Regulation of Early Light-Inducible Protein Gene Expression ......1168 Adamska Plant Physiol. Vol. 107, 1995 and accumulation of the protein in mature green plants (Adamska et al.,

Plant Physiol. (1 995) 107: 11 67-1 175

Regulation of Early Light-Inducible Protein Gene Expression by Blue and Red Light ira Etiolated Seedlings

lnvolves Nuclear and Plastid Factors’

lwona Adamska*

lnstitut für Botanik, Universitat Hannover, Herrenhauser Strasse 2, 3041 9 Hannover, Germany

fluence responses have a threshold of approximately 10-’ PE m-’ and are fully reversible by far-red The very- low-fluence responses have a threshold of approximately l O P 3 pE m-’ and are not reversible by far-red light; in addition, these responses can be induced by far-red sources (Kaufman et al., 1984). Cryptochrome regulation occurs via both low- and high-fluence systems (Warpeha et al., 1989). The blue low-fluence responses have a threshold of approximately lo-i m-z and below, whereas the blue high-fluence responses have a threshold between 10’ and 103 pE m-*. The nature of the receptor and the signal

1981) and pterins (Galland and 1988) have been suggested as the Possible photoreceptors. Since blue li@ induces Phos- phorylation of a 120-kD plasma membrane protein, it was proposed that this protein might be a part of a blue light receptor system (Gallagher et al., 1988). Recently, Ahmad and Cashmore (1993) proposed that a gene product with significant homology to microbial DNA photolyases most likely represents a blue light photoreceptor in plants.

ELIPs are nuclear-encoded proteins localized in the thylakoid membranes and related to the cab (light-harvesting

Among severa1 environmental factors known to affect Chl a / b binding) gene family ( G ~ ~ ~ ~ et al., 1989), ELIP plant gene expression, light is the most important. Quite a transcription OCCUrS in etiolated pea (Pisum sativum) few receptors are involved in the reception Of the signal. (Meyer and Kloppstech, 1984) and barley (Hordeum vulgare) A1though the phytochrome family and the holo- (Grimm et al., 1989) during the first 2 to 4 h of the greening chrome are activated by red light, separate photoreceptors process. The induction of ELIP mRNA synthesis and accu- exist for blue/UVA and UVB r%ions of the sPectrum mulation of its translation product in developing plants (ThomPson and White~ 1991). PhYtochrome is a well-char- was shown to be under diurna1 and circadian control (Otto acterized cytosolic plant photoreceptor that has a co- et al., 1988). valently linked linear tetrapyrrole chromophore and exists No definite function has yet been described for ELIPs in in two forms that are interconvertible by light. The inactive higher plants. A protein family with high similarity to the Pr form ~ b ~ o r b s primarily red light (wavelength at maxi- higher plant ELIPs and apparently related to carotene bio- mum = 660 nm) and is transformed to the functional Pfr synthesis has been reported in the green alga Dunaliella form, which has a main absorption peak in the far-red bardawil (Lers et al., 1991). A dessication-related ELIP-like region (wavelength at maximum = 730 nm) of the spec- protein has been described for the resurrection plant Cra- trum (Thompson and White, 1991). terostigma plantagineum (Bartels et al., 1992). An intrinsic

Most plants examined contain multiple phytochrome 22-kD protein (psbS gene product) closely related to LHC isoforms. In Arabidopsis, five phytochrome genes have been 1/11, CP24/CP29 apoproteins, and ELIPs has been discov- identified (Sharrock and Quail, 1989). Phytochrome regu- ered in spinach plants (Kim et al., 1992; Wedel et al., 1992). lation occurs over two fluence ranges of red light. Low- Recently, it was reported that strong light causing photo-

inhibition of photosynthesis induces ELIP transcription ’ This work was supported by Deutsche Forschungsgemein-

Abbreviations: cab, mRNA for PSII light-harvesting Chl a/b- * E-mail [email protected]; fax binding complex; ELIP, early light-inducible protein; LHC 11, PSII

Early light-inducible proteins (ELIPs) are nuclear-encoded chloroplast proteins whose genes are transiently transcribed during the greening process of etiolated plants. In the present work the regulation of ELlP gene expression by blue and red light has been investigated in plumulas of etiolated pea plants (Pisum sativum). l h e results show that the steady-state leve1 of ELlP transcripts is controlled by a combined action of phytochrome and blue light receptor systems and, in addition, depends on the age of the seedlings. Both a low-light fluence system of blue and a very-low-fluence system of red light are involved in ELlP induction. l h e threshold for accumulation of ELlP transcripts was as low as 10F5 p E m-’ s-‘ for

obtained in blue and in red light. Blue light not only acts at the level of transcription but also regulates the stability of the ELlP transcripts in a light intensity-dependent manner. Moreover, it is shown that product(s) of nuclear gene(s) negatively regulate the steady- state level of ELlP transcripts during the 1 s t h of illumination with red light. Preillumination of seedlings with white light abolishes this repression. Accumulation of ELlP transcripts requires “plastid factors” in both blue and red light qualities.

both light qualities but a different pattern of accumulation was transduction for any Of these blue light effects remain unknown. F1avins (Leong et

schaft, Bonn, Germany.

49 -51 1-762-3992. light-harvesting Chl a/b-binding complex. 1167

https://plantphysiol.orgDownloaded on May 27, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

1168 Adamska Plant Physiol. Vol. 107, 1995

and accumulation of the protein in mature green plants (Adamska et al., 1992b; Potter and Kloppstech, 1993). We propose that ELIPs are light-stress response proteins involved in protection against light stress.

Very little is known about factors controlling expression of ELIP genes in etiolated plants. In the present work the cellular and molecular mechanisms responsible for blue light- and red light-induced changes in ELIP genes activity were investigated in plumulas of etiolated pea plants. The results show that the photoregulation of ELIP genes involves the action of blue light and phytochrome receptors activated by the low-fluence system for blue and very-low- fluence system for red Iight, respectively. The steady-state level of ELIP transcripts depends on the age of the seedlings and is controlled at the level of mRNA stability by blue light. Finally, it is shown that abundance of ELIP transcripts is negatively regulated by nuclear gene product(s) induced by red light. Signals originating from the plastids are necessary for accumulation of ELIP transcripts in both blue and red light qualities.

MATERIALS AND METHODS

Growth of Plants and lllumination

Pea plants (Pisum sativum L. cv Rosa Krone) were allowed to imbibe and were grown on vermiculite in complete darkness for 7 d at 25°C. AI1 manipulations of dark- grown plants were performed in complete darkness without safety light.

The illumination was performed on plumulas detached with the epicotyl hooks that were floated on water. Differ- ent light qualities were obtained by using projectors equipped with interference filters (Balzer, Balzers Aktien- gesellschaft, Liechtenstein): blue (410-550 nm) and red (625-680 nm). Photon fluence rates of red, blue, and white light were measured using a photometer (Tektronix, Bea- verton, OR). The different light intensities were obtained by varying the distance between the seedlings and the light sources or with the aid of a neutral-density filter (Balzer). Illumination was carried out at room temperature (22- 25°C) and plant material was immediately frozen in liquid nitrogen and stored at -70°C for further analysis. A11 experiments were carried out at least in duplicate and with at least 30 to 50 plumulas per data point.

lsolation and Assay of RNA

Poly(A+) RNA was isolated as described by (Potter and Kloppstech (1993) using oligo(dT)-cellulose chromatogra- phy. In vitro translation was performed using the wheat germ system according to the method of Roberts and Patterson (1973) in the presence of [35SlMet (Amersham). Samples containing equal amounts of radioactivity were separated by SDS-PAGE, and the gels were treated for fluorography and exposed to x-ray film at -70°C.

Dot blot hybridization was performed using a 32P-labeled randomly primed homologous cDNA insert of ELIP (Scharnhorst et al., 1985) or cab (Otto et al., 1988) according to the Amersham protocol. The probes recognized specifi- cally the ELIP or LHC I1 mRNAs as verified by northern

analysis (data not shown). After autoradiography, signals linear in intensity with exposure time (A600 < 13.8) were scanned at 600 nm for quantification. To ensure the repro- ducibility of the data, some of the filters were cut into pieces of identical size and bound radioactivity was measured by Cerenkov counting. To determine background radioactivity, a piece of RNA-free filter was alscl counted aiid the value was subtracted from the final counts.

lsolation of Nuclei and in Vitro Transcription

Nuclei were isolated using the method described by Gallagher and Ellis (1982). The final nuclei pellet was re- suspended in 50 mM Tris-HCI, pH 8.5,5 mM MgCl,, 10 mM p-mercaptoethanol, and 50% (v/v) glycerol at a concentration of 10* nuclei/mL and stored at -80°C.

To assess overall transcriptional activity, a stanc ard transcription assay (60 pL) contained 106 nuclei in 50 mM Tris-HC1, pH 8.5, 5 mM MgCl,, 10 mM P-mercapt3ethano1, 20% (v/v) glycerol, 50 mM (NH4)2S04, 2.5 p~ creatine phosphate, 25 k g of creatine phosphokinase, 0.5 I ~ M ATP, CTP, and UTP, and 2 pCi of [a-32PlGTP (400 Ci/mmol). The assay mixture was incubated for 30 min at 25°C and transcription was terminated by spotting aliquots onto filter discs. The filter discs were washed five times ibr 5 min with 5% TCA and then twice in ethanol. The discs were then dried and counted in a scintillation couníer. As a control, an aliquot of transcription mixture was íaken before starting the incubation and this value was subtracted from the final counts.

To assess the rate of transcription specific for the ELIP gene, preparative scale assays (1.2 mL) with 10' niiclei and 150 pCi of [cx-~*P]GTP were used. Transcription was stopped by the addition of 20 pg/mL DNase free), and the incubation was continued at 25°C for an atlditional 10 min. Nuclear RNA was isolated according to thti method of Deng et al. (1987) and the final ethanol precipi tate was dissolved in 100 mM Tris-HC1, pH 7.6, and 50 mrn EDTA. Hybridization of Iabeled nuclear transcripts to dot blots containing 500, 250, and 130 ng of ELIP insert "as performed according to the Amersham protocol. Thr! labeled RNA was heated to 100°C for 5 min before hybridization.

Treatment with lnhibitors

For inhibition of plastid or cytosolic protein synthesis, chloramphenicol or cycloheximide (Sigma) were used at final concentrations of 200 or 20 Fg/mL, respecth ely. Ap- proximately 50 plumulas with epicotyl hooks were used for three independent experiments. Detached plumu [as were incubated in the appropriate solutions for 2 h at room temperature in darkness. To increase transpiration the plumulas were exposed to cool air blown from a vcntilator. After preincubation the plumulas were illuminai ed with different light qualities and poly(A+) RNA was extracted as described above. The action of inhibitors on protein synthesis was assayed in in vivo labeling experirnents as described below.

To inhibit nucleic acid synthesis (transcription aiid chain elongation), actinomycin D (Sigma) was used during illu-

\ https://plantphysiol.orgDownloaded on May 27, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


Regulation of Early Light-lnducible Protein Expression in Etiolated Seedlings 1169

mination at the final concentration of 30 ju,g/mL. Inhibitionof transcriptional activity by actinomycin D was tested inrun-off transcription experiments by determination of ra-dioactivity incorporated into transcripts synthesized in iso-lated nuclei.

In Vivo Labeling, Isolation, and Assay of ProteinsFor in vivo labeling of proteins, 30 plumulas with epi-

cotyl hooks were floated on water in the presence of 100ju,Ci/mL [35S]Met (1220 Ci/mmol, Amersham) and in thepresence or absence of protein synthesis inhibitors. After 2h of pretreatment in complete darkness, plumulas wereexposed to blue or red light for 2 h at 25 or 42°C (heat-shock conditions). At the end of illumination the plantmaterial was frozen in liquid nitrogen and stored at -70°C.Thylakoid membranes were isolated as described byAdamska et al. (1993). Proteins were separated by SDS-PAGE according to the method of Laemmli (1970) using theHoeffer minigel system. Radioactive gels were dried andexposed to x-ray film.

RESULTSConstant Fluence Rates for Accumulation of BLIPTranscripts in Plumulas of Etiolated Pea Seedlings

Constant fluence rate for accumulation of ELIP tran-scripts was investigated by dot blot hybridization in etio-lated pea plumulas exposed for 2 h to continuous blue orred light of various intensities. As shown in Figure 1 ELIPtranscripts were not detected in dark-grown seedlings. Thethreshold for induction of ELIP transcripts was approxi-mately 10~5 /J.E m~2 s"1 (total fluence 7.2 X 10~2 /xE m~2)for both blue and red light but different patterns of accu-mulation were obtained in both light qualities. During

light intensity[pE/m2s]

5 ™ ™S 5 2 ,- S

zaat

0.130.250.5

"0.130.250.5

• 4

• • • •

• * * *

* • * • •

UJ

_iGO

QUJDC

[pE/m28]

Figure 1. Blue and red light constant fluence rates for accumulationof ELIP transcripts in etiolated pea plumulas. Plumulas detached frometiolated pea seedlings were illuminated with blue (410-550 nm),red (625-680 nm), or white light for 2 h at different light intensities.Poly(A+) RNA was isolated and used for dot blot hybridization withthe labeled insert of an ELIP cDNA clone. Results are the averages ofthree independent experiments.

illumination with blue light only traces of ELIP transcriptswere present in seedlings exposed to 10~5 to 10~3 piE m~2

s"1 and a gradual increase in the steady-state level of ELIPtranscripts was observed as the light intensity increasedfrom 1 to 250 jaE m~2 s"1. In red light the response reacheda maximum between 10~3 and 1 ;aE m~2 s^1 (total fluencebetween 0.72 and 72 X 102 juE m~2) and the amount of ELIPtranscripts decreased with increasing light intensity. Nota-bly, the maximal amount of ELIP transcripts induced byred light was lower than that accumulated during illumi-nation with blue light.

The threshold for ELIP induction during exposure ofplumulas to 2 h of continuous white light was the same asfor blue or red light but accumulation of ELIP transcriptsdid not increase between 1 and 250 /u,E m~2 s"1 (Fig. 1,right).

Control of Steady-State Level of ELIP Transcripts byCombined Action of Phytochrome and Blue Light Systemsand Its Dependence on the Seedling's Age

It is known that long-term irradiation of plants with bluelight can cause high irradiance responses of phytochrome(Marrs and Kaufman, 1989). Saturation of phytochrome bycontinuous illumination with red light and additional illu-mination with blue light usually helps to distinguish be-tween phytochrome and cryptochrome responses. For thisreason the expression of ELIP genes was investigated inetiolated plumulas that were preilluminated for 30 minwith blue or red light and then transferred to another lightquality for 2 h. Control plumulas were illuminated withonly one light quality, blue or red, for the same time. Thelight intensity was 250 fiE m~2 s"1 for both light qualities.The results of such an experiment (Fig. 2) show that accu-mulation of ELIP transcripts assayed by dot blot hybrid-ization is higher in plumulas exposed to both light qualitiesthan in plants illuminated with blue or red light alone. Thetranscript level for cab increased considerably during illu-mination but the level of response was similar in all lightqualities used.

To test whether photoresponses of ELIP genes depend onthe age of etiolated plants, plumulas of pea seedlings ofincreasing age (3, 7, and 12 d) were illuminated for 2 h withblue or red light and the abundance of ELIP transcripts wasassayed by dot blot hybridization as shown in Figure 3.Independent of light quality, the highest level of ELIPtranscripts accumulated in the plumulas of the oldest seed-lings. A similar accumulation pattern was obtained for cabtranscripts (data not shown).

Blue Light Regulates ELIP Transcript Stability in aLight Intensity-Dependent Manner

It was observed previously that in etiolated (Meyer andKloppstech, 1984) or mature green plants (Adamska et al.,1993) ELIP mRNAs have a high turnover and are continu-ously degraded even under conditions that induce theiraccumulation. To assay whether blue or red light of variousintensities can influence the stability of ELIP transcripts thefollowing experiment was performed. Plumulas detached




R Bt t

B R B R

zDCO)

0.130.250.5

0.130.250.5

CL-ILU

Q.O

Figure 2. Effect of various combinations of blue and red light onaccumulation of ELIP transcripts in etiolated pea plumulas. Plumulasdetached from etiolated pea seedlings were exposed to blue (B) orred (R) light (250 /xE m~2 s~') for 30 min and then either transferredto another light quality for an additional 2 h or allowed to remainunder the same light quality for the same time. Poly(A+) RNA wasisolated and used for dot blot hybridization with labeled inserts ofELIP and LHC protein (LHCP) clones. Data represent the averages ofthree independent experiments.

from etiolated pea seedlings were preilluminated with bluelight for 2 h to induce ELIP transcription and then trans-ferred to blue or red light of various intensities for anadditional 4 h (Fig. 4). During this time one set of plumulaswas treated with actinomycin D to prevent further tran-scription and to assay the degradation of accumulatedELIP transcript (Fig. 4, B and D). Control plumulas wereilluminated under the same conditions in the absence ofactinomycin D (Fig. 4, A and C). The rate of total transcrip-tion was estimated for each time by run-off transcriptionassays (Fig. 4, A and C) and the abundance of ELIP tran-script was determined by dot blot hybridization (Fig. 4, Band D). The radioactivity incorporated into in vitro synthe-sized transcripts measured by scintillation counting showsthat there were no significant differences between the rateof overall transcription in plumulas exposed to 1 or 250 pEm~2 s"1. The graphs shown in Figure 4 represent theaverage values that are representative for both light inten-sities. The data demonstrate that the rate of total transcrip-tion increases about 6-fold after 2 h of preillumination withblue light as compared with etiolated controls and is 2-foldgreater in plumulas further exposed to blue than to redlight (Fig. 4, cf. A with C). Addition of actinomycin D topreilluminated plumulas inhibits drastically the transcrip-tional activity in nuclei (Fig. 4, B and D). Only 1 h after theaddition of actinomycin D the transcriptional activity ofisolated nuclei was reduced to 10 to 20% of the initial valuereached in blue light-preilluminated plumulas.

In control plumulas exposed to blue (Fig. 4A) or red light(Fig. 4C) in the absence of inhibitor, the steady-state levelof ELIP transcripts induced during preillumination re-

mained stable or accumulated in a time-dependent mannerand reached the highest levels in plumulas exposed to 250ju,E m~2 s"1 of blue light. Blocking transcriptional activityby addition of actinomycin D to plumulas exposed to bluelight (Fig. 4B) and subsequent quantification of dot blotdata by scanning of the autoradiograms showed that thestability of this transcript is much lower at 1 /j,E m~2 s"1

(10% of an initial value after 4 h of illumination) than at 250H.E m~2 s"1 (55% of initial value after 4 h of illumination).The initial value (100%) represented the amount of ELIPtranscripts accumulated in plumulas during preillumina-tion (lane P) prior to actinomycin D treatment. In seedlingsexposed to red light (Fig. 4D) the degradation rate of ELIPtranscript was independent of light intensity.

High intensity of blue light could modulate transcrip-tional activity of specific genes. To determine whether thehigh abundance of ELIP transcripts at 250 /nE m~2 s"1 ofblue light might result from a high rate of transcriptionspecific for the ELIP gene, preparative run-off transcriptionassays were carried out. For this purpose the nuclei wereisolated from etiolated plumulas or plumulas that werepreilluminated with blue light of various intensities (1 or250 nE m~2 s~J) for 30 min for the induction of ELIPtranscription. The nuclei were used for in vitro transcrip-tion assays and isolation of radioactively labeled ELIP tran-scripts. Results of such an experiment (Fig. 5) show that theELIP transcripts were not detected when nuclei from etio-lated plumulas were used. The transcription rate specificfor ELIP genes increased rapidly after illumination withblue light but was very similar at 1 and 250 fiE m~2 s"1.These data indicate that the blue light intensity-dependentabundance of ELIP transcripts is regulated at the level ofthe ELIP transcript stability.

Role of Protein Synthesis in the Accumulation of ELIPTranscript in Plumulas of Etiolated Pea Plants

To determine whether the blue light- and red light-induced accumulation of ELIP transcripts requires concom-itant synthesis of either nuclear- or chloroplast-encodedproteins, etiolated plumulas were treated with the proteinsynthesis inhibitors cycloheximide or chloramphenicolprior to illumination. The abundance of ELIP transcripts

DARK BLUE RED

3 7 12 3 7 12 3 7 12 days

ZGCO)

"0.130.250.5

* . 0 '

* • •

0

• • •Figure 3. Expression of ELIP genes in plumulas detached from etio-lated seedlings of different ages. Plumulas detached from etiolatedpea seedlings of increasing age (3, 7, and 12 d) were harvestedimmediately (dark) or exposed to blue or red light for 2 h at a lightintensity of 100 /J.E m~2 s~' . Poly(A+) RNA was isolated and used fordot blot hybridization with the labeled insert of the ELIP cDNA clone.The experiment was repeated two times.




BLUE

oc

inO

O.O

250 -i200 -150 -100 -50 -0

RED

D P 124 hour*

o

inO

"ifQ.O

r 250 -200 -150 -100 -50 -0

+ 8

— Actinomycin DD P 1 2 4 hours

|___________j

— Actinomycin D

oc2

<oc2

0.130.250.5

0.130.250.5

COO <Vm Ew U

coCM

*~ UJ

0.13

0.25

.0.5

0.13

0.25

.0.5

• • • •

• • • •

• • • •

• • • •

COOinCM

CM

UJ

CM*

UJ2.

BLUE REDX

"5oc

ino

ao

r 250i200 -150 -100 -50 -0 -

±7

D P 1 2 4 hours

zoc0.130.250.5

+ Actinomycin D

CM

UJ

CM*"

UJ

zocO)

0.13

0.25

.0.5

• • •

• • • •

OmCM

X

'55o3C

inO

"Eao

r 25° i200 -150 -100 -50 -

- 0 -

+ 7

-1D P

+ 1 + 2 + 2

1 2 4 hour*

+ Actinomycin Dr

zEC2

0.13

0.25

.0.5

zDC

2

0.13

0.25.0.5

.• • *

:..

COo «Vin Ew u7

2,

COCM

Y— fi

UJ

BFigure 4. Stability of BLIP transcripts in etiolated pea plumulas exposed to blue or red light of various intensities. Plumulasdetached from etiolated pea seedlings were harvested immediately (lanes D) or preilluminated with blue light (50 fiE m~2

s~1) for 2 h to induce BLIP transcripts (lanes P). Such pretreated plumulas were then transferred to different intensities of blue(A and B) or red (C and D) light for an additional 4 h in the absence (A and C) or presence (B and D) of actinomycin D (atthe final concentration of 30 ng/ml_). Top panels, Nuclei were isolated from approximately 200 plumulas per time point andused for run-off transcription assays as described in "Materials and Methods." The overall transcription rate was estimatedby scintillation counting of the incorporated l«-32P]CTP into TCA-precipitable material. Since there were no significantdifferences between the rates of overall transcription in plumulas exposed to 1 or 250 pf. m~2 s~', the graphs show theaverage values that represent both light intensities. Each bar represents the average of six independent run-off transcriptionexperiments (three for each light intensity tested). The numbers over the bars represent SE. Bottom panels, Poly(A+) RNA wasisolated for each time point and used for dot blot hybridization with the labeled insert of the BLIP cDNA clone.




1 250.•*

,•*

0.13

0.250.5

<D

.£ 'o>0.3_jHI

Figure 5. Hybridization of transcripts synthesized by nuclei isolatedfrom etiolated plumulas preilluminated with various intensities ofblue light to ELIP-specific probe. Nuclei isolated from etiolatedplumulas or plumulas exposed for 30 min to blue light of variousintensities were used for run-off transcription assays as described in"Materials and Methods." The transcription rate specific for the ELIPgene was determined by hybridization of isolated labeled transcriptswith the different concentrations of the ELIP gene-specific probebound to a nylon membrane.

was assayed after 2 h of illumination either by in vitrotranslation (Fig. 6A, top) or by dot blot hybridization (Fig.6A, bottom). The inhibition of protein synthesis by chlor-amphenicol or cycloheximide was tested by in vivo label-ing of proteins carried out at ambient (Fig. 6B, top) orelevated temperatures (Fig. 6B, bottom). The results showthat the inhibition of cytoplasmic protein synthesis by cy-cloheximide did not alter significantly the blue light-in-duced accumulation of ELIP transcripts (Fig. 6A). Cyclo-heximide treatment, however, had a significant effect on

the accumulation of ELIP transcripts in seedlings exposedto red light. In such plants the steady-state level of ELIPtranscripts was much higher in the presence of this inhib-itor than in untreated control seedlings (Fig. 6A). Theseresults suggest that the red light-induced accumulation ofELIP transcripts occurs with concomitant synthesis of nu-clear-encoded polypeptides that negatively regulate thelevel of responses. Notably, treatment of seedlings with thechloroplast protein synthesis inhibitor chloramphenicolprevented the accumulation of ELIP transcript in both lightqualities (Fig. 6A). These results demonstrate that transla-tional activity in the plastid is necessary for normal accu-mulation of ELIP transcripts. In contrast, inhibitors of cy-toplasmic and chloroplastic protein synthesis have noeffect on accumulation of ELIP transcripts when applied topreilluminated seedlings (data not shown). Different pat-terns of radioactively labeled proteins obtained in the pres-ence or absence of inhibitors demonstrate that the inhibi-tion of protein synthesis was very efficient (Fig. 6B, top). Inaddition, the synthesis of nuclear-encoded heat-shock pro-teins as a response to exposure of plumulas to elevatedtemperature shows that the cytoplasmic translation systemremains intact even under conditions when translationalactivity in plastids is arrested (Fig. 6B, bottom).

DISCUSSION

It has been reported (Kloppstech et al., 1984) that inetiolated pea and barley seedlings ELIP transcription canbe induced by red light pulses. Recently, it was shown thataccumulation of ELIP transcripts in dark-grown plants oc-

i <0 0

i0

o m a m E oc cc

kDa £

30 -pELIP

ELIP

BkDa69-

45-

Q. _ Q. —< I < Ioo oo+ + + +

m m m DC oc oc

30-

+ HS +HS

kDa30-

14-

S E0 0

CD ffi CD OD OC

ao

OC OC Ft+C

HI

PI:

Iff* -Figure 6. Accumulation of ELIP transcripts in etiolated plumulas exposed to blue or red light in the presence or absence ofprotein synthesis inhibitors. A, Plumulas of etiolated pea seedlings were harvested immediately (DARK) or exposed for 2 hto blue (B) or red (R) light at a light intensity of 250 /iE m~2 s"1 in the absence or presence of chloramphenicol (CAP; 200/xg/mL) or cycloheximide (CHI; 20 /ig/mL). Poly(A+) RNA was isolated and used for in vitro translation, followed byfluorography (top) or for dot blot hybridization with labeled the insert of the ELIP cDNA clone (bottom). pELIP, Position ofELIP precursor. B, Plumulas were treated as in A but additionally the proteins were labeled with [35S]Met (100 /xCi/mL)during incubation with inhibitors. Illumination was performed either at 25°C (top) or at 42°C (bottom). Equal amounts ofthylakoid proteins were assayed by SDS-PAGE, followed by autoradiography.




curs not only in response to illumination with red light but also by illumination with blue light (Adamska et al., 1992a).

The fluence-response studies presented in this work show that the accumulation of ELIP transcripts is charac- teristic of very-low-fluence responses of phytochrome and low-fluence responses of cryptochrome receptors. To date, the red very-low-fluence response has been observed only for a limited number of genes. Kaufman et al. (1984) reported that in etiolated pea buds cub transcripts have both very-low- and low-fluence responses. Such sensitive responses were reported also for phytochrome (phyA), Pchlide reductase (Pcr), and P-tubulin genes (Thompson and White, 1991). Interestingly, a11 of these transcripts decreased in abundance during illumination.

The activity of the blue light low-fluence system in plants is more common than the very-low-fluence responses mediated via phytochrome. Excitation of the blue low-fluence system induces transcription of cab, plastocyanin (petE), subunit I1 of PSI (psaD), and rbcS (Marrs and Kaufman, 1989; Oelmiiller et al., 1989) genes. Activation of ELIP genes in response to very low fluences of light will have biological significance. Such low light fluxes might be present during germination of seeds below the soil surface and accumulation of ELIP transcript would be a way of preparing for rapid synthesis of the corresponding protein after emergence of seedlings. This view would be in agree- ment with the proposed ELIP function in protection of plants against light stress (Adamska et al., 1992a, 1992b).

Blue light can affect both the rate of transcription and the steady-state level of RNA as was reported for the cub gene family in pea (Marrs and Kaufman, 1989). In the present report, it is shown that blue light intensity-dependent accumulation of ELIP transcript is regulated at the level of mRNA stability. Such posttranscriptional control is very common for plastid gene expression. For nuclear genes, however, most of the evidence for posttranscriptional mechanisms is based so far on discrepancies between in vitro transcription data and in vivo mRNA levels (Briggs et al., 1988; Warpeha et al., 1989).

It was reported previously (Adamska et al., 1992a, 1992b) that in mature green pea plants ELIP transcription is spe- cifically induced by blue and UVA light. Neither ELIP transcripts nor their translation products could be detected in plants exposed to red or far-red light. However, the level of blue light-induced ELIP transcripts was significantly repressed by low-intensity red light, indicating the regulatory role of phytochrome in green plants.

In etiolated seedlings two photoreceptor systems, cryptochrome and phytochrome, are able to induce accumulation of ELIP transcripts and to regulate their abundance. This suggests that the signal transduction chains originating from the blue light receptor and phytochrome leading to accumulation of ELIP transcript in etiolated plants have some steps in common. The fact that both blue and red light qualities applied to seedlings in succession increase the magnitude of the responses support this contention.

A number of genes are known to be controlled by combined action of both receptors. A requirement of both phytochrome and cryptochrome has been reported for the

induction of genes for enzymes of anthocyanin synthesis (Oelmiiller and Mohr, 1985) and induction of cab (Marrs and Kaufman, 1989) and rbcS transcript levels (Fluhr and Chua, 1986). Also, the accumulation of chloroplast-encoded transcript psbD-psbC was shown to be regulated by both receptors (Gamble and Mullet, 1989). However, in many of these cases the blue light was an essential factor for transcription and phytochrome modulated the ampli- tude of the responses.

Inhibition of chloroplast protein synthesis arrests almost completely the accumulation of ELIP transcripts in plumulas of etiolated pea seedlings exposed to either blue or red light. A certain degree of plastid development appears necessary for formation of the plastid factors, since chloramphenicol can prevent their appearance when applied to etiolated plumulas to block plastid development but is ineffective when applied after preillumination. Plastid factors have been postulated to influence the expression of some light-regulated nuclear genes coding for chloroplast proteins as well as the accumulation of enzymes that are related to chloroplast functions (Mayfield and Taylor, 1984; Oelmiiller, 1989). In this context one should mention that in barley plants plastid factors have been suggested to regu-

ETIOLATED SEEDLINGS

plastid factors

BLUE LIGHT PHYTOCHROME RECEPTOR

red light blue /ight Figure 7. Working model for regulation of ELlP expression in etiolated plumulas of etiolated pea seedlings. Photoregulation of ELlP genes in plumulas of etiolated pea plants involves the action of cryptochrome and phytochrome receptors. Severa1 factors seem to be required for ELlP gene expression: red or blue light that activate ELlP transcription and influence mRNA stability and positive signals originating from the plastids. Additionally, nuclear-encoded gene products negatively modulate the steady-state level of ELlP transcripts during the 1 st h of illumination. Preillumination of seedlings abolished this repression.



1 174 Adamska Plant Physiol. Vol. I07, 1995

late the level of ELIP and LHC I1 transcripts under conditions of light stress (Potter and Kloppstech, 1993). The nature of these factors is still unknown.

Inhibition of cytoplasmic protein synthesis resulted in an increased accumulation of ELIP transcripts during illumination of plumulas with red light, whereas in blue light the ELIP transcripts accumulated to the same level as in untreated control plumulas. Also, in this case the preillumination of plumulas abolished this effect. These results (summarized in Fig. 7) suggest that during the 1st h of illumination with red light d e novo synthesized nuclear- encoded gene product(s) negatively regulate ELIP gene expression. This mechanism of negative regulation of ELIP genes in red light appears to be unique. Many reports have been published concerning the involvement of nuclear gene products in the regulation of both nuclear and chloroplast gene expression. In contrast to ELIP, however, a n arrest of cytoplasmic translation prevented the accumulation of these gene products. Lam et al. (1989) have reported that in etiolated wheat induction of cab and rbcS genes is sensitive to cycloheximide. Gamble and Mullet (1989) dem- onstrated that blue light-responsive nuclear genes are involved in the light-induced accumulation of psbD-psbC transcript.

The negative control of ELIP expression in red light might occur a t the level of transcription or mRNA stability or might influence one step in the signal transduction chain leading from receptor to activation of ELIP genes. The mechanism of this control is still unknown and further efforts will be concentrated on identification and characterization of this repression operating in red light.

ACKNOWLEDCMENTS

I am grateful to Drs. Bertil Andersson, Maus Kloppstech, and Itzhak Ohad for comments concerning the manuscript. I also thank K. Debel for helping with computer graphic programs.

Received August 29, 1994; accepted December 14, 1994. Copyright Clearance Center: 0032-0889/95/107/1167/09.

LITERATURE ClTED

Adamska I, Kloppstech K, Ohad I (1992a) UV light stress induces the synthesis of the early light-inducible protein and prevents its degradation. J Biol Chem 267: 24732-24737

Adamska I, Kloppstech K, Ohad I (1993) Early light-inducible protein in pea is stable during light stress but is degraded during recovery at low light intensity. J Biol Chem 268:

Adamska I, Ohad I, Kloppstech K (199213) Synthesis of the early light-inducible protein is controlled by blue light and related to light stress. Proc Natl Acad Sci USA 89 2610-2613

Ahmad M, Cashmore AR (1993) HY4 gene of Arabidopsis thaliana encodes a protein with characteristics of a blue-light receptor. Nature 366: 162-166

Bartels D, Hanke C, Schneider K, Michel D, Salamini F (1992) A desiccation-related ELIP-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. EMBO

Briggs WR, Mosinger E, Schafer E (1988) Phytochrome regulation of greening in barley. Effects on chlorophyll accumulation. Plant Physiol86 435-440

Deng XW, Stem DB, Tonkyn JC, Gruissem W (1987) Plastid run-on transcription. Application to determine the transcrip-

5438-5444

J 11: 2771-2778

tional regulation of spinach plastid genes. J Biol Chem 262:

Fluhr R, Chua NH (1986) Developmental regulation of two genes encoding ribulose-bisphosphate carboxylase small subunit in pea and transgenic petunia plants. Phytochrome responses and blue light induction. Proc Natl Acad Sci USA 83: 23Ei8-2362

Gallagher S, Short T, Ray PM, Pratt LH, Briggs WR (1'?88) Light- mediated changes in two proteins found associated with plasma membrane fraction from pea stem sections. Proc Natl Acad Sci USA 85 800315007

Gallagher TF, Ellis JR (1982) Light-stimulated transcription of genes for two chloroplast polypeptides in isolated p:a leaf nuclei. EMBO J 1: 1493-1498

Galland P, Senger H (1988) The role of pterins in the photorecep- tion and metabolism of plants. Photochem Photobiol48: 811-820

Gamble PE, Mullet JE (1989) Translation and stability of proteins encoded by the plastid psbA and psbB gene are regulated by a nuclear gene during light-induced chloroplast development in barley. EMBO J 8: 2785-2794

Grimm 8, Kruse E, Kloppstech K (1989) Transiently expressed early light-inducible proteins share transmembranc: domains with light-harvesting chlorophyll binding proteins. Plant Mo1 Biol 13: 583-593

Kaufman LS, Thompson WF, Briggs WR (1984) Different red light requirements for phytochrome induced accumulati on of cab RNA and rbcS RNA. Science 226 1447-1449

Kim S, Sandusky P, Bowlby NR, Aebersold R, Greeri BR, Vla- hakis S, Yokum CF, Pichersky E (1992) Characterization of a spinach psbS cDNA encoding the 22 kDa protein of photosystem 11. FEBS Lett 314: 67-71

Kloppstech K, Meyer G, Bartsch K, Hundrieser J, Link G (1984) Control of gene expression during the early phase of chloroplast development. I n W Wiesner, DG Robinson, RC Starr, eds, Com- partments in Alga1 Cells and Their Interaction. Springer Verlag, New York, pp 36-46

Laemmli UK (1970) Cleavage of structural proteins during assem- bly of the head of bacteriophage T4. Nature 227: 680--685

Lam E, Green PJ, Wong M, Chua NH (1989) Phytochroae activation of two nuclear genes requires cytoplasmic protein synthesis.

Leong TY, Vierstra RD, Briggs WR (1981) A blue light-sensitive flavin-cytochrome complex from corn coleoptiles: fuIther characterization. Photochem Photobiol 3 4 697-703

Lers A, Levy H, Zamir A (1991) Co-regulation of a geri'? homologous to early light-induced genes in higher plants and p-carotene biosynthesis in the alga Dunaliella bardawil. J Biol Chem 266:

Marrs KA, Kaufman LS (1989) Blue light regulation of transcription for nuclear genes in pea. Proc Natl Acad Sci USA 86: 4492-4495

Mayfield SP, Taylor WC (1984) Carotenoid deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/ b binding protein (LHCP) mRNA. Eur J Biochem 1 4 4 79-84

Meyer G, Kloppstech K (1984) A rapidly light-induced chloroplast protein with a high turnover coded for by pea nuclear DNA. Eur J Biochem 138: 201-207

Oelmiiller R (1989) Photooxidative destruction of chloroplast and its effect on nuclear gene expression and extraplastidic enzyme levels. Photochem Photobiol 49: 229-239

Oelmiiller R, Kendrick RE, Briggs WR (1989) Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato. Plant Mo1 Biol 13: 223-232

Oelmiiller R, Mohr H (1985) Mode of coaction between blue/UV light and light absorbed by phytochrome in light-metliated anthocyanin formation in the mil0 (Sorghum vulgare Pers.) seedlings. Proc Natl Acad Sci USA 82: 6124-6128

Otto B, Grimm B, Ottersbach P, Kloppstech K (1988) IZircadian control of the accumulation of mRNAs for light- and heat- inducible chloroplast proteins in pea (Pisum sativum L.). Plant Physiol 88: 21-25

9641-9648

EMBO J 8: 2777-2783

13698-13705




Potter E, Kloppstech K (1993) Effects of light stress on the expression of early light-inducible proteins in barley. Eur J Biochem 214: 779-786

Roberts BE, Patterson BM (1973) Efficient translation of tobacco mosaic virus RNA and globin 9s RNA in a cell-free system from commercial wheat-germ. Proc Natl Acad Sci USA 70: 2330-2334

Schamhorst C, Heinze H, Meyer G, Kolanus W, Bartsch K, Heinrichs S, Gudschun T, Moller M, Herzfeld F (1985) Molec- ular cloning of a pea mRNA encoding early light induced, nuclear coded chloroplast protein. Plant Mo1 Biol 4 241-245

Sharrock RA, Quail PH (1989) Nove1 phytochrome sequences in Arabidopsis thaliuna: structure, evolution and differential expres-

sion of plant regulatory photoreceptor family. Genes Dev 3:

Thompson WF, White MJ (1991) Physiological and molecular studies of light-regulated nuclear genes in higher plants. Annu Rev Plant Physiol Plant Mo1 Biol 4 2 423-466

Warpeha KMF, Marrs KA, Kaufman LS (1989) Blue light regulation of specific transcript levels in Pisum sutivum. Plant Physiol

Wedel N, Klein R, Ljungberg U, Andersson 8, Herrmann RG (1992) The single copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem 11. FEBS Lett 314: 61-66

1745-1 757

91: 1030-1035



Documents

Regulation of Early Light-Inducible Protein Gene Expression ......1168 Adamska Plant Physiol. Vol. 107, 1995 and accumulation of the protein in mature green plants (Adamska et al.,