et al., 1993), comparison of white light-grown phyAphyBphyDphyE quadruple mutants with phyBphyDphyE triple mutants revealed considerably elongated leaves in the quadruple mutant, suggesting a redundant role for phyA in inhibiting leaf elongation (Franklin et al., 2003a).

The marked insensitivity of phyB seedlings to continuous red light (Rc) has established phyB to be the predominant R-sensing phytochrome in Arabidopsis (Devlin et al., 1992; Reed et al., 1994). Minor additional roles in R-sensing have since been proposed for phytochromes C, D and E (Aukerman et al., 1997; Franklin et al., 2003a,b; Monte et al., 2003), although phyC function in R is more obvious in the presence of phyB (Franklin et al., 2003b; Monte et al., 2003). Analysis of phyAphyB double mutants grown in Rc showed seedlings to display modestly longer hypocotyls, increased hook opening and larger cotyledons than phyB monogenic mutants (Casal and Mazzella, 1998; Neff and Chory, 1998; Reed et al., 1994). Such observations provided early evidence of phyA function in R and redundancy of function between different phytochromes. However, the photon irradiances of R used in these analyses were relatively low (<50 µmol m−2 sec−1). Using densely packed monochromatic light-emitting diodes and strict temperature-control measures, we observed considerable phyA activity at high photon irradiances of Rc. Under these conditions, a pool of phyA displayed markedly increased stability in de-etiolating WT seedlings. Observations showing prolonged fluorescence of nuclear phyA:YFP in these conditions additionally suggest irradiance-dependent photoprotection of nuclear-localized phyA.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Mature plant architecture in Rc is regulated predominantly by the redundant activity of phytochromes A, B and D

The growth of a range of phytochrome-deficient mutants to maturity in Rc of high photon irradiance (160 µmol m−2 sec−1) revealed novel roles for phytochromes A and D in mediating plant development. Red light was provided by densely packed light-emitting diodes (600–700 nm, λmax 665 nm). The spectral energy distribution of this light source is shown in Figure 1. At this photon irradiance, all mutant combinations, including phyBphyCphyDphyE quadruple mutants containing only functional phyA, survived to flowering. Mutants deficient in phyA resembled WT plants and displayed downward leaf curling, characteristic of the phot1phot2 double mutant grown in white light (Figure 2; Sakamoto and Briggs, 2002). As expected, phyB-deficient mutants grown to maturity in R displayed elongated petioles and reduced leaf expansion (Figure 2). Mutants deficient in phytochromes A and B displayed visibly elongated internodes, characteristic of phyAphyBphyE and phyAphyBcry1 triple mutants grown in white light at temperatures above 20°C (Devlin et al., 1998; Halliday and Whitelam, 2003;Mazzella et al., 2000). Most interestingly, phyAphyBphyD triple mutants grown in Rc showed developmental retardation, similar to that observed in phyAphyBphyDphyE and phyBphyCphyDphyE quadruple mutants (Figure 2). Triple mutants deficient in phytochromes A, B and E, however, remained phenotypically similar to phyAphyB double mutants and displayed a considerably larger stature, increased mass and increased leaf expansion when compared with phyAphyBphyD triple mutants (Figure 2). Such observations suggest R-mediated leaf expansion and mature plant architecture to be regulated predominantly by phytochromes A, B and D, acting in a redundant manner, with no obvious role for phyE (Figure 2). The phenotypic similarity between phyBphyD double and phyBphyDphyE triple mutants grown in R support this finding (data not shown). The survival of phyBphyCphyDphyE quadruple mutants to flowering also suggests phyA-mediated de-etiolation in R in the absence of other phytochrome family members.

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Figure 1.  Spectral energy distribution of the red LED light source used for all experiments.

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Figure 2.  Phytochromes A, B and D act redundantly to regulate plant development in Rc. WT and phy mutant combinations were grown on soil in Rc at 160 µmol m−2 sec−1 at 21°C for 6 weeks. Scale bar, 10 mm. Mean plant biomass and mean area of leaf 5 (leaf 4 was measured for phyAphyBphyDphyE and phyBphyCphyDphyE mutants) are shown below each image with SE values.

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Phytochrome A is an effective R sensor at high photon irradiances

To investigate further the role of phyA in R-sensing, seedling de-etiolation was examined in phyB and phyAphyB double mutants grown in R at a range of photon irradiances. In accordance with previous studies, when grown at low (<30 µmol m−2 sec−1) photon irradiances of R, phyB mutant seedlings displayed gross insensitivity to the R signal with respect to the inhibition of hypocotyl elongation (Figure 3a; Reed et al., 1993, 1994). However, as the photon irradiance was increased beyond 100 µmol m−2 sec−1, a striking inhibition of hypocotyl elongation was observed, with phyB mutant seedlings grown at 180 µmol m−2 sec−1 displaying almost 50% inhibition when compared with dark-grown controls (Figure 3b,c). By comparison, phyAphyB double mutants displayed considerably longer hypocotyls in all experimental conditions, supporting a significant role for phyA in inhibiting hypocotyl growth at higher photon irradiances. The small response observed in the phyAphyB double mutant at 180 µmol m−2 sec−1 of R implicates the activity of the residual phytochromes C, D and E. In agreement with previous observations, phyB mutants displayed greater cotyledon expansion than phyAphyB double mutants at all photon irradiances (Figure 3a,b,d; Neff and Chory, 1998). As seen for hypocotyl inhibition, phyAphyB double mutants displayed a small increase in cotyledon area at 180 µmol m−2 sec−1, suggesting that phytochromes C, D and/or E function at this photon irradiance (Figure 3d).

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Figure 3.  Phytochrome A displays significant activity in high photon-irradiance Rc. (a, b) Phenotypes of WT, phyB and phyAphyB mutant seedlings grown for 5 days at 21°C in (a) 0.1; (b) 180 µmol m−2 sec−1 Rc. Scale bars, 10 mm. (c, d) Hypocotyl lengths (c) and cotyledon area (d) in seedlings grown for 5 days at 21°C in different photon irradiances of Rc. Bars represent SE values.

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R of high photon irradiance appears to ‘photoprotect’ a pool of phyA from degradation

The first-order degradation kinetics of phyA suggest that rapid proteolysis of the protein should occur following saturation of the Pr-to-Pfr conversion (Hennig et al., 1999a). It would therefore be expected that, following an initial increase in phyA activity with increasing photon irradiance of R, any further increases in photon irradiance would result in diminishing phyA activity. To investigate the nature of the considerable phyA activity observed at 180 µmol m−2 sec−1 of R, phyA degradation was examined in WT and phyB mutant plants using Western blotting. Seedlings were grown in the dark for 4 days before transfer to R at different photon irradiances. Total protein was extracted at 1-h intervals and blots were probed with the phyA-specific monoclonal antibody AS32 (Whitelam et al., 1993). Consistent with previously published observations, in 1 µmol m−2 sec−1 R, phyA appears to display first-order degradation kinetics in both WT and phyB mutants, with no detectable phyA observed beyond 2 h of R-treatment (Figure 4a; Hennig et al., 1999a). However, when seedlings were transferred to R at 180 µmol m−2 sec−1, a detectable pool of phyA was observed to persist in both WT and phyB seedlings throughout the 5-h time-course (Figure 4a). Furthermore, phyA was still detectable for up to at least 8 h after the initiation of R treatment (data not shown). Such behaviour suggests the occurrence of phytochrome ‘photoprotection’, a phenomenon previously demonstrated in a variety of dark-grown species treated with high photon-irradiance white light (Kendrick and Spruit, 1972; Smith et al., 1988). Photoprotection was also observed in seedlings transferred to continuous blue light (Bc) at 180 µmol m−2 sec−1, although the degradation of phyA at the lower photon irradiance was considerably slower than in Rc (Figure 4b).

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Figure 4.  Photoprotection of phyA occurs at high photon irradiances of Rc. (a) Immunoanalysis of phyA degradation in WT and phyB seedlings grown for 4 days in the dark at 21°C and transferred to Rc at 1 and 180 µmol m−2 sec−1 for 5 h. (b) Immunoanalysis of phyA degradation in similarly grown WT seedlings transferred to Bc at 1 and 180 µmol m−2 sec−1 for 10 h. (c) Rapid (2 h) time-courses of phyA degradation in WT and phyB seedlings. Crude extracts were prepared (1 g tissue mL−1 buffer) and resolved on 7.5% polyacrylamide gels. Immunoblotted proteins were probed with the phyA-specific monoclonal antibody AS32, which detected a single band of approximately 120 kDa. (d) Representative phyA half-life calculations obtained by densitometry of blots from 2 h time-courses. Mean half-life value from three independent experiments is displayed with SE values.

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Mutants deficient in phyB display retarded phyA degradation on initial transfer to high photon-irradiance R

The degradation behaviour of phyA at 1 and 180 µmol m−2 sec−1 of R was analysed in more detail through a set of shorter time-course experiments. Seedlings were grown for 4 days in the dark and transferred to R at 1 and 180 µmol m−2 sec−1 for up to 2 h (Figure 4c). The first two samples were harvested at 15-min intervals and remaining samples every 30 min thereafter. Multiple blots were scanned and band densities were used to calculate approximate phyA degradation rates (Figure 4c,d). In WT seedlings transferred to R at both photon irradiances, and in phyB seedlings transferred to Rc at 1 µmol m−2 sec−1, phyA displayed an initial half-life of approximately 40 min (Figure 4c,d). At 180 µmol m−2 sec−1 of R, however, phyB mutants consistently displayed a longer phyA half-life of approximately 118 min (Figure 4c,d). Such observations suggest a role for phyB in modulating the initial stages of phyA degradation at high photon irradiances of R.

Nuclear-localized phyA:YFP displays increased stability in R of high photon irradiance

The cellular localization of photoprotected phyA was investigated through epifluorescence analysis of phyA:YFP in transgenic plants. Dark-grown seedlings were transferred to Rc at 1 and 200 µmol m−2 sec−1, and hypocotyl cells were imaged at a range of time points for up to 7 h. The data shown represent the accumulation and cellular distribution of phyA:YFP after (a) 10 sec, 3 min, 20 min, and (b) 90 min of R at 1 and 200 µmol m−2 sec−1 (Figure 5). After 10 sec of Rc treatment, speckles of phyA:YFP were observed in the cytosol at both photon irradiances (Figure 5a, A,D). After 3 min, cytosolic speckles and nuclear fluorescence were observed at both photon irradiances (Figure 5a, B,E). Following 20 min of Rc treatment, however, cytosolic speckles were no longer visible at either photon irradiance (Figure 5a, C,F). At this time point, the majority of detectable fluorescence was associated with nuclei at both photon irradiances. Considerably increased nuclear epifluorescence was clearly visible following 90 min of Rc in seedlings transferred to R at 200, but not 1 µmol m−2 sec−1 (Figure 5b). Taken together, these data suggest that a proportion of photoprotected phyA resides within cell nuclei.

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Figure 5.  Cellular localization of photoprotected phyA. (a) Epifluorescent analysis of the cellular distribution of phyA:YFP in 3-day-old dark-grown seedlings transferred to Rc at 1 (A, B, C) and 200 µmol m−2 sec−1 (D, E, F) for different periods of time. (b) Epifluorescent analysis of the cellular distribution of phyA:YFP in 3-day-old dark-grown seedlings transferred to Rc at 1 (A) and 200 µmol m−2 sec−1 (C) for 90 min. B and D represent differential interference contrast images of A and C, respectively. Positions of nuclei (nu) are indicated. Scale bar, 20 µM.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Phytochrome A is one of the most extensively characterized angiosperm phytochromes. The most striking phenotype of phyA mutants, and the basis on which they were identified, is a complete insensitivity to continuous FR. When grown in FR, phyA mutants are etiolated in appearance and display none of the de-etiolation phenotypes (hypocotyl inhibition, hook opening or cotyledon expansion) observed in WT controls (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993). In contrast to other family members, phyA accumulates to relatively high levels in etiolated seedlings, but is rapidly degraded on conversion to its active Pfr form (Clough and Vierstra, 1997; Quail et al., 1973). Such behaviour has led to suggestions that phyA functions predominantly upon initial exposure of plants to light (Smith and Whitelam, 1990). However, a pool of phyA exists in de-etiolated tissues and performs a variety of functions throughout the development of light-grown plants. In Arabidopsis, these include the inhibition of elongation growth during shade avoidance (Yanovsky et al., 1995), detection of photoperiod (Johnson et al., 1994; Lin, 2000), the suppression of internode elongation (Devlin et al., 1998), and the regulation of leaf expansion (Franklin et al., 2003a). Physiological analyses of phytochrome-deficient mutants have established that phyA functions in at least two distinct response modes: the very low-fluence response (VLFR), and the high-irradiance response (HIR) (for review see Casal et al., 1998).

The action of phyA has been studied predominantly in FR, the waveband at which the HIR displays action maxima and phyA levels remain relatively high (Hennig et al., 2000). The modest insensitivity of phyA mutants to blue wavelengths has also established a role for phyA as a blue-light sensor (Neff and Chory, 1998; Whitelam et al., 1993). Studies of phyA action in R are limited. Mutant analyses have revealed the R-enhancement of phototropic curvature in blue light to be regulated primarily by phyA (Parks et al., 1996). In addition, a role for phyA was identified in the R-induced positive phototropism response of Arabidopsis roots (Kiss et al., 2003). In this study, phenotypic analysis of phy-deficient mutants grown to maturity in high photon-irradiance R revealed redundancy of function between phytochromes A, B and D in regulating leaf development and plant stature. Triple mutants deficient in phytochromes A, B and D were developmentally retarded and displayed considerably reduced leaf numbers, leaf expansion and plant stature when compared with phyAphyB double and phyAphyBphyE triple mutants. The severely reduced production of leaves in R-grown phyAphyBphyD and phyAphyBphyDphyE mutants suggests phytochromes A, B and D may perform key roles in regulating meristem activity in R.

Redundancy of function between phytochromes A, B and D has not been reported previously in the regulation of any plant developmental process. Phytochromes A, B and E have been shown to regulate the suppression of internode elongation redundantly in Arabidopsis, with no apparent role for phyD (Devlin et al., 1998). Functional redundancy has, however, been reported between phytochromes B and D, with no reported role for phyA. Examples include the inhibition of hypocotyl elongation in R (Aukerman et al., 1997) and the white-light enhancement of hypocotyl inhibition by pulses of R (Hennig et al., 1999b). In the latter study, the inhibition of hypocotyl elongation by pulses of R was shown to be amplified by a white-light pretreatment, requiring the presence of either phyB, phyD or cry1. Redundancy of function between phytochromes A, B and D has, however, been reported for the R-mediated turnover of the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3) (Bauer et al., 2004). In this study, phyA protein abundance was determined in dark-grown phy mutant combinations treated with 1 h of R. Degradation of PIF3 was observed in R-treated phyAphyB and phyAphyD double mutants, but not in phyAphyBphyD triple mutants, suggesting regulatory roles for these phytochromes in PIF3 turnover. The PIF3 transcription factor interacts with phytochrome in the Pfr form (Ni et al., 1998, 1999), and has been shown to bind to G-boxes in the promoters of phytochrome-regulated genes (Martinez-Garcia et al., 2000). More recently, analyses of PIF3 mutant and overexpressing lines have suggested a negative regulatory role for PIF3 in phyB signalling (Kim et al., 2003). The developmental retardation observed in R-grown phyAphyBphyD triple mutants therefore presents an intriguing correlation with PIF3 stability.

The biological function of phyA in R was examined here through detailed analysis of seedling de-etiolation at different photon irradiances. Previously published analyses using photon irradiances of R < 50 µmol m−2 sec−1 recorded modest phenotypic differences between phyB and phyAphyB mutant seedlings (Reed et al., 1994), differences that could, in theory, be attributed to phyA action in the VLFR mode or FR contamination of the filtered incandescent light source. At relatively low photon irradiances of R, significant FR contamination could establish a sufficiently low Pfr concentration to elicit detectable phyA action. Here we have shown that phyA effectively mediates a variety of responses, characteristic of HIRs, in high photon-irradiance R. In addition to demonstrating considerable inhibition of hypocotyl elongation (approximately 50% of dark-grown length in phyB mutants), we have also shown photon irradiance-dependency of phyA-mediated cotyledon expansion in R. It should be noted, however, that the significant hypocotyl inhibition recorded in phyB mutants grown in high photon-irradiance R was observed only if growth temperature was maintained below 22°C. This was achieved not only by controlling air temperature, but also by filtering light through 20 mm water, thus eliminating the radiant heating of seedlings by densely packed light-emitting diodes. In the absence of these control measures, R-grown phyB mutant seedlings frequently displayed longer hypocotyl lengths than dark-grown controls (data not shown), a frequently published, yet apparently paradoxical, phenomenon (Monte et al., 2003; Reed et al., 1994). These observations are consistent with the temperature-dependent auxin-mediated promotion of hypocotyl elongation reported in Arabidopsis seedlings (Gray et al., 1998). The inhibition of hypocotyl elongation and promotion of cotyledon expansion observed in phyAphyB double mutants grown in R at180 µmol m−2 sec−1 suggest the activity of phytochromes C, D and/or E at this photon irradiance. Phytochrome C has been demonstrated to exert a small inhibition of hypocotyl elongation in R at 30 µmol m−2 sec−1 in the absence of all other phytochromes (Franklin et al., 2003b), suggesting that this phytochrome may perform an important role in modulating seedling de-etiolation in the absence of phytochromes A and B at higher photon irradiances.

The abundance of phyA at high photon irradiances of R was investigated through Western blot analysis of phyA degradation. Spectrophotometric studies have reported the R-induced degradation of phyA in Arabidopsis seedlings to proceed with a half-life of approximately 30 min (Hennig et al., 1999a). The same study reported no phyA to be immunochemically detectable beyond 3 h of R-treatment. Our Western blot analyses of phyA degradation using R at 1 µmol m−2 sec−1 support these findings with a half-life of approximately 40 min. At 180 µmol m−2 sec−1 of R, however, phyA was still detectable in both WT and phyB seedlings up to 8 h of R treatment (Figure 4a; data not shown). The increased stability of phyA at higher photon irradiances resembles the phenomenon of phytochrome photoprotection, recorded in a variety of species exposed to high photon-irradiance white light (Kendrick and Spruit, 1972; Smith et al., 1988). In the former study, Kendrick and Spruit spectrophotometrically demonstrated delayed phytochrome degradation in Amaranthus caudatus seedlings exposed to high-irradiance white light. In the latter study, the authors immunochemically demonstrated that white light treatment at 957 µmol m−2 sec−1 retarded the degradation of phytochrome in etiolated seedlings in Amaranthus, Phaseolus radiatus, Pisum sativum and Avena sativa, when compared with seedlings treated with a lower (3 µmol m−2 sec−1) photon irradiance. The possibility therefore exists that some of the published phyA functions observed in white light-grown plants (e.g. Devlin et al., 1998; Franklin et al., 2003a) may result from photoprotection of the photoreceptor under these conditions.

A further intriguing observation from this work was the retarded initial degradation rate of phyA recorded in phyB seedlings transferred to R at 180 µmol m−2 sec−1. In these experiments, the half-life of phyA was more than twice that recorded in WT plants. Such observations suggest a role for phyB in regulating the initial rate of phyA degradation upon transfer of etiolated seedlings to R of high photon irradiance. In accordance with the findings of Hirschfeld et al. (1998), we observed similar levels of phyA in dark-grown WT and phyB seedlings, suggesting that loss of phyB does not affect phyA synthesis. Mutants deficient in phyB have been reported to display greater PHYA synthesis than WT controls following 24 h of Rc treatment (Cantón and Quail, 1999). The possibility therefore exists that increased PHYA transcription in phyB mutants contributes to the increased protein levels observed. Co-immunoprecipitation studies in planta have shown heterodimerization to occur between light-stable phytochromes (phyB–E) in Arabidopsis, but not between phyA and other family members (Sharrock and Clack, 2004). The inability of phyA to form heterodimers with other phytochromes thereby excludes heterodimerization as a potential mechanism for the apparent phyB-induced enhancement of phyA degradation in R of high photon irradiance. One mechanism by which light signals regulate photomorphogenesis is via the specific targeting of proteins for ubiquitination and proteasome-mediated degradation. Phytochrome A has been shown to interact directly with the E3 ubiquitin protein ligase cop1 (constitutive photomorphogenesis 1) following sequestration in nuclear bodies (speckles), leading to its ubiquitination and subsequent degradation (Seo et al., 2004). The apparent delay in phyA degradation in etiolated phyB seedlings transferred to R at 180 µmol m−2 sec−1 could therefore suggest a role for phyB in the sequestration and/or ubiquitination of phyA at this photon irradiance in planta.

The nuclear translocation of phytochrome upon absorption of light is a waveband- and irradiance-dependent process (Gil et al., 2000; Kim et al., 2000; Nagy et al., 2000). Import of phyA to the nucleus is rapid, occurring within minutes of exposure to R, FR or blue light (Kim et al., 2000). Here we investigated the stability of nuclear phyA during prolonged exposure to R at low (1 µmol m−2 sec−1) and high (200 µmol m−2 sec−1) photon irradiances through analysis of a phyA:YFP fusion protein in hypocotyl cells. Cytosolic speckles of phyA:YFP appeared following 10 sec of Rc treatment at both photon irradiances. By 20 min of Rc treatment, however, cytosolic speckles had disappeared at both photon irradiances, and the majority of detectable fluorescence was associated with nuclei. Following 90 min of R treatment, little fluorescence was observed in the nuclei of cells transferred to R at 1 µmol m−2 sec−1. At 200 µmol m−2 sec−1, considerable nuclear epifluorescence was observed after 90 min and was still visible up to 7 h (data not shown). This was largely diffuse but contained discrete speckles, similar to those observed on transfer of transgenic plants to lower photon irradiances (Bauer et al., 2004). Taken together, these data suggest that photoprotection of nuclear-localized phyA occurs at high photon irradiances of Rc.

The use of mutants deficient in multiple phytochromes has been paramount in elucidating the redundant functions of phyA throughout the life cycle of plants (Devlin et al., 1998; Franklin et al., 2003a; Johnson et al., 1994). However, the activation of phyA activity by red, far-red and blue wavelengths precludes the accurate dissection of phyA-signalling pathways in white light. We have established that phyA is an effective R sensor, and that treatment of etiolated seedlings with Rc of high photon irradiance leads to a greater stability of phyA protein than similar treatment at a lower photon irradiance. Under daylight conditions, despite the establishment of a high relative Pfr concentration, photoprotection ensures maintenance of a phyA pool (Kendrick and Spruit, 1972; Smith et al., 1988). The considerable biological activity observed from a small pool of photoprotected phyA in this study suggests a much greater role for this photoreceptor in the development of daylight-grown plants than has previously been considered.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material and growth conditions

All experiments were performed using the La-er accession of Arabidopsis thaliana. The majority of phytochrome mutant alleles used in this study have all been described previously (Halliday et al., 2003; Halliday and Whitelam, 2003). The phyBphyCphyDphyE quadruple mutant was created from existing mutants using standard screening procedures. For analyses of hypocotyl length and cotyledon area, seeds were surface-sterilized in 10% commercial bleach and sown directly onto Lehle medium (Lehle Seeds, http://www.arabidopsis.com) supplemented with 0.8% agar. Following 4 days’ stratification in darkness, seeds were transferred to Rc at different photon irradiances for 5 days. For Western blotting experiments, seed was treated with a white-light pulse following stratification and returned to the dark for 4 days before receiving Rc treatments. In experiments with mature plants, seed was sown directly onto 5 × 5 × 5-cm pots containing a 3 : 1 compost–horticultural silver sand mix and stratified for 4 days in darkness before transfer to Rc at 160 µmol m−2 sec−1. For microscopy analyses, transgenic La-er seed containing 35S:PHYA:YFP was used. The generation of this construct been described previously (Bauer et al., 2004).

Light sources

Red light was provided by densely packed light-emitting diodes at λmax 665 nm (Farnell, http://www.farnellinone.com), filtered through 20 mm water to eliminate radiant heating of seedlings. Blue light was provided similarly by custom-made ceramic-mounted LEDs at λmax 468 nm. All light measurements were performed using a EPP2000 fibre-optic spectrometer (Stellarnet, http://www.stellarnet-inc.com). Ambient temperature at all photon irradiances was maintained at 21°C.

Plant growth analyses

For hypocotyl length measurements, seedlings were photographed and measured using ImageJ software (National Institutes of Health, http://www.nih.gov ). For cotyledon area measurements, cotyledons were excised following 5 days’ growth in Rc, photographed and measured similarly. Data represent means ± SE from at least 40 seedlings. Plants grown in Rc to maturity were photographed at 6 weeks, shortly before bolting occurred in WT controls. Plant biomass and leaf area were recorded from a minimum of 12 plants at 6 weeks. Leaf 5 was chosen for analysis and measured as above (leaf 4 was measured in phyAphyBphyDphyE and phyBphyCphyDphyE mutants).

Protein extraction and immunoblotting

Crude protein extracts (1 mL buffer to 1 g tissue) were prepared as described previously (Devlin et al., 1992). Samples of 40 µL were resolved on 7.5% polyacrylamide gels and electroblotted on to PROTRAN nitrocellulose membrane (Schleicher and Schuell, http://www.whatman.com). Membranes were probed overnight at 4°C with the phyA-specific monoclonal antibody AS32 (Whitelam et al., 1993). Protein bands were visualized by secondary incubation with horseradish peroxidase-anti-mouse immunoglobulin antibodies and chemiluminescence (Amersham International, http://www.amersham.com/). Blots were scanned and optical density values were obtained using QuantiScan (Biosoft, http://www.biosoft.com).

Microscopy

Seedlings were grown in the dark for 5 days, then transferred to R (1 or 200 μmol m−2 sec−1) for different periods of time. Seedlings were mounted onto microscope slides under R and imaged immediately. Fluorescence imaging was performed on a Nikon TE-2000U inverted fluorescence microscope with an Exfo X-cite 120 fluorescence illumination system (Exfo, http://www.exfo.com) and a filter for yellow fluorescent protein (YFP) (exciter HQ500/20, emitter S535/30) (Chroma Technologies, http://www.chroma.com). All images were captured using a Hamamatsu Orca ER cooled CCD camera. Openlab software (Improvision, http://www.improvision.com) was used to capture images at time points 10 sec to 20 min. Volocity II software (Improvision) was used to capture 0.3-µm Z-sections through the samples and to generate 3D images at time point 90 min. The exposure time was constant for all YFP images. After YFP imaging, samples were viewed using Nomarski differential interference contrast microscopy. The experiment was repeated with multiple seedlings and yielded similar results.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Ferenc Nagy (University of Szeged), Eberhard Schäfer and Stefan Kircher (University of Freiburg) for provision of 35S: PHYA:YFP transgenic seed. We also thank the UK Biotechnology and Biological Sciences Research Council and the Royal Society for financial support. K.A.F. is a Royal Society University Research Fellow.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References