Journal of Experimental Botany, Vol. 51, No. 345, pp. 703-711,
April 2000
© 2000 Oxford University Press
Phytochrome regulation of phytochrome A mRNA levels in the model short-day-plant Pharbitis nil
1 Plant Genetics and Biotechnology Department, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
2 Department of Botany, School of Plant Sciences, University of Reading, Whiteknights, Reading RG6 2AS, UK
3 Nicholas Copernicus University, Institute of Biology and nvironmental Protection, Depatment of Plant Physiology and Morphogenesis, Torun, Poland
4 Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada
Received 6 December 1999; Accepted 10 December 1999
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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The exposure of dark-grown Pharbitis nil seedlings to continuous R induces a rapid decrease in PHYA mRNA abundance with a half-life of about 2 h. A 5 min R pulse also induces this decline, and the effect is partially reversible by subsequent FR irradiation, confirming that the regulation of expression is mediated via the Pfr form of a phytochrome. When de-etiolated seedlings are returned to darkness after a W photoperiod, PHYA mRNA slowly reaccumulates from 20% to 50% of the dark level within 24 h. The rate of reaccumulation is greatly accelerated by the removal of Pfr with a FR pulse, resulting in reaccumulation to 100% within approximately 11 h. Without FR irradiation PHYA mRNA expression remains fully repressed for at least 11 h after the end of the photoperiod, suggesting that the controlling Pfr is highly stable.
Key words: Phytochrome A, Pharbitis nil, photoperiodism, short-day plant, far-red light.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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The photoperiodic control of floral induction in higher plants has been extensively studied with a view to understanding the molecular mechanisms involved (for a review see Vince-Prue, 1994
). Much of the research has concentrated on understanding the mechanisms of photoperiodism in SDP, particularly Pharbitis nil, a model SDP (Vince-Prue et al., 1978; Lumsden et al., 1982; King et al., 1982; Vince-Prue and Lumsden, 1987). It is now widely accepted that the plant measures photoperiod via the interaction of light with a circadian rhythm in light-sensitivity (a theory first suggested by Bunning, 1960).
The phytochrome system family of R/FR photoreceptors, has been shown to be involved in both the initiation of the timing rhythm at dusk (Lumsden and Vince-Prue, 1984), and the interaction of light with sensitive phases of the rhythm (Vince-Prue, 1975; Vince-Prue and Lumsden, 1987). Five members of the phytochrome gene family have now been identified (Clack et al., 1994; Pratt, 1995). Different phytochrome types differ in their expression patterns, stability in the active Pfr form and sensitivity to various light qualities. While specific photomorphogenic responses have been attributed to specific phytochromes, for example, de-etiolation via the FR-HIR has been shown to be mediated via phytochrome A (Parks and Quail, 1993; Nagatani et al., 1993; Whitelam et al., 1993)), and the shade-avoidance response principally via phytochrome B (Nagatani et al. 1991; Whitelam and Smith, 1991), little is known about which phytochrome species are involved in photoperiodism in SDP. Phytochrome B is thought to affect the photoperiodic control of flowering, as the PHYB-deficient mutants of the LDP Arabidopsis thaliana display an early flowering phenotype (Goto et al., 1991). Phytochrome B also affects the photoperiodic sensitivity of tuber formation in potato (Jackson et al., 1996). Phytochrome A has been shown to play a central role in LDP photoperiodic floral induction in Arabidopsis thaliana (Johnson et al., 1994) and in pea (Weller et al., 1997).
PHYA mRNA is the most abundant transcript in dark-grown (etiolated) tissue and is the only phytochrome transcript to be regulated by light (Clack et al., 1994), being down-regulated upon exposure to R or W. The effect of exposure to a R pulse can be reversed by treatment with FR showing that this light regulation is mediated through phytochrome. The down-regulation of PHYA mRNA by light has been shown in several species including Arabidopsis and pea (Sharrock and Quail, 1989; Sato, 1988), however, the degree and speed of the down-regulation varies between species, being more pronounced and rapid in monocots than in dicots. In Avena, for example, just 5 s of R are sufficient to repress transcription of the PHYA gene and reduce transcript levels by around 90% (Lissemore and Quail, 1988), however, a R pulse of 5 min has little effect in Arabidopsis where 3 h of continuous W are necessary to cause a strong reduction in transcript level (Sharrock and Quail, 1989). Unlike Arabidopsis, pea responds to a R pulse, although not to the same extent as Avena, 3 min of R resulting in a 40% reduction in transcript levels (Tomizawa et al., 1989).
It has been suggested that PHYA, or a similar phytochrome with unstable Pfr, may have a role in photoperception in SDP floral induction (Thomas, 1991; Vince-Prue, 1994), but the nature of the regulation of PHYA in SDP has not yet been demonstrated. The aim of the work described in this paper is to determine the extent to which PHYA expression is regulated by light in the model SDP Pharbitis nil.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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Plant material and light treatments
Seeds of Pharbitis nil Choisy cv. Violet (Marutane, Kyoto. Japan) were pretreated with concentrated sulphuric acid for 40 min, rinsed in cold water, and allowed to imbibe for 24 h in darkness. Seeds were then sown on moist vermiculite, 10 seeds per 8 cm square pot, covered to a depth of 2 cm in moist vermiculite and placed in darkness, 25 °C. After 4 or 5 d in the dark, seedlings were subjected to different light treatments, cotyledons harvested at various time points (40 seedlings per sample point), frozen in liquid nitrogen and stored at -80 °C until subsequent RNA extraction.
Red light was supplied by filtering the light from Grolux fluorescent lamps through one layer of Plexiglas R 501 (Rohm und Haas; supplied by Westlake Plastics, Lenni Mills, Pa., USA) to give a fluence rate of 11.5 µmol m-2 s-1 between 600 nm and 700 nm. Far-red light was supplied by filtering the light from 60 W tungsten filament strip lights through three layers of Plexiglas blue 627 and one layer of Strand Cinemoid deep orange No. 58 (Rank Strand Electric, Kingsway, London, UK) to give 9.2 µmol m-2 s-1 between 700 nm and 780 nm.
RNA extraction
Total RNA was isolated from frozen cotyledon tissue (pooled material from 40 seedlings per sample point) using the method of Jordan et al. (Jordan et al., 1992), with the additional step of a 2-butoxyethanol precipitation (as described in Manning, 1991), to remove carbohydrates and phenolics, which may inhibit PCR. Prior to reverse transcription RNA samples were treated with RQ1 RNase-free Dnase. For northern analysis of poly A+ RNA, total RNA was isolated from frozen cotyledon tissue (using the method of Logemann et al., 1987). Poly A+ RNA was subsequently isolated using Dynabeads oligo (dT)25 (Dynal Ltd.).
Reverse transcription
Five µg total RNA was reverse transcribed using the Promega Reverse Transcription System (Promega Corporation), in 20 µl reactions with 0.5 µg oligo(dT)15 primer and 15 U AMV reverse transcriptase, according to the provided protocol, with an extended reaction time of 30 min at 42 °C followed by a 5 min 99 °C denaturation step. Reactions were then rapidly cooled to 0 °C and stored at -80 °C prior to PCR.
Polymerase chain reaction
Products from each 20 µl reverse transcription reaction were diluted by at least 1 in 5 in water. Ten µl of this dilution was used as template in 100 µl PCR reactions. Each PCR reaction mixture also contained 2.0 mM MgCl2, 10 mM TRIS-HCl pH 8.8 at 25 °C, 50 mM KCl, 0.1% Triton X-100, 200 µM of each dNTP, 0.5 µM sense primer, 0.5 µM antisense primer, and 2.5 U Taq polymerase (Gibco BRL). Where appropriate a master mix of PCR reaction components was made and aliquoted into individual reactions. Products were amplified by 35 repeated cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min. Ten µl aliquots of each reaction were subjected to electrophoresis on a 1.2% agarose (w/v) TAE gel.
Primers for PCR
Degenerate PHYA gene specific primers were designed from regions in the sequence which are highly conserved among species. Sense primer BT5 5'-GTGCTTCARGAYGARAARCT-3'; Antisense primer BT2 5'-CTTGATCTCRAAYTGNACATThyphen;3'. These primers were used to amplify and clone a 1.2 kb fragment of Pharbitis PHYA sequence enabling a Pharbitis nil PHYA-specific sense primer to be designed, PHA1 5'-TCAATAGCGTCACTGGTAATGG-3'.
Semi-quantitative PCR
The method of cDNA equalization described by Kolls et al. was used to standardize the amount of reverse transcription product in each sample prior to PCR (Kolls et al., 1993). This method involves the incorporation of [
hyphen;32P]dCTP during the reverse transcription reaction and quantitation of the subsequent total cDNA by liquid scintillation counting. Equal amounts of cDNA are then added to subsequent PCR reactions, hence eliminating quantitation problems due to variations in reverse transcription efficiencies between samples. PCR reactions were performed as described above. Aliquots of 10 µl were removed from reactions after successive cycles of amplification (between 14 and 20), and subjected to electrophoresis on a 1.2% agarose (w/v) TAE gel. Southern blots were produced and prehybridized for 16 h at 65 °C in hybridization solution (5xSSC, 1xDenhardt's solution, 1% SDS, and 100 µg ml-1 denatured salmon sperm DNA). An [-32P]dCTP-labelled PHYA probe was produced by the random primer labelling of the purified plasmid pAPH2 insert, using the Rediprime kit and protocol (Amersham Life Science Plc.). Hybridization was allowed to proceed for 18 h at 65 °C. Blots were washed twice at 65 °C in 1xSSC, 0.1% SDS (v/v) for 15 min. Autoradiographs were scanned by densitometry and product amounts expressed as peak area (OD mm). The amount of initial template was extrapolated from plots of log[product amount] versus cycle number (as described in Chelly et al., 1988).
Northern blot procedures
Poly A+ RNA (0.5 µg) for each sample were subjected to formaldehyde denaturing gel electrophoresis in 1.5% agarose (w/v), and the RNA blotted on to nylon membrane. Blots were prehybridized for 2–4 h at 42 °C in hybridization solution, and hybridized with a radiolabelled PHYA probe for 18 h at 42 °C. Blots were washed three times at 62 °C in 3xSSC, 0.1% SDS (v/v) for 10 min. Phosphor screens were exposed to blots, and scanned on a phosporimager (Molecular Dynamics). The intensity of each signal was determined using ImageQuant software (Molecular Dynamics). Autoradiography film was exposed to blots for 24–48 h at -70 °C. Blots were stripped using boiling water and reprobed with a radiolabelled probe for the constitutively expressed S4 ribosomal protein gene (Braun et al., 1994).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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The BT5/BT2 PCR product of Pharbitis nil PHYA cDNA was cloned into the TA site of pCRII (Invitrogen) to create the plasmid pAPH2. The nucleotide sequence and the polypeptide translation of the insert is shown in Fig. 1
. The annealing site of Pharbitis PHYA specific primer PHA1 is indicated, as well as recognition sites for some diagnostic restriction sites. The sequence encodes a 1203 bp partial cDNA with 78% homology to Solanum tuberosum Désirée PHYA mRNA (accession number S84872) and 75% homology to Arabidopsis thaliana PHYA mRNA (accession number X17341), starting at nucleotide 1084 in the Arabidopsis sequence. At the protein level the translated polypeptide shows most homology to Nicotiana tabacum PHYA1 (84%) (accession number P33530), and is 55% homologous to Pharbitis nil phytochrome E (accession number P55004).
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Primers PHA1 and BT2 were used in RT-PCR reactions to amplify a single 1097 bp fragment from Pharbitis nil total RNA (prepared from the cotyledons of 4-d-old dark-grown (etiolated) seedlings). The PCR product was confirmed to be PHYA by digestion with restriction enzymes Nsi I, Mse I and Hind III, and by hybridization with a 32 P-labelled PHYA specific probe on Southern blots, under high stringency conditions.
Initial experiments were performed to determine whether PHYA expression was light regulated, and if so whether expression was under phytochrome control. Semi-quantitative RT-PCR was used to assess changes in PHYA mRNA levels in Pharbitis nil seedlings under various light regimes.
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Down-regulation of PHYA mRNA in continuous R light following dark growth |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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Four-day-old etiolated seedlings were placed in continuous R light, and cotyledons were harvested after 0 (dark), 60, 120, and 240 min. Control plants were left in darkness and samples were harvested from these after 240 min. Total RNA was prepared from each sample and the relative amount of PHYA mRNA in each sample determined by semi-quantitative RT-PCR using primers PHA1 and BT2.
Two major factors are important to consider when using RT-PCR as a quantitative tool. Firstly it is important to ensure that differences in reverse transcription reaction efficiency are accounted for. In this case cDNA equalization was used to eliminate this as a potential source of error. Secondly, PCR is only quantitative during exponential amplification. After a certain number of cycles, however, the reaction efficiency decreases dramatically, after which further cycling fails to increase product yield due to substrate limitation and end-product inhibition. Therefore, in order to use PCR quantitatively product amounts must be determined during the exponential amplification phase.
Aliquots were removed from the PCR reactions during exponential amplification after 14, 15, 16, 17, and 18 cycles. Negative controls consisting of non-reverse transcribed RNA, produced no amplification products after 35 cycles of amplification, confirming that the RT-PCR products were derived from mRNA. Figure 2a shows autoradiography signals of the time-course RT-PCRs after 14–18 successive amplification cycles. The amount of product, which approximately doubled in each successive cycle, was determined by densitometry and the amount of initial PHYA template in each PCR was extrapolated from plots of log[product amount] versus cycle number (as described in Chelly et al., 1988).
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) Continuous R; (•) continuous dark control.The relative amount of PHYA template in each PCR was expressed in terms of percentage PHYA mRNA relative to the initial dark expression level. Figure 2b
shows a plot of percentage PHYA mRNA versus time of exposure to continuous R light. The dark control level is also plotted and remains high throughout the period of the experiment. In continuous R light, however, the amount of PHYA mRNA declines rapidly, after an initial lag phase, with an estimated half-life of just under 2±1 h in this case. Such a rapid decrease was also observed in subsequent experiments (see below) and is in good agreement with spectrophotometric measurements of the rate of decrease of total phytochrome levels in dark-grown Pharbitis upon exposure to continuous R (Rombach et al., 1982).
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PHYA mRNA levels after the interruption of darkness by R, FR or R followed by FR light pulses |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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Four-day-old etiolated seedlings were subjected to a 5 min R pulse, a 10 min FR pulse, or a 5 min R followed by a 10 min FR pulse, and then returned to darkness. Cotyledons were harvested from each treatment and from untreated seedlings (continuous dark), 3 h after the light treatments. Total RNA was prepared from each sample and the relative amount of PHYA mRNA in each sample determined as above by semi-quantitative RT-PCR. Negative controls showed no amplification products, confirming that the RT-PCR products are derived from mRNA. Figure 3a
shows autoradiography signals from RT-PCRs after 15–19 successive amplification cycles. The amount of product in successive cycles was determined by densitometry, and the amount of initial PHYA template in each PCR was extrapolated, as previously described.
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The relative amount of PHYA template in each PCR was expressed in terms of percentage PHYA mRNA relative to the dark expression level. Figure 3b
shows a histogram of % PHYA mRNA 3 h after the treatments. The results show that PHYA mRNA levels decrease dramatically within 3 h of a saturating R light pulse, consistent with the effect of R on transcript levels described above (Fig. 2b). A FR pulse leads to a relatively small decrease in the amount of PHYA mRNA relative to the dark level. The down-regulation caused by the R pulse was partially reversed by a subsequent FR pulse. This profile of responses to R and FR light was observed in two independent experiments using different sets of seedlings and is consistent with a phytochrome (Pfr) controlled process, confirming that the abundance of PHYA mRNA in Pharbitis nil is controlled via Pfr.
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Reaccumulation of PHYA mRNA levels in darkness following light induced down-regulation |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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Experiment 1: Five-day-old etiolated seedlings were placed in W light for 24 h and then returned to darkness, with or without an end-of-day 5 min FR pulse. Experiment 2: Five-day-old etiolated seedlings were placed in W light for 24 h and then returned to darkness. The dark period was then interrupted after 8 h by a 5 min R night break, with or without a subsequent 15 min FR pulse.
Cotyledons were harvested at various times during the dark period in both experiments, and the relative amount of PHYA mRNA at each time point determined by Northern blotting. Northern blots were used for these more detailed analyses of PHYA expression changes, as although the RT-PCR procedure is a useful method for assessing the expression of low abundance transcripts, it is limited by the number of samples which can be assessed for each assay. In order to detect the low levels of PHYA expression after the light treatment, poly A+ RNA was isolated from the cotyledons of the treated plants and used for Northern analysis.
Figure 4a and b shows autoradiographs of Northern blots probed with a Pharbitis PHYA probe. Signals were analysed by phosphorimagery, and quantified using ImageQuant software. The amount of PHYA mRNA from each sample was and is expressed as a percentage of the dark level. This was plotted against time (Fig. 4c). The Northern blots were subsequently probed with a constitutively expressed S4 ribosomal protein gene to verify equal lane loading.
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) R night-break +FR; (
) R night-break –FR control. Arrow indicates time of R night-break in Experiment 2 (8 h).In experiment 1 after 24 h W light the amount of PHYA mRNA remaining was about 20% of the dark level (0 h). Without an end-of-day FR pulse PHYA mRNA reaccumulated slowly in the dark, reaching 50% of the dark level within 24 h. During the first 11 h darkness no significant reaccumulation of PHYA mRNA was observed. However, when a FR pulse preceded the dark period the rate of reaccumulation of PHYA mRNA was greatly increased, reaching dark levels within the first 11±2 h of darkness after a lag period of up to 3 h. This is consistent with PHYA mRNA levels being controlled via phytochrome (Pfr), and shows that the Pfr responsible for this control remains stable and active for at least 11 h after its formation. The removal of this Pfr by a saturating FR pulse results in the rapid release of the repression mechanism, either at the transcription or post-transcription level. Without a FR pulse at time zero Pfr remains for a considerable time in darkness, preventing PHYA mRNA accumulation, until levels of Pfr fall below a threshold, due to either gradual destruction or dark reversion to the inactive Pr isoform.
In experiment 2 the R night-break after 8 h darkness prevented significant reaccumulation of PHYA mRNA for at least a further 24 h. However, when the R night-break was followed immediately by a FR pulse reaccumulation was more rapid, reaching dark levels within 16±3 h of the night-break. The effect of the R night-break was to increase the amount of Pfr, and hence continue the repression of PHYA mRNA levels. If the R is followed immediately by FR the Pfr is converted back to Pr alleviating the repression and allowing PHYA mRNA levels to reaccumulate. As with a FR treatment at the end of the day in experiment 1, there is only a small increase in the amount of transcript within the first 3 h of the FR treatment; after this lag period the reaccumulation is much more rapid. The lag period before reaccumulation and the rate of reaccumulation following a FR treatment was similar whether the FR was given at the end of the day or following an R night-break 8 h later. Similar profiles of reaccumulation have been observed in two independent repeats of these experiments, however, in those cases the poorer quality of the Northern blots prevented quantification of the whole range of time points.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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The work presented in this paper shows that in Pharbitis nil the amount of phytochrome A is controlled in the light-grown plant, not only by destruction of the Pfr form of the PHYA protein, but also by the down-regulation of PHYA transcript abundance. Since this down-regulation is inducible by a R pulse and reversible (at least partially) by subsequent FR the control of transcript levels has been shown to be mediated via the Pfr form of a phytochrome.
Earlier work (Rombach et al., 1982; Jabben et al., 1980) showed that phytochrome reaccumulation in darkness was enhanced by FR in Pharbitis nil light-grown seedlings, suggesting that Pfr represses the expression of type I phytochrome. However, these results did not distinguish between phytochrome types, as they were based on spectrophotometric quantitation of phytochrome protein, nor did they give any indication of whether control was being exerted at transcriptional or post-transcriptional levels.
Autoregulation of phytochrome expression is well characterized for Avena sativa (Colbert et al., 1985; Quail et al., 1986; Lissemore and Quail, 1988). When dark-grown Avena seedlings are exposed to either continuous W light, or given a R pulse and returned to the dark, the amount of phytochrome mRNA decreases rapidly with a half-life of about 1 h (Colbert et al., 1985; Quail et al., 1986). Ten hours after a R pulse mRNA begins to reaccumulate, reaching a level of 40% after 24 h (Colbert et al., 1985). Using run-on transcription techniques, it has been shown that the phytochrome transcription rate decreased to less than 20% of the dark level within the first 15 min of continuous W (Quail et al., 1986). In addition, it has been shown that phytochrome mRNA is unstable and degrades rapidly in a cell-free system (Byrne et al., 1993), however, there was no observable difference in stability of mRNA from etiolated and R-light treated seedlings. Taken together these two results suggest that the control of phytochrome mRNA levels is principally at the transcriptional level.
In Pharbitis dark-grown seedlings exposed to continuous W or R, the half-life of PHYA mRNA decrease is just under 2 h (Fig. 2b), and repression is sustained in excess of 11 h in darkness following irradiation (Fig. 4c). Although in Pharbitis nil, the down-regulation of PHYA mRNA abundance does not appear to be as extreme as in Avena, it is much stronger than in most dicots where measurements have been made. The only other dicot species, known to date, where such strong regulation of phytochrome transcript levels has been observed is Pisum sativum, where the RNA1 transcript is strongly down-regulated by W or R light with a half-life of about 2 h (Sato, 1988; Tomizawa et al., 1989), and repression is sustained for 16 h (Furuya, 1989). In cucumber, the abundance of PHYA mRNA is regulated, but to a lesser extent, the mRNA decreasing to 40% of that in the dark within 2 h of an R pulse, but reaccumulating rapidly to 100% within 10 h (Cotton et al., 1990). However, in other dicots that have been analysed, for example, tomato and Arabidopsis (Lissemore and Quail, 1988; Quail, 1994), the regulation of PHYA mRNA abundance by R or W light is much weaker or undetectable.
FR light alone also caused a small reduction in Pharbitis PHYA mRNA (Fig. 3b). This was also observed to a much greater extent in Avena, where a FR pulse produced sufficient Pfr to decrease PHYA transcription to 25% of the dark rate in the first 3 h after irradiation (Lissemore and Quail, 1988).
Down-regulation of phytochrome mRNA in R light does not in itself provide enough evidence to prove that the control is being exerted via Pfr without evidence of FR reversibility. In Pharbitis, the effect of R is only partially reversed by FR. Failure to reverse completely the effect of the R pulse by the FR pulse may be due to partial ‘escape’ (Fukshansky and Schäfer, 1983), where the Pfr formed during the R pulse has already induced irreversible responses before the FR pulse is given.
In fully green light-grown Avena and pea seedlings returned to darkness, phytochrome mRNA reaccumulates slowly; if a FR pulse is given prior to darkness the rate of reaccumulation is much faster (Colbert et al., 1985; Tomizawa et al., 1989). FR-enhancement of PHYA mRNA reaccumulation in the dark was also observed in de-etiolated Pharbitis seedlings (Fig. 4c). Without the FR pulse the Pfr remaining after the light period continues to repress transcription in the subsequent dark period, reaccumulation only occurring when the Pfr amount falls below a threshold level due to slow destruction or reversion. This does not occur until at least 11 h after the light–dark transition in Pharbitis suggesting that the controlling Pfr is highly stable. The removal of the Pfr by a FR pulse derepresses the system and allows a more rapid reaccumulation of PHYA mRNA, which reaches dark levels between 8–11 h, and this is consistent with spectrophotometric measurements of phytochrome protein levels made by others (Jabben et al., 1980).
A single photoperiod is sufficient to set the phase of a photoperiodically sensitive rhythm in Pharbitis nil (Lumsden et al., 1982). In these experiments the seedlings were given a 24 h light treatment to initiate such a rhythm. FR light given at the end of such a light period is inhibitory for flowering in Pharbitis (Vince-Prue, 1981) and, as has been shown, causes much higher levels of PHYA mRNA throughout the subsequent dark period. A night-break of R given 8 h into the dark period is also inhibitory for flowering in Pharbitis (King et al., 1982), however, in this case the R night-break represses PHYA mRNA levels throughout the subsequent dark period. FR given immediately after the R night-break reverses this repression of PHYA transcript levels and allows them to reaccumulate, but it has been shown that such a FR treatment given within the first 10 h of a dark period is inhibitory for flowering (Takimoto and Hamner, 1965). Thus whilst all these R and FR treatments inhibit flowering in Pharbitis they have dramatically different effects on PHYA transcript accumulation. There is therefore no evidence to support the hypothesis that the transcript levels of PHYA play a direct role in the flowering response of Pharbitis. Other factors such as the control of nuclear import of PHYA, or the availability of proteins interacting with phytochrome may be much more important in regulating PHYA action and its role in the control of flowering.
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Acknowledgments |
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This work was supported by BBSRC (BT, SJ, CER), The Royal Society (MH), and Nicholas Copernicus University, Torun, Poland (AS-J). We would like to thank Dr Mick Partis, Horticulture Research International, Wellesbourne, Warwickshire, for his help with primer design and sequence database tuition.
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Notes |
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5 To whom correspondence should be addressed. Fax: +44 1789 470552. E-mail: stephen.jackson{at}hri.ac.uk
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Abbreviations |
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R, red light; FR far-red light; W, white light; Pr, red light-absorbing form of phytochrome; Pfr far-red light-absorbing form of phytochrome; SDP, short-day plant; LDP, long-day plant; HIR, high irradiance response..
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TopAbstractIntroductionMaterials and methodsResultsDown-regulation of PHYA mRNA...PHYA mRNA levels after...Reaccumulation of PHYA mRNA...DiscussionReferences |
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