Apr 24, 1987 - black cloth except during weighing, cutting, and addition of sol- vent. The scintillation vials ..... acid in mustard seedlings. Planta 126: 111-117.
Plant Physiol. (1988) 86, 435-440 0332-0889/88/86/0435/06/$O1 .00/0
Phytochrome Regulation of Greening in Barley-Effects on Chlorophyll Accumulation' Received for publication April 24, 1987 and in revised form October 5, 1987
WINSLOW R. BRIGGS,*2 EGON MOSINGER3, AND EBERHARD SCHAFER Biologisches Institut II, Albert-Ludwigs-Universitdt D-7800 Freiburg i. Br., Schiinzlestrasse 1, Federal Republic of Germany ABSTRACT Red light treatment of dark-grown 6-day-old barley seedlings (Hordeum vulgare L.) strongly reduces the lag in chlorophyll accumulation in subsequent white light over that found in dark control seedlings placed under white light. Fluence-response studies show that the effect has both very low fluence and low fluence components. Kinetic studies indicate that the reduction in lag begins immediately following either a low fluence or a very low fluence red irradiation, with the initial rate of change significantly lower after the very low fluence treatment and showing sharp far redabsorbing form of phytochrome dependence. In both cases, the effect is maximal after roughly 4 hours, either remaining fairly constant (very low fluence) or declining somewhat (low fluence) thereafter. Saturating far red light alone yields a response equivalent to very low fluence red, and will reverse only the low fluence component of the red response. Escape from far red reversibility occurs gradually over about a 3 hour period. Since the kinetics described here differ from those in the literature related to phytochrome effects on transcription of the mRNA for the light-harvesting chlorophyll a/b-binding protein, we conclude that the phytochrome-regulated component of chlorophyll accumulation is not limited by transcription of the mRNA for its major apoprotein. Leaf segments vacuum-infiltrated with water retain the capacity to green in white light. If they are infiltrated with mannitol solutions of various concentrations, their capacity to green declines sharply at concentrations above 0.2 molar. These results bear on interpretation of run-on transcription experiments with isolated nuclei: preparation of the nuclei involves enzymic digestion of the tissue in the presence of 0.7 molar mannitol for 2.5 hours, to obtain protoplasts prior to breaking the cells. The results here make it unlikely that normal transcriptional regulation is occurring during this procedure.
It is well known that a brief pulse of R4 administered to darkgrown seedlings can eliminate the normal lag period in Chl accumulation encountered when such seedlings are placed for the first time under white light. Virgin (30) has reviewed earlier literature on the phenomenon. These first studies all failed to I Carnegie Institution of Washington Department of Plant Biology Publication No. 971. 2 Department of Plant Biology, Carnegie Institution of Washington, 290 Panama St., Stanford, CA 94305, U.S.A. 3 Pflanzenphysiologisches Institut der Universitat Bern, Bern. Altenbergrain 21, CH-3013 Bern, Switzerland. 4 Abbreviations: R, red light; Cab mRNA, transcripts coding for LHCP polypeptides; FR, far red light; LF, low fluence; LHCP, light-harvesting, chlorophyll a/b-binding polypeptide(s); Pchl-reductase, NADPH: protochlorophyllide oxidoreductase; Pfr, far red-absorbing form of phytochrome; Pr, red-absorbing form of phytochrome; VLF, very low fluence. 435
find more than weak FR reversibility (30). More recently, Raven et al. (24-26) have reported the results of detailed studies with pea seedlings. In pea, R sensitivity covers eight orders of magnitude of R fluence (24). In agreement with other workers, Raven and Spruit found only weak FR reversibility (26). The involvement of the plant photoreceptor phytochrome was made plausible largely on the basis of the action spectrum (25) which resembles the absorption spectrum of Pr, the R-absorbing form of phytochrome. In contrast to the above reports, a recent study (13) reports significant photoreversibility with etiolated peas. There is now considerable evidence that phytochrome can control a wide range of responses both through low fluence (LF, far red-reversible) and very low fluence (VLF, not far red-reversible) responses (7). The properties of the greening response to R strongly suggest that phytochrome regulation may occur both through LF and VLF responses. The earlier studies may have chosen conditions under which the VLF response predominated (the kinetics of appearance of the VLF and LF components in pea are not identical [13]) and hence failed to find more than weak photoreversibility. Greening involves far more than Chl accumulation, and there is now a large body of evidence that light, acting through phytochrome, regulates the abundance of numerous chloroplast polypeptides and their mRNAs (9, 28). In several cases, the experiments suggest regulation at the level of transcription (6, 8, 21, 22; see 28). For barley seedlings (Hordeumn vulgare L.), light regulation of two important chloroplast proteins has been studied at the level of translatable mRNA (1, 2), mRNA abundance as assessed by dot blot hybridization (4), and by in vitro transcription by isolated nuclei (21, 22). These proteins are those of the LHCP and the Pchl-reductase. With respect to the LHCP, phytochrome regulation is positive, with light-induced increases in protein, mRNA abundance, and in specific transcriptional activity of isolated nuclei. The reductase, by contrast, shows negative regulation in all cases. Kinetic studies are available in both cases for mRNA abundance (4) and transcriptional activity following a saturating pulse of red light (21, 22). In most cases mentioned above, phytochrome involvement has been inferred from experiments showing induction by a saturating pulse of R and prevention of induction by a saturating pulse of FR that follows immediately. Kaufman et al. (14, 15), studying the abundance of a number of light-regulated mRNAs in pea seedlings, have done detailed experiments at the level of mRNA abundance (as determined by slot blot hybridization), to investigate fluence-response relationships, far red reversibility (14, 15), and kinetics for induction by R and also for escape from FR reversibility (16). For the Cab gene product, the mRNA for LHCP, a transcript one might expect to be closely linked to the greening process, Kaufman et al. found both LF and VLF responses. Likewise, Mosinger et al. (22) recently showed in barley
436
BRIGGS ET AL.
that at the transcriptional level, a saturating pulse of FR regulated both the LHCP and the reductase mRNAs, results indicative of a VLF response (7). These studies taken together suggest that phytochrome regulation of processes involved in greening is occurring both at the LF and VLF levels of induction. However, in no one plant are detailed fluence-response relationships, induction kinetics, and escape kinetics available for each stage from transcription through Chl accumulation. To understand the transduction chain from phytochrome phototransformation to potentiation of the greening process, and to learn at which level(s) light regulation might be limiting, we carried out studies with barley to complement those already in the literature for this plant. The present paper describes studies on phytochrome regulation of Chl accumulation itself. A second paper (23) considers regulation at the level of mRNA abundance, as well as at the level of transcription as assayed with isolated nuclei. A brief summary of this work has appeared elsewhere (8).
MATERIAL AND METHODS Plant Material. Dry seeds of barley were sown on lightly packed moist vermiculite in plastic boxes 5 cm deep and 10 cm on a side (about 75 seeds per box) and covered with 1 cm additional moist vermiculite. Twelve such boxes were placed in a larger plastic box containing water a few mm deep to maintain humidity at saturation. The containers were covered, wrapped in heavy black cloth, and placed in a constant temperature (25°C) dark room for 6 d. At the end of this growth period the shoots were 10 to 12 cm in height, with primary leaves extending 6 to 9 cm beyond the coleoptile tips. For one experiment (Fig. 4), 5-d-old plants were used. At this age, the shoots were 5 to 8 cm in height, with the primary leaves only just beginning to break through the
coleoptiles. Light Sources and Irradiation Treatments. For preliminary experiments on the effect of saturating R on the time course of Chl accumulation in a subsequent white light period, and for experiments involving mannitol, seedlings were exposed to 10 min of a standard R field (7 W M2; 19). For all other experiments, R and FR were obtained from modified 500 W Leitz Prado projectors (12). R was obtained with a Balzers wide-band red filter, K65, and FR with a Schott RG9 filter. The fluence rate was varied by the use of neutral density filters (Schott). All R treatments were for 1
was
min, FR for 5 min. The FR fluence
rate
12 W m -2. For greening, 4 W M- 2 white light from flu-
orescent tubes was used.
Harvest and Chl Extraction. For the experments with 6-d-old seedlings, primary leaves that extended at least 7 cm beyond the coleoptile tips were selected at the appropriate time, and the apical 7 cm was excised. For the experiment with 5-d-old plants, the apical 4 cm of the primary leaves were harvested. Eight such leaves or shoots were obtained for each sample (12 for 5-d-old plants); and four samples, two from each of two different boxes, were obtained for each data point in any given experiment. The harvested leaves or shoots were placed quickly on plastic film on ice, and covered to exclude further light. Harvest of four samples required approximately 5 min and times indicated in the results below are times for the beginning of harvest for each
point. Samples were weighed, cut into roughly 1 cm lengths, placed in scintillation vials, and then covered with N,N-dimethylformamide for Chl extraction according to the method of Moran and Porath (20). For each 100 mg tissue 1 ml of solvent was used. Sample weight was rarely less than 350 mg or greater than 420 mg. Although these manipulations were done under normal laboratory illumination, the samples were kept covered by heavy black cloth except during weighing, cutting, and addition of solvent. The scintillation vials were
tightly capped and then kept
Plant Physiol. Vol. 86, 1988
at 4°C in darkness for at least 2 d before Chl assay. This time period was sufficient to permit Chl extraction to go to completion, and longer times could be used without Chl degradation (20). Chl was determined by measuring the OD in the solvent with a Zeiss PMQ spectrophotometer at 664 nm (20). All experiments were done on at least three occasions. Results from similar experiments were normalized and pooled as shown below. Standard errors, actually indicated only in Figures 3 to 6, rarely exceeded 5% of the mean. RESULTS
Effect of R on Chi Accumulation in White Light. A saturating pulse of R given 2 to 8 h prior to transferring barley seedlings into continuous white light significantly reduces the lag period in Chl accumulation (Fig. 1) relative to the dark control (0 h dark in Fig. 1, open squares). A pulse of R given 4 to 6 h prior to the onset of white light is maximally effective (Fig. 1, open triangles and open circles, respectively). A 2 h dark period still shows some lag (Fig. 1, diamonds) although the rate of accumulation achieved eventually parallels those for 4 and 6 h. An 8 h dark period, by contrast, shows a reduction in lag comparable to that obtained with 4 and 6 h dark periods but the rate of Chl accumulation declines after 2 to 4 h in white light (Fig. 1, solid squares). Full expression of the effect of R on Chl accumulation was reached after 4 h white light. Hence. for all following experiments, we assayed Chl after 4 h of white light treatment. Since a 4 h dark period gave a maximal effect, we used it in those subsequent experiments addressing fluence dependence of the R effect and the extent to which FR would reverse it. Fluence-Response Relationships. With a constant dark period of 4 h and a 4 h white light treatment, R fluence was varied over eight orders of magnitude and Chl was assayed as described above. The results are shown in Figure 2. The response shows both VLF and LF components. The threshold for the VLF response is near 10- pLmol m 2 with saturation near 10 ' pumol m-'. The threshold for the LF response, by contrast, is at 10 umol m-2, with saturation not quite obtained at the highest 20
Dark period, h c
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White light, hours FIG. 1. Effect of saturating R fluences administered at various times prior to the onset of continuous white light on the time course of Chl accumulation in 6-d-old seedlings during white light treatment. Each point is the average of at least three separate experiments, replicated four times on each occasion. Standard errors rarely exceeded 5'Xc. All experiments normalized to Chl obtainable at t 0 in white light. Actual optical densities at 664 nm were near 0.03 at t = 0. and near 0.5 for the highest values at t = 6 h.
437
GREENING IN BARLEY-CHLOROPHYLL ACCUMULATION 220
6-Day-old seedlings
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Log fluence R, jumol m-2 FIG. 2. Fluence-response relationships for the effect of R on Chl accumulation in 6-d-old seedlings. Protocol: R irradiation, 4 h dark, 4 h white light, harvest for Chl determination. Replication as for Figure 1. Actual optical densities in the different experiments: dark control, near 0.2 OD; highest red fluence, above 0.4 OD.
fluence that could be administered in 1 min, 104 ,umol m-2. A plateau extending over two decades separates saturation of the VLF from threshold for the LF, a pattern commonly found when both responses occur (7). Kinetics for R-Induced Changes. Mosinger et al. (22) have recently presented kinetics for changes in in vitro transcription by isolated barley nuclei following saturating R (LF) or saturating FR (the equivalent of VLF [17]) for transcripts hybridizing with a probe for LHCP mRNA (as well as for the Pchl-reductase). During the first 1.5 h following the onset of irradiation, the rate of increase in LHCP transcription was the same for both light treatments, and hence was saturated by the very small amount of Pfr produced by the far red light. The duration of the increase following FR was shorter than that following R, however, and hence not yet saturated by the amount of Pfr produced by FR. (By contrast, neither the initial rate of decrease in transcription for the Pchl-reductase nor the duration of the decrease were saturated by FR.) Since accumulation of Chl is reported to be tightly linked to accumulation of LHCP (e.g. 3), it was of interest to determine whether the kinetics for potentiation of greening by R treatments in the VLF and LF ra-nge paralleled those for transcriptional activity for LHCP mRNA (22). Therefore groups of plants were given fluences in the low VLF 1 m -2), the high VLF range (10 ,umol m-2), or range (10 -wmol the LF range (103 ,umol m-2) (Fig. 2). After different dark periods, the various groups of plants were placed in white light for 4 h before harvest for Chl assay. The results are shown in Figure 3. In contrast to LHCP mRNA transcription by isolated nuclei, where the initial rates of change are identical for 1.5 h following either saturating R or FR (22), the initial rate of potentiation of Chi accumulation shows a sharp Pfr dependence. The curves for the various fluences diverge from each other beginning at time zero, and remain well separated for the full 8 h dark period investigated. The differences in response to the VLF low and VLF high fluences shown in Figure 3 are greater than those shown in the fluence-response curve illustrated in Figure 2 (the same discrepency is seen in the studies of FR reversiblity shown in Fig. 5, below). The reason for these differences is not clear, but the values seen in Figures 3 and 5 for fluences in the low and high VLF ranges lie within the extremes of the variability seen in
2
4
6
8
Dark period, h, after red light FIG. 3. Time course for potentiation of greening in white light by R fluences in the low and high VLF range and the LF range (see text for details). Six-d-old seedlings were given the various R treatments, returned to darkness for the times indicated, and then given 4 h white light prior to harvest for Chl assay. Replication as for Figure 1. Actual optical densities in different experiments: dark control, near 0.2 OD; maximum response, near 0.38 OD. r-
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Dark period, h, after red light FIG. 4. Treatments as in Figure 3 for various fluences as indicated, but with 5-d-old shoots. Actual optical densities in different experiments: dark control, near 0.2 OD; maximum response, near 0.4 OD.
Figure 2. Since the experiments done by Mosinger et al. (22) were done with 5-d-old shoots rather than on the 6-d-old leaves used here, we repeated measurement of these kinetics on 5-d-old shoots to determine whether the discrepency between greening kinetics and transcription kinetics was based on plant age or reflected intrinsic differences in the ways in which Pfr was regulating transcription of LHCP on the one hand and Chl accumulation on the other. The results for the first 3 h are shown for six different fluences in Figure 4. The relationships here are very similar to those found with the older leaves (Fig. 3). Even at the highest VLF irradiation tested (10' ,umol m-2) the initial rate of change differs significantly from that for a fluence in the LF range (103 umol m -2 s -'): the curves diverge significantly by 1 h after irradiation. Note that a fluence-response curve constructed from the 3-h data in Figure 4 will closely resemble that presented in Figure 2 for leaves of older plants measured after a 4 h dark
period. A plateau from 10 - 1 to 101 ,Mmol m -2 separates the VLF and LF ranges in both cases. The differences in developmental status of the primary leaves between d 5 and 6 is hence not reflected by any significant changes in Pfr-potentiated greening. FR Reversibility. If the responses obtained here are typical of VLF and LF responses in other systems (7), the LF response should be reversible by FR, the VLF should not be reversible by FR, and saturating FR alone should produce a response equal to that obtained with a R fluence sufficient to saturate the VLF response. Separate groups of 6-d-old plants were given the VLF or LF doses described above for Figure 3 ( + FR), or FR, returned to darkness for 4 h, placed in white light for 4 h, and then harvested for Chl assay. The results, shown in Figure 5, are as predicted: the LF response is largely photoreversible while the VLF is not, and FR alone yields a response equivalent to that obtained with a saturating VLF R fluence. Escape from FR Reversibility. Mosinger et al. (22) have also examined escape from FR reversibility for the Cab gene product (and the reductase) at the level of in vitro transcription, and it was therefore of interest to investigate the same phenomenon at the level of Chl accumulation. Groups of plants were given a saturating fluence of R (LF) and then returned to darkness for from 0 to 3.5 h before being given FR. Four h after R treatment they were placed in white light for the usual 4 h prior to harvest and Chl assay. The results are shown in Figure 6. After a lag of about 1.5 h, photoreversibility is gradually lost, with the respon-se approaching that from R alone after 3.5 h. The time course for escape found here is somewhat more rapid than that found for Mosinger et al. (22) for in vitro transcription of LHCP mRNA where reversibility was still complete after 3 h but completely gone after 6 h. For reasons that are not entirely clear, the amount of photoreversibility obtained in these experiments was somewhat less than that obtained in the initial photoreversibility experiments illustrated in Figure 5. Since the experiments for Figure 6 were done almost a year after all other experiments, this discrepancy may be related to duration of seed storage. Rapid escape could have occurred in the latter case but not in the former. Influence of Osmoticum on Greening. A principal aim of this paper is to obtain data on Chl accumulation under various conditions of light treatment for comparison with data for mRNA abundance and transcriptional activity of isolated nuclei with 220 L-
o 200 L-
a
A-o
Plant Physiol. Vol. 86. 1988
BRIGGS ET AL.
438
FR Reversibility
180
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Low
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R alone
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Dark period, h, between R and FR FIG. 6. Escape from FR reversibility. Groups of 6-d-old seedlings were given a saturating R fluence (LF) and then given FR immediately or after the dark periods shown. Four h after the R treatment the plants were placed in white light for 4 h prior to harvest for Chl assay. Replication as for Figure 1. Actual optical densities in different experiments: dark, near 0.2 OD; R alone, near 0.4 OD.
comparable light treatments. The technique for isolating nuclei from barley leaves involves their digestion with cell wall-degrading enzymes for 2.5 h in the presence of 0.7 M mannitol (21). It was therefore important to know whether R-induced changes could be occurring during this digestion period, or whether normal regulatory processes were suspended by osmotic shock, as suggested by the studies with tobacco protoplasts (10, 11, 29). Seedlings were given a saturating dose of R (LF) and returned to darkness for 4 h. At the end of the dark period, lots of primary leaf tissue weighing 1 g were quickly harvested and vacuuminfiltrated as described elsewhere (22) either with 10 ml distilled water or various concentrations of mannitol. They were then placed under standard white light conditions for 4 h. The liquid was then removed and the Chl extracted as usual. (Preliminary experiments indicated that inclusion of the digestion enzymes had no effect.) The results are shown in Figure 7. Substantial greening occurred in the distilled water controls (about half that obtained in intact seedlings) and greening was increasingly inhibited by increasing concentrations of mannitol. Thus, a strong osmoticum clearly inhibits some aspect of the greening process. A precise quantitative comparison of these results with those obtained from intact seedlings is not possible, because the absolute amount of greening was less. However, it is noteworthy that 0.7 M mannitol reduces Chl accumulation during 4 h white light to about 60% of the value with distilled water, a difference not dissimilar to that seen between intact seedlings given near saturating R fluences prior to 4 h white light and those given no prior red treatment. Hence, the mannitol treatment has the effect of inhibiting the R-induced enhancement of greening, although it may simply be inhibiting all of the biosynthetic activities associated with greening, R-induced or not.
i~~~iIUi:Uz~~~
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Irradiation treatment FIG. 5. FR reversibility. Separate groups of 6-d-old seedlings were given R fluences in the low and high VLF range and in the LF range as for Figure 3, with FR as shown. They were returned to darkness for 4 h, then exposed to white light for 4 h, and harvested for Chl assay. Replication as for Figure 1. Actual optical densities in different experiments: dark, near 0.2 OD; maximum LF response, near 0.4 OD.
DISCUSSION In accord with studies in other plants, Pfr reduces the lag period for Chl accumulation in white light in barley. Also con-
sistent with studies with other plants (13, 26, 30), FR reversibility is incomplete, and the response to R contains both LF and VLF components. Barley primary leaves show this pattern of phytochrome regulation whether they are harvested 5 or 6 d after imbibition.
GREENING IN BARLEY-CHLOROPHYLL ACCUMULATION I~~
Il
.2
Water control ----a}---
E
C
----------------------
0
0
(0 .15
F
a
0 R, 4 h dark, 4 h white light 0
1 0
.2
.4
.6
[Mannitol], M FIG. 7. Influence of osmoticum on greening of isolated and vacuuminfiltrated sections of primary leaves. Six-d-old seedlings were treated with R and then returned to darkness for 4 h. Leaves were then harvested under dim green light, cut into segments, and vacuum-infiltrated as described in the text prior to white light treatment for 4 h. Replication as for Figure 1.
The kinetics for changes in Chl accumulation following various fluences (Figs. 3 and 4) do not parallel comparable kinetics for production by isolated nuclei of RNA hybridizable to an LHCP probe. Mosinger et al. (22) found a rapid increase in transcription for the LHCP during the first 1.5 h following light treatment, with the R (LF) and FR (VLF) curves showing identical slopes. Therefore, the initial increase in capacity for LHCP mRNA transcription was saturated when less than 0.1% of the total phytochrome is transformed to Pfr by FR (27). After 1.5 h. the two curves diverged sharply. Following R (LF), the capacity of the nuclei to yield counts hybridizable to the LHCP mRNA probe continued to increase for an additional 2.5 h before falling to a level well above the dark control. By contrast, following FR (VLF), this capacity begins to decline sharply after 1.5 h, reaching the dark control level by 6 h. Hence, the duration of the effect is Pfr limited though the initial rate of response is not. For Chl accumulation, Pfr does limit the rate of the initial response. The potentiation of greening in barley by R. like that in pea (13), shows the partial FR reversibility (Figs. 5 and 6) that is to be expected of a phytochrome response with both LF and VLF components. It clear from Figure 3 that after any dark period between 1 and 8 h, prior to the 4 h of greening in white light, some FR reversibility will be detectable. Such is not the case, however, with in vitro transcription of LHCP mRNA where the amount of FR reversibility obtained will depend strongly on when after the light treatment the nuclei were isolated (cf. Fig. 4. here, and Fig. 1 [22]). After a 1 h dark period, scarcely any photoreversibility will be detectable, since the response after VLF and LF light treatments is indistinguishable at that time. After a 2 h dark period, the response will appear more than 50% reversible as the LF response continues to climb and the VLF responses decays. After 6 h, the response will be completely reversible by FR as all that remains is an LF component. An explanation for the discrepancy between the kinetics for change in Chl accumulation and those for LHCP mRNA transcription during the first 1.5 h following R treatment is that some step in the greening process other than LHCP mRNA transcription limits the rate of Chl accumulation during this time period. A possible candidate is the synthesis of 6-aminolevulinic acid, known to limit Chl accumulation (5). Synthesis of this Chl precursor is known to be under phytochrome control (17, 18). The
439
difference encountered after longer dark periods is not really a discrepancy in that one would expect the consequences of a VLF response, measured at the level of product accumulation, to persist after a transient VLF transcriptional response itself had decayed. Proper interpretation of kinetic data obtained from nuclei prepared from protoplasts requires some knowledge of the events which may be occurring during their preparation. The strong inhibition of the greening response by 0.7 M mannitol, described here, makes it unlikely that normal transcriptional regulation is occurring during the treatment with the plasmolysing osmoticum used during the preparation of nuclei. Experiments investigating both mRNA abundance and transcriptional activity of isolated nuclei, done with plants grown under the conditions used in the present work, and of similar age, are reported in another paper (23). Further discussion of the levels at which phytochrome regulation of greening per se in barley might occur are therefore deferred to that paper. A companion study to this one (13), involving greening of etiolated pea seedlings, addresses this same problem. Acknowledgments-The work described in this paper was largely carricd out while the senior author was on sabbatical leave at the University of Freiburg. Federal Republic of Germany. in 1984-1985 as a U.S. Senior Scientist Awardee of the Alexander von Humboldt-Foundation. The authors are grateful to Renate Wiehe for skilled technical help. This research was supported bv the Deutsche Forschungsgemeinschaft (SFB 206 to E. Schafer).
LITERATURE CITED 1. APEL K 1979 Phytochrome-induced appearance of mRNA activity for the apoprotein of the light-harvesting chlorophyll alb protein of barley (Hordelwn vulgare L.). Eur J Biochem 97: 183-188 2. APEI K 1981 The protochlorophyllide holochrome of barley (Hordeuml vulgare L.). Phvtochrome-induced decrease of translatable mRNA coding for the NADPH: protochlorophyllide oxidoreductase. Eur J Biochem 120: 89-93 3. APEL K. K KLOPPSTECHi98X( The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein. Evidence for the requirement of chlorophyll a for the stabilizattion of the apoprotein. Planta 150: 426-430 4. BATSCHAUER A. K APEL 1984 An inverse control by phytochrome of the expression of two nuclear genes in barley (Hordeurn vulgare L.). Eur J Biochem 143: 593-597 5. BEALE SI 1978 8-Aminolevulinic acid in plants: its biosynthesis, regulation, and role in plastid development. Annu Rev Plant Physiol 29: 95-120 6. BERRY-LoWE SL. RB MEAGHER 1985 Transcriptional regulation of a gene encoding the small submit of ribulose-1,5-bisphosphate carboxylase in soybean tissue is linked to the phytochrome response. Mol Cell Biol 5: 19101917 7. BRIGGS WR, DF MANDOLI, JR SHINKLE, LS KAUFMAN. JC WATSON, WF THOMPSON 1985 Phytochrome regulation of plant development at the whole plant. physiological, and molecular levels. Itn G Columbetti. P-S Song. eds, Sensory Perception and Transduction in Aneural Organisms. Plenum, New New York, pp 265-280 8. BRIGGS WR, E MOSINGER. A BATSCHAUER. K APEL, E SCHAFER 1987 Molecular events in photoregulated greening in barley leaves. In EJ Fox. M Jacobs. eds. Molecular Biology of Plant Growth Control. Alan R. Liss, New York, pp 413-423 9. ELLis RJ 1981 Chloroplast proteins: synthesis. transport, and assembly. Annu Rev Plant Physiol 32: 111-137 10. FLECK J, A DURR. C FRITSCH, T VERNET, L HIRTH 1982 Osmotic-shock stress proteins' in protoplasts of Nicotiana sylv'estris. Plant Sci Lett 26: 159-165 11. FLECK J, A DURR, MC LETT, L HIRTH 1979 Changes in protein synthesis during the initial stage of life of tobacco protoplasts. Planta 145: 279-285 12. HEIM B, E SCHAFER 1984 The effect of red and far-red light in the high irradiance reaction of phytochrome (hypocotyl growth in dark-grown Sinapis alba L.). Plant Cell Environ 7: 39-44 13. HORWITz BA, WF THOMPSON, WR BRIGGS 1988 Phytochrome regulation of greening in Pisum: chlorophyll accumulation and abundance of mRNA for the light-harvesting chlorophyll a/b-binding proteins. Plant Physiol. In press 14. KAUFMAN LK, WR BRIGGS, WF THOMPSON 1985 Phytochrome control of specific mRNA levels in developing pea buds. The presence of both very low fluence and low fluence responses. Plant Phvsiol 78: 388-393 15. KAUFMAN LK. LL ROBERTS. WR BRIGGS. WF THOMPSON 1986 Phytochrome control of specific mRNA levels in developing pea buds. Kinetics of accumulation, reciprocity. and escape of the low fluence response. Plant Physiol 81: 1033-1038 16. KAUFMAN LK, WF THOMPSON. WR BRIGGS 1984 Different red light requirements for phytochrome-induced accumulation of cab RNA and rbcS RNA.
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