Regulation of chloroplast-encoded chlorophyll-binding protein ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

VOl. 261, No. 24, Issue of August 25, pp.

Regulation of Chloroplast-encoded Chlorophyll-binding Protein Translation duringHigher Plant Chloroplast Biogenesis* (Received for publication, February 11, 1986)

Robert R. Klein and John E. Mullet From the Department of Biochemistry and Biophysics, Texas A&M Uniuersity, College Station, Texas 77843

Etioplasts of 5-day-old dark-grown barley seedlings synthesize most of the soluble and membrane proteins found in chloroplasts of illuminated plants. Prominent among these proteins are the large subunit of ribulose bisphosphate carboxylase and the a- and &subunits of the chloroplast ATPase. However, etioplasts donot synthesize four chloroplast-encodedproteins which are major constituents of the chloroplast thylakoid membrane: two chlorophyll apoproteins of photosystem I (68 and 6 5 kDa) and two chlorophyll apoproteins of photosystem I1 (47 and 43 kDa). Pulse-labeling experiments show that the lack of radiolabel accumulation in the chlorophyll apoproteins in etioplasts is due to inhibition of synthesis rather than apoprotein instability. Illumination of 5-day-old dark-grown barley selectively induces synthesis of the plastid-encoded chlorophyll apoproteins and proteins of 32, 23, and 21 kDa. Synthesis of the chlorophyll apoproteins was significant in plants illuminated for 15 min andwas near maximum by 1 h. The induction of photosystem I chlorophyll apoprotein synthesis was not accompanied by an increase in mRNA for these proteins. These results demonstrate that the synthesis of the plastid-encoded photosystem I chlorophyll apoproteins is blocked at the translational level in dark-grown barley. Translation of the chlorophyll apoproteins is induced rapidly by light with a time course which is similar to the lightdependent formation of chlorophyll from protochlorophyllide.

14). RNA coding for the chlorophyll a/b-binding proteins is low in dark-grown tissue, and transcript accumulation can be induced by activating the phytochrome system (15-18). Accumulation of the chlorophyll a/b-binding proteins may also involve protein stabilization by chlorophyll since in the absence of chlorophyll the apoproteins do not accumulate (15, 16). In oats, bean, and spinach plastid-encoded the photosystem I (PSI)’ chlorophyll apoproteins were reported to accumulate in dark-grown plants (8). In contrast, these proteins did not accumulate in dark-grown barley ( 7 ) .When dark-grown barley seedlings were illuminated, synthesis of PSI chlorophyll apoproteins was observed within 15 min. It was hypothesized that,aswiththe chlorophyll a/b-bindingproteins,lightinduced transcription of the genes coding forthe PSI chlorophyll apoproteins and association with chlorophyll may be required for Stabilization of the apoproteins in the thylakoid membrane ( 7 ) .In maize, mRNA from thegene coding for the PSI chlorophyll apoproteins increased 2-4-fold upon illumination of dark-grown plants (19) consistent with the transcription activation hypothesis (20). However, in spinach a lack of correspondence between changes in mRNAlevels and PSI chlorophyll apoprotein accumulation was reported (11). In this paper we have examined the effect of light on the synthesis of the plastid-encodedchlorophyll-binding proteins during light-induced development. Our results demonstrate that etioplasts are translationally competent and synthesize most of the soluble and membrane proteins found in mature chloroplasts. Our results further indicate that light rapidly and specifically increased the synthesis of PSI chlorophyll apoproteins while transcript levels for the apoproteins did not During leaf formation chloroplasts develop from small un- increase. differentiated organelles known as proplastids (1-3). In monMATERIALS ANDMETHODS ocotyledons such asbarley, leaf development and many steps in chloroplast maturationoccur in theabsence of light. These Plant Growth-Barley (Hordeum uulgare L. var Morex) seedlings include increases in plastid number/cell (1, 4), increases in were grown and maintainedfor allexperimentsin controlled environvolume/plastid (1, 2, 5), synthesis of most stromal proteins mentchambers at 23 “C. Seedswere planted in vermiculiteand watered with full-strength Hoagland’s nutrient solution. For devel(4, 6-9), and accumulation of thylakoid membrane proteins studies seeds were germinated and grown for 5 days in a such as the ATPase subunits( 7 , 8, 10, ll),cytochrome f and opmental dark chamber located ina light-tight room. At this stage of developcytochrome bs (8, 11, 12). When dark-grown plants are illu- mentseedlingswere 9-10 cm tall. After 5 days,seedlingswere minated, protochlorophyllide is reduced to chlorophyll, chlo- transferred to an illuminated chamber witha light intensityof 10,000 rophyll-protein complexes are synthesized and assembled, and lux (fluorescentplus incandescent bulbs).All manipulations of darkgrown plants were performed when possible in complete darkness. photosyntheticelectrontransport is activated.Theevents involved in the transformationof etioplasts (plastids in dark- However, when required, light was provided by a dim green safelight which was unable t o photoconvert measureable amounts of protogrown plants) to chloroplasts are controlled by a t least two chlorophyll. photoreceptors, protochlorophyllide and phytochrome (13). Plastid Isolation-Approximately 50 g ofbarleyprimaryleaves Noticeably absent from etioplast membranes are the nuwere cut and immediately placed in iced water. After approximately replaced witha solution clear-encoded chlorophyll a/b-binding antennae proteins(13, 10 min, water wasdrained from the tissue and ~.

~~~~

* This work was supported by National Science FoundationGrant PCM-8316431 to J. E. M. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

The abbreviations used are: PSI, photosystem I; PSII, photosystem 11; LS, large subunit of ribulose bisphosphate carboxylase; S S , smallsubunit of ribulosebisphosphatecarboxylase;Hepes, 4-(2hydroxyethy1)-1-piperazineethanesulfonicacid NaDodS04, sodium

dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

11138

Regulation of Chlorophyll-binding Synthesis Protein containing 2.1% (v/v) sodium hypochlorite, 0.25% (v/v) Tween 80, and 1.0% (w/v) NaCI. After 5 min, the sterilization fluid was drained and leaves thoroughly rinsed with 5-10 changes of iced water. Leaves were ground in 300 ml of chilled grinding media containing 0.33 M sorbitol, 50 mM Hepes-KOH (pH 8.0), and 2 mM EDTA (pH 7.5 with NaOH). Thehomogenate was filtered through Miracloth and centrifuged at 4000 X g for 1 min. The pellet was gently resuspended in 6 ml of grinding media, and intactplastids isolated by Percoll gradient centrifugation as in Ref. 21 except that sodium pyrophosphate, MnCl,, and MgCl, were eliminated from the gradients. Intact plastids from Percoll gradients were washed once with grinding media, centrifuged at 2500 X g for 5 min, and gently resuspended in chilled sorbitol-Hepes (0.33 M and 50 mM, pH 8.0, with KOH). Generally, the preparation yielded 90-95% intact plastids. All manipulations were performed in a cold room (2-4 "C) in the presence of a dim safelight with wavelength emission of 515-560 nm. Protein Synthesis in Isolated Intact Etioplasts and Plastids from Illuminated Tissue-Conditions for ATP-driven protein synthesis by isolated intact chloroplasts have been previously reported (22). To find optimum incubation conditions for protein synthesis by isolated intact etioplasts, some parameters of the incubation mediawere investigated. Total [35S]methionine incorporation (membrane plus soluble) was greatest at 5 mM ATP, 5 mM MgCl, for etioplast and 10 mM ATP, 10 mM MgCl, for chloroplast translation. Optimum translation conditions for other parameters (amino acid concentration, pH, K+ concentration) were similar for intact etioplasts and intact chloroplasts. The optimized protein synthesis mixture (75 p1, final volume) contained 0.33 M sorbitol, 50 mM Hepes-KOH (pH 8.0), 40 p~ of each amino acid (minus methionine), 10 mM dithiothreitol, 25 pCi of [35S]methionine(specific activity, 1098 Ci/mmol), and 5 mM ATP, 5mM MgC1,. Intact plastids were added at a final concentration of approximately 1.25 X lo' plastids/translation assay. The suspension was warmed to room temperature (23 "C) and incubated with gentle shaking in the light (10,000 lux, for plastids isolated from illuminated tissue) or in the dark (etioplastsfrom dark-grown tissue). Plastids were incubated for 30 min at which time unlabeled methionine (8.5 mM final concentration) was added to block further incorporation of [35S]methionineand to allow chain elongation of incomplete nascent polypeptides (the chase period was 30 min). SorbitolHepes was added toterminatethe reaction. In one experiment, etioplasts were pulse labeled for 5 min in the dark, and thensorbitolHepes was added to terminate thereaction. Following the labeling period, plastids were fractionated into membrane and soluble polypeptides as previously described (22). Measurements of trichloroacetic acid-insoluble radioactivity were obtained (23), and samples were subsequently electrophoresed and autoradiographed (22). In Vivo Labeling-Excised barley seedlings were labeled with [35S] methionine inthe absence or presence of cycloheximide as previously described (22) but with several modifications. Briefly, barley leaves of 5-day-old dark-grown seedlings or leaves of 5-day-old dark-grown seedlings illuminated for 8 h were cut underwater at thebase of their stems and quickly placed in iced water. Leaves were surface sterilized as described above, cut under water at the base of the leaves, and quickly immersed in 300 pl of40 mM Hepes-KOH (pH 8.0) in shortened nitrocellulose vials (10 leaves/vial). When appropriate, cycloheximide was added to a final concentration of 20 pg/ml. Seedlings were preincubated for 20 min at 23"C with the evaporative demand increased by cool air from a hair drier. After 20 min, 300 pCi of [35S]methioninewas added to each vial, and leaves were allowed to incorporate radioactivity under the same conditions for an additional 3 h. When solutions became low during this period, 40 mM HepesKOH (plus cycloheximide when appropriate) was added to each vial. Following incubation, leaves were placed in chilled water and subsequently macerated in 6 ml of grinding media with a razor blade. The suspension was filtered through a 20-pm nylon mesh filter, layered directly onto Percoll gradients, and intact plastids were isolated and fractionated as described above. Determination of Plastid Number and Volume-For quantitation of plastid number (plastids/pl of suspension volume) aliquots of isolated plastids were diluted and plastids counted in a hemocytometer with a X 20 phase contrast lens on a Nikon photomicroscope. Plastid dimensions (length and width) of approximately 50 plastids/ treatment were measured on photographic prints of negatives enlarged to give a final magnification of 10,000. Plastid sections with the largest values for length and width were recorded since the longest value for each dimension should represent the median plane section of the organelle and thus give an accurate approximation of the

11139

dimensions of the plastid (5). Measurements of plastid dimensions obtained in this manner are in close agreement with previously reported estimates of length and width of barley plastids (1).Based on the observation that plastids of dark-grown barley (5-7 day-old) are ellipsoidal (5) and that plastids do not change shape during the early phase of illumination, we have estimated the volume of plastids by the volume of rotation equation for elliptical objects,

where L is the long axis of ellipsoid and W is the short axis of ellipsoid. Isolation and Quuntitation of Plastid rRNA and mRNA-Pea chloroplast RNA wasisolated by phenol extraction as previously described (24). To ensure high yield and reproducibility the phenol phase of each extract was re-extracted twice. Variation of nucleic acid recovery/plastid volume was less than 5% (based on 2 extractions done in triplicate). Detection of rRNA was done by separating total nucleic acid isolated from a known plastid volume (approximately 0.5 pg/ lane) on methyl mercury gels (25). Separated RNA was detected by ethidium bromide staining. A portion of the total nucleic acid extract was treated with DNase I (24) and the RNA reisolated after phenol extraction. RNA concentration was calculated using one absorbance unit at 260 nm equal to 40pgof RNA/ml. Northern analysis was done using chloroplast RNA separated on glyoxal gels (26) (approximately 0.3 pg of nucleic acid/lane). Methods used for DNA labeling and hybridization were as described previously (24). The Northern probe for rbcL mRNA was a 1.3-kbp PstI-Hind111 internal fragment from barley rbcL (27). The Northern probe for psaA-psaB was a 1.4kbp BamHI-BamHI DNA fragment from spinach which contains a portion of the open reading frames of psaA andpsaB(28). The Northern probe for psbA was a 0.55-kbp EcoRI-PstI internal DNA fragment of pea psbA (29). RESULTS

Tissue Selection-The growth of the barley primary leaf is only marginally different for the first5 days whendark-grown plants are compared to light-grown plants. Since the leaves of grasses have basal meristems, the variouscells of the leaf represent an ontogeneticsequence with themost mature cells situated at the apex and the youngest cells at the base at any point in time (3,4). aIn 5-day-old dark-grownbarley seedling, plastidsinthebasalpart of theprimary leaf are typical (1).Plastids in proplastids and contain almost no membranes more mature regions (5th cm from the base to the leaf tip) are larger, more numerous per cell, and have a well defined prolamellar body (1). Mature fully developed etioplasts require a relatively brief period of illumination to be transformed into mature functional chloroplasts while the undifferentiated proplastids (basal leaf sections) require a much longer period of illumination to develop into a mature chloroplast (1). Thisis because proplastids have notalready synthesized the membranes, pigments, and enzymes that are present in mature etioplasts.Based on these factswe decided to concentrate investigations on the 4-cm apical section of 5 day-old dark-grown barleyleaves whose plastids represent near fully developed etioplasts. Protein Synthesis in Vivo in Plastids of Dark-grown and Illuminated Plants-It has been proposed by Siddell and Ellis (6) that plastids at different stages of development may synthesizea differentspectrum of proteinsthanthose from mature chloroplasts. Based on this and the observation that chlorophyll-binding proteins of PSI did not accumulate in dark-grown barley ( 7 ) ,we characterized the patterns of protein synthesis of plastids from dark-grown and illuminated plants. To do this [35S]methionine was used to label plastid proteins in uivo in the presenceor absence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. After the labelingperiod, plastids were isolatedfrom the apical4cm of barley leaves, and radiolabeled polypeptides were examined

11140

Regulation of Chlorophyll-binding Protein Synthesis

by autoradiography of NaDodSO, gels (Fig. 1). This experiment indicated that major light-induced changes in the synthesis of plastid pol-ypeptides occur in the membrane fraction. Both thelarge subunit (LS) and small subunit (SS)of ribulose bisphosphate carboxylase were synthesized in mature etioplasts and in plastids from illuminated seedlings (Fig. 1, lanes 5-6). As expected, cycloheximide inhibited synthesis of SS and mostof the soluble plastid polypeptides but did not inhibit synthesis of LS and two proteins which comigrated with the (Y- and B-subunits of the chloroplast ATPase (Fig. 1, lanes 78).It was noted that illumination of dark-gown barley altered the synthesis of only a few nuclear-encoded soluble proteins (Fig. 1, lanes 5 uersus 6). In contrast numerous differences were observed when radiolabeled membrane proteins from dark-grown plants were compared to illuminated plants (Fig. 1, lane 1 u e r s u 2 or 3 versus 4 ) . Of the nuclear-encoded membranepolypeptides(synthesisinhibited by cycloheximide), light induced the synthesisof chlorophyll a/b-binding proteins (i.e. LHCII) and inhibited the synthesis of several unidentified high molecular weight (270 kDa) polypeptides of etioplast membranes(Fig. 1,lane 1 versus 2). Of the plastidencoded membrane polypeptides (synthesized in the presence

Soluble

Membrane

?1 .2

3 4 7 8

5 6

924-

u68. -:PSI:

8"cF'c 43.

18.1

14.2

ua :PsII:

==

ais

"LS.

L.

-

-ss

of cycloheximide), etioplasts synthesize the(Y- and &subunits of the ATPase, a low level of a 32-kDa polypeptide (possibly the gene product of psbA), andseveral lower molecular weight polypeptides (Fig. 1, lane 3). Etioplasts, in general, synthesized a nearly complete set of chloroplast-encoded polypeptides with the exception of the chlorophyll-binding proteins of PSI and PSII(Fig. 1, lane 3 uersus 4 ) . Net synthesis of the chlorophyll apoproteins of PSI and PSII was not obtained until seedlings were illuminated. In addition,the relative accumulation of label in a 32-kDa protein increased significantly in illuminated plants (Fig. 1, lane 3 uersus 4 ) . These results indicated that while etioplasts are translationally active and are capableof synthesizing a nearly complete set of plastid-encoded polypeptides, light selectively stimulated the synthesis of alimited number of membrane polypeptides which included chlorophyll apoproteins of PSI and PSII. The resultsin Fig. 1 indicate that theexpression of plastidencoded chlorophyll-binding proteins was tightly coupled to early events of light-induced plastid development. However, it was unclear whether expression was regulated at the transcriptional or post-transcriptionallevel. Post-transcriptional regulation could involve apoproteinor mRNA stabilityor inhibition of apoprotein mRNA translation. Unfortunately, complications associated with in vivo labeling methods concerningthe secondaryeffects of plant excision, inhibitor application, and plant-to-plant variation in label uptake make quantitativestudies of plastid gene expressiondifficult. Therefore, to examine the regulation of chlorophyll apoprotein synthesis in a more quantitative way, we examined protein synthesis, protein turnover, and RNA levels in plastids isolated from dark-grown and illuminated seedlings. Changes in PIastid Size-Light-induced changes in the size of plastids from the primary leaf of barley were determined during the first 16 h of illumination (Table I).A 31% increase in plastid volume occurred when 5-day-old dark-grown barley seedlings were illuminated for 16 h. Other workers have also noted significant increases inplastid size and plastid number/ cell when dark-growntissue was illuminated (1, 30-32). Therefore, to account for the observed increasein plastid volume during light-induced development, data obtainedwith isolated plastids(proteinsynthesis, RNA content, protein composition and turnover)was expressed on an equal plastid volume basis. Data expressedin this way separate plastid events from other cellular changes yet allow expression on a per cell basis using published values for plastid number/cell (3, 4, 33,34). Polypeptide Composition, rRNA Content, and Protein Synthesis of Plastids Isolated during Light-induced DeuelopmentThe time course of polypeptide accumulation during lightinduced development is represented in Fig. 2. When expressed on an equal plastid volume basis, light-induced changes in

TABLE I Average plastid length, width, and estimatedp h t i d volume from 5-day-old dark-grown and illuminatedseedlings Five-day-olddark-grownbarleyseedlings were transferred to a FIG.1. In vivo-labeled plastid polypeptides from 5-day-old dark-grown and illuminated barleyseedlings. Barley leaves lighted chamberand plastids isolated after 0,1,4, or 16 h. Dimensions were labeled in the absence (lanes I, 2 , 5 , 6 ) or presence of 20 pg/ml (length and width) of approximately 60 plastids/treatment were cycloheximide (lanes 3 . 4 , 7,R). Seedlings labeled included 5-day-old measured on photographic prints of plastids a t a final magnification as mean 2 S.E. dark-grown barley (lanes I , 3 , 5 , 7) and 5-day-old dark-grown barley of 10,000 times. Results are expressed-~ illuminatedfor 8 h prior to labeling (lanes 2, 4, 6, 8). Following Hours of Plastid Plastid Plastid labeling,plastids were isolated,fractionated into membraneand length illumination volume width soluble polypeptides, and separated on NaDodS0.-PAGE gels (loaded pm wn p l x 10-8 on equal cpm/lane basis). Numbers to the left indicate mobility of 6.5 f 0.1 4.9 f 0.1 10.7 ? 0.6 0 M , standards (kDa). CFI refers to the n- and &subunits of the 1 6.4 2 0.1 4.8 f 0.1 10.3 f 0.4 chloroplast ATPase,D marks the position of a 32-kDa protein which 6.8 f 0.1 4 5.1 f 0.1 12.8 f 0.4 comigrates with the psbA gene product, LHCII marks the position of 16 7.1 f 0.1 5.4 f 0.1 14.0 2 0.5 the chlorophyll a/b-binding antennae proteins of PSII. ~- ~~~~

~

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~~~

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~~~~

"~~

~

~

Regulation of Chlorophyll-bindingSynthesis Protein

Membrane

Soluble

2oc

“LS

I P S II

43.

11141

of the thylakoid membraneoccur. The protein synthesis activity of intact plastids isolated from plants illuminatedfor 0-16 h is shown in Fig. 3. A slight increase in the activity of soluble polypeptide synthesis by isolated plastids was observed during the firsthour of illumination followed by a decrease in synthesis during the next3 hours of illumination. In agreement with previous reports (6, 22), the LS of ribulose bisphosphate carboxylasewas the major soluble product synthesized by isolated etioplasts and illuminated plastids. In contrast, a 3-4-fold increase in synthesis of membrane polypeptides was observed within 1 hour afteronset of illumination (Fig. 3). Thereafter, agradual decline in synthesis of membrane polypeptides was observed. The increase in membrane protein synthesis activity during light-induced plastid development could be due to anincrease in chloroplast ribosome number. T o examine this possibility nucleic acidwasisolatedfrom plastids of dark-grown and illuminated seedlings. The nucleicacid was treated with DNase I, and the RNA content/plastid was determined after phenol extraction and precipitation (Table 11). This analysis

25.; LHC II

189 14.3

0 1 4 1 6 Timeilluminated, hr

u 1 4 1 t j

FIG. 2. Accumulation of plastid polypeptides during the first 16-h illumination of barley seedlings. Five-day-old dark-

1

4

16

Time illuminated. hr

grown barley seedlings were transferred to a lighted chamber and plastids isolated after 0.1.4, or16 h. Plastid concentrations (plastids/ pl of solution) and plastid volumes (pl/plastid) were estimated as described under “Materials andMethods.” Plastids were fractionated into membrane andsoluble polypeptides and loaded onto NaDodS04PAGE gels on an equal plastid volumebasis.Polyacrylamidegels were fixed and silver stained. Numbers to the left indicate mobility of M ,standards (kDa). Pchlrd marks the position of the protochlorophyllide reductase protein. CFZ and LHCZZ are defined in thelegend to Fig. 1.

stainable levels of polypeptides were restricted to membrane proteins (Fig. 2, lanes 1-4). Little change in stainable levels of solublepolypeptides (includingLSand SS of ribulose bisphosphate carboxylase) were observed during greening (Fig. 2, lanes 5-8).This is in agreement with reports that light is notrequired for the accumulationof ribulose bisphosphate carboxylase in barley (4, 7, 35). In contrast, after 16 h of illumination marked accumulation of nuclear encoded chlorophyll a/b-binding proteins (LHCII) and polypeptides in the region of the chlorophyll-binding proteins of PSI and PSI1 was observed (Fig. 2, lanes 1-4). A decrease in the level of a 36-kDa polypeptide, tentatively identified asprotochlorophyllide reductase, was also observed. Little change was observed in the stainablelevels of the 0-and P-subunits of the ATPase. This experiment established the time frame during which major light-induced changes in the proteincomposition

Time illuminated, hr

FIG.3. Quantitation of [S5S]methionineincorporation into soluble and membrane polypeptides by isolated plastids. Plastids were isolated from seedlings after 0,1,4, or 16 h of illumination and incubated with [3sS]methionine in optimized protein synthesis mixtures for 30 min. After 30 min, excess unlabeled methionine (8.5 mM) was added to each mixture and incubated for 30 min. Plastids were subsequently fractionated into membrane and solule polypeptides, and 1-pl aliquots were processed for measurement of trichloroacetic acid-insoluble radioactivity.Plastid concentrations(plastids/ pl of solution) and plastid volumes (pllplastid) were estimated and incorporation of label expressed on a equal plastid volume basis.


11 142

TABLE I1 R N A content of plastids from 5-day-old dark-grown and illuminated barley seedlings . " . . ~

Hours of illumination

Soluble

1 2 3 4 5 6

7 8 9 10 11 12

~

RNA/plastid

RNA/plastid ~

pg/2 X

5.7

Membrane

0 0.25 0.5 1.0 16.0

~

.~

.

-

~

IO9plastids

P.c/lpl

5.9 1224 1200 1224 1158 ~

5.6 5.7 4.1 -~

18.4-

14.3-

-

~

--

"

-

" "

0

1

4

13

24

FIG.5. Increased synthesis of PSI and PSII chlorophyll apoproteins, a 32-kDa polypeptide ( D ) ,and proteins of 23 and 21 kDa (open arrow) during the first 1 6 h of illumination. FIG. 4. Ribosomal RNA content of plastids during the first Plastids were isolated from seedlings illuminated for 0 (lanes 1 and 2 4 hofseedlingillumination. Five-day-old dark-grown barley 7), 0.25 (lanes 2 and 8),0.5 (lanes 3 and 9),1 (lanes 4 and l o ) , 4 seedlings were transferred to a lighted chamber, and plastids isolated (lanes 5 and 11 ), or 16 h (lanes 6 and 12). Isolated plastids were after 0 , 1, 4, 13, and 24 h. Plastid concentrations (plastids/pl) and plastid volumes (pl/plastid) were determined, and plastid RNA was incubated with ["S]methionine for 30 min and chased for 30 min isolated by phenolextractions. RihosomalRNAisolated from an with excess unlabeled methionine. Plastid concentrations (plastids/ were equal volume of plastids was loaded on methyl mercury gels (approx- pl) and volumes (pl/plastid) were determined,andplastids imately 0.5 pg/lane), and separated RNA was detected by ethidium fractionated into membrane and soluble polypeptides. Protein samples were loaded on NaDodS0,-PAGE gels on a equal plastid volume bromide staining. Arrows mark ribosomal RNA. basis. Gels were fixed, fluorographed, and exposed to Kodak x-ray film (Type BB-1) for 72 h. Numbers to the left indicate mobility of showed that the total RNA content/plastid remained constant M, standards (kDa).CFI and D are defined in the legend to Fig. 1. Time illuminated, hr

during the first hour of seedling illumination with a small decrease in RNA content apparent after 16 hours of illumination. Examination of plastid nucleic acid on methyl mercury agarose gels revealed that rRNA (marked by arrows in Fig. 4), which makes up the bulk of plastid RNA, did not change significantly in composition or content in response to illumination. These analysessuggest that changesin ribosome number do not account for the increase in plastid membrane protein synthesis in illuminated plants. To determine whether t.he increase in synthesis of membrane polypeptides during illumination resultsfrom a general increase in membrane polypeptide synthesis orfrom increased synthesis of selected light-induced polypeptides, the pattern of proteins synthesized during the first 16 h of illumination was examined (Fig. 5). Theprofile of membrane polypeptides synthesized by isolated intact etioplasts was very similar to that, obtainedin vivo plus cycloheximide (Fig. 1, lane 3 uer.ws Fig. 5 , lane I). No net synthesis of either PSI or PSII chlorophyll apoproteins was observed in plastids isolated from dark-grown plants. After 15 min of illumination, however, synthesis of either PSI and PSII apoproteins was observed, and synthesis increased to an apparent maximum a t 1 hour. No further qualitative changes in pol?lpeptide synthesis were observed out to 16 h of tissueillumination.Theseresults demonstrate thatwhile etioplasts do not synthesize thechlorophyll apoproteins of PSI and PSII, the light-regulated induction of apoproteinsynthesis occurs within 15min of illumination. I t was also noted that induction of chlorophyll apoprotein synthesis was selective. Light did not induce a

general increase in the synthesis of all membrane polypeptides. Rather, exceptfor the chlorophyll apoproteinsand proteins of 32,23, and21 kDa, light did not alter the synthesis of membrane polypeptides. The resultsin Fig. 5 verified the changes in protein synthesis observed in uiuo and provided a more quantitative analysis of the time course and magnitude of the changes in protein synthesis which occurwhendark-grown plants are illuminated. However, it was not clear whether thelack of detectable label in chlorophyll apoproteins was due to the inability of etioplasts to synthesize chlorophyll the apoproteins or to rapid turnover of newly synthesizedapoproteins. To determine which of these mechanisms accountsfor the lack of detectable label in chlorophyll apoproteins, isolated plastids from darkgrown plants were pulse-labeled to detect transient polypeptides synthesizedwithin etioplasts (Fig. 6 , lanes 1 and 4 ) . During a 5-minpulse label etioplasts synthesized measurable levels of full length polypeptides along with incomplete nascent polypeptides (marked by open arrows). No PSI or PSII chlorophyll apoproteins were detected in either themembrane or soluble phase of pulse-labeled etioplasts (radiolabeled polypeptides of isolated chloroplasts are shown in lanes 3 and 6 for comparison). When pulse-labeled etioplasts were chased with unlabeled methionine, incomplete nascent polypeptides full length (marked by openarrows) read outtomature polypeptides (22), yetagain the chlorophyll apoproteins were not radiolabeled (Fig. 6, lunes 2 and 5 ) . These results, therefore, demonstrate that the lack of radiolabel in chlorophyll

Regulation of Chlorophyll-binding Protein

12

Soluble Membrane 1

2

3

4

53

4Ps I

-mu

4CF I

-

43'

4

25.7.

D 1 8.4.

34

psaA/B)

.PS II

C h

--

5 6

6

l4LS

h

D

5

11143

4

D

L;

Synthesis

"B

D 4D

psbA)

--

--

C FIG. 7. Northern blot of rbcL, psbA,and psaA-psaB mRNA. RNA was isolated from an equal volume of plastids isolated from plants grown in the dark ([ones I , 3, 5 ) or from plants illuminated for 1 h (lanes 2, 4, 6 ) . The RNAwas separatedon glyoxal gels, transferred tonylon membranes, and hybridized with nick-translated DNA from rbcL (lanes I , 2 ) , psbA (lanes 3, 4 ) or psaA-psaB (lanes 5 , 6 ) . RbcL encodes LS, psbA encodesa32-kDa quinone-binding protein, and psaA-psaH encode chlorophyll apoproteins of PSI.

the ratio of the two rbcL transcripts (Fig. 7, lunes 1 and 2). In contrast to LS, the PSI chlorophyll apoproteins and a 32-kDaprotein show a dramatic light-inducedincreasein synthesis (Figs. 1,5, and 6). Thelight-induced increase could be a consequence of activated transcription or translation of mRNA. To address this question the mRNA levels for the PSI chlorophyll apoproteins anda 32-kDa protein (psbAgene FIG. 6. Pulse-chase labeling of etioplast polypeptides. Five- product) were determined in etioplasts andin plastids isolated day-old dark-grown barley seedlings were transferred to illuminated from illuminatedplants (Fig. 7). The genes for thePSI chambers and plastids isolated after 0 or 1 h. Etioplast polypeptides chlorophyll apoproteins (psaA-psaB) and the32-kDa protein were pulse-labeled with ["SSjmethionine for 5 min and then chased (psbA) have not been mapped on the barley chloroplast gefor 0 (lanes I and 4 ) or 30 min (lanes 2 and 5 ) with excess unlabeled nome. Therefore, for psbA we used an internal 5'-end probe methionine. Plastids from 1-h illuminated seedlings were also pulsefrom peas to detect barley mRNA in the two plastid populalabeled for5 min and chasedfor 30 min (lanes3 and 6) with unlabeled methionine. Reactions were terminated by the addition of 0.375 ml tions. This probe hybridized to an RNA of a size similar to of chilled sorbitol-Hepes to reaction vessels. Plastids were fraction- that foundin peas (1.3 kbp) (Fig. 7, lanes 3 and 4 ) . This ated and protein samples loaded on NaDodS0,-PAGE gels on an analysisalso showed that psbA transcript levels did not equal cpm basis. Gels were fixed, fluorographed, and exposed to xincrease during the first hour of plant illumination. During ray film for 1 week. CFI and D are defined in the legend to Fig. 1. this same period a significant increase in synthesis of a 32kDa protein comigrating with the psbA gene product was apoproteins is not due tochlorophyll apoprotein turnover but observed. T o analyze psaA and psaB mRNA levels we used rather to theirlack of synthesis in etioplasts. an internal psaA probe from spinach (KpnI-BarnHI) and a Transcript Levels in Plastidsof Dark-grown and Illuminated probe which containedportions of both psaA andpsaB BarleyPlants-It has previouslybeen reportedthat two (BamHI-BamHI) (28, 37). These probes as well as a psaBmRNAs (1.8 and 1.6 kbp), differing attheir5'-ends,are specific probehybridized to abarleychloroplastRNA of formed from barley rbcL (the gene coding for ribulose bisapproximately 5.8 kbp (data not shown). This suggests that phosphate carboxylase LS(36)).Theshorter of the rbcL psaA and psaB are cotranscribedin barley as has been found transcriptspredominatesinetioplasts of 7-day-old dark- in spinach (28) and peas (37). The spinach psaA-psaB probe grown barley, and the relative amount of the larger transcript was used to analyze RNAsextracted from etioplastsand illuminatedplants.Thisanalysis increases upon illumination of these plants (36). Our results plastids isolatedfrom show that LS synthesis is active in etioplasts of 5-day-old showed that barley psaA-psaB transcript levels did not inof illumination of dark-grown dark-grown barley, and illumination of the plants for 1 h has crease during the first hour 7, lanes 5 and 6). During this same period PSI barley (Fig. little effect on LS synthesis(Fig. 5). Northern blots were done to determine if the level of rbcL mRNA or ratio of the two chlorophyll apoprotein synthesis went from undetectable to transcripts changed inresponse to illumination. Fig. 7 shows near maximum levels. DISCUSSION that both rbcL transcripts are present in the5-day-old darkgrown barley plastids used in this study. In addition, illumiEtioplasts in 5-day-old dark-grown barley synthesize most nation of plants for 1 h does not alter the concentration or of the soluble and membrane-bound polypeptides found in

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chloroplasts (Figs. 1and 5). Prominent among these proteins are the LS of ribulose bisphosphate carboxylase and the aand &subunits of the chloroplast ATPase. Illumination of dark-grown barley does not significantly alter the synthesis of most plastid-encoded proteins but selectively enhances the synthesis of 7 thylakoid polypeptides: two PSI proteins of 68 and 65 kDa which bind chlorophyll a (7,38), two PSII proteins of 47 and 43 kDa which bind chlorophyll a (39-43), a 32-kDa protein which comigrates with the psbA gene product, and polypeptides of 23 and 21 kDa. It has recently been suggested that the psbA gene product is a PSII reaction center protein and also binds chlorophyll (44). The induction of the synthesis of these proteins occurred within 15 min and was near maximum by 1hour of illumination. The present results and those of Vierling and Alberte (7) indicate that the synthesis of PSI and PSIIchlorophyll apoproteins and a 32-kDa polypeptide is tightly coupled to an early event of light-induced plastid development. One such early event is the light-dependent conversion of protochlorophyllide to chlorophyllide with subsequent conversion to chlorophyll (45). In 5-day-old dark-grown barley the formation of chlorophyll from protochlorophyllide was 90%complete after 15 min of illumination (7). This time course agrees closely with the induction of apoprotein synthesis which suggests that formation of chlorophyll a may be a key regulatory point for chlorophyll apoprotein synthesis. A tight coupling between chlorophyll formation and chlorophyll apoprotein synthesis provides several advantages to the plant. First, the build-up of a pool of chlorophyll precursors (protochlorophyllide) and chlorophyll apoprotein transcripts in dark-grown plants allows rapid synthesis and assembly of PSI complexes once plants areilluminated. This is consistent with reports of rapid activation of PSI activity upon illumination of etiolated plants (40, 46). Second, it is known that chlorophyll which is not associated with its normal protein-carotenoid complex can photooxidize membranes (47). Therefore, lack of coordination between chlorophyll synthesis and chlorophyll apoprotein synthesis could have deleterious consequences. We also note that these early light-induced events precede the major increase in chlorophyll and chlorophyll a/b apoprotein accumulation which occurs between 2 and 16 hours of illumination ((7, 48) see Fig. 2). Theselater light-induced events are influenced by phytochrome (13). Investigation of transcript levels showed that transcripts for the PSIchlorophyll apoproteins were present in etioplasts and that their levels did not increase during the first hour of illumination. Therefore, light rapidly and specifically increased the synthesis of these polypeptides at a time when transcript levels did not change indicating that synthesis is regulated at thepost-transcriptional level. Pulse-labeling experiments clearly show that the failure to radiolabel PSI chlorophyll apoproteins in etioplasts was not due to protein turnover but rather due to the lack of synthesis of the apoproteins. These results show that, at the stage of seedling development investigated here, a marked effect of light upon plastid protein synthesisis exerted at thetranslational level. In addition to activating PSI apoprotein synthesis, light also caused increased synthesis of two PSII chlorophyll apoproteins (products of the psbB and psbC genes), a 32-kDa protein which comigrates with the psbA gene product and proteins of 23and 21 kDa. RNA from the psbC gene is present in barley etioplasts as part of a polycistronic RNA which suggests that this PSIIapoprotein is also under translational control? The same is true forpsbA; however,further analysis is needed to test whether the 32-kDa polypeptide which shows T. Berends and J. E. Mullet, unpublished data.

light-induced synthesis is a product of the psbA gene.Finally, the 23- and 21-kDa polypeptides which show light-induced synthesis arethe products of unknown genes. Previous reports have shown that qualitative and quantitative changes of plastid-encoded polypeptides during lightinduced development correlated with corresponding alterations in the abundance of specific transcripts (19). This led to the idea that light-dependent increases in transcription played a central role in light-induced chloroplast development. The genes whose transcript levelswere reported to increase upon illumination included psaA-psaB, psbA, and genes for subunit I11 and the a-subunit of the chloroplast ATPase (19). However, Herrmann et al. (11)and Altman et al. (49) have shown in spinach andmaize that plastid-encoded genes are not shutoff in etiolated tissues. When expressed on a total cellular RNA basis, illumination of spinach leaves increased the steady state mRNA concentrations of all plastid-encoded genes, not aselective set of photogenes (11).Even then, there was a lack of correspondence between changes in mRNA levels and polypeptide accumulation. Our results, in contrast to those of Rodermel and Bogorad (19), show that changes in synthesis of the chlorophyll apoproteins of PSI and a 32-kDa polypeptide occur rapidly in response to light and that these changes precede large changes in steady-state mRNA levels. These conflicting results may be resolved by the fact that plants of different developmental stages had been used in these experiments. We have observed changes in transcript levels of plastid-encoded genes when older (710-day-old) etiolated tissue is illuminated? However, even though changes in transcript levels of plastid-encoded genes were observed in the older tissue, light-induced control of gene expression also appears to be exerted at thetranslational level? Further, reported observations of light-dependent regulation of transcript levels and the reported low level of plastid-encoded transcripts in etiolated tissue may, in part, be related to themanner of data expression. When expressed on a whole tissue basis (mg of fresh weight, mg of total protein, leaf area basis, or total cellular RNA basis), the influence of light on plastid gene expression is confounded by effects of light on leaf development and associated increases in plastid number/cell and increased volume/plastid. Finally we should note that the selective regulation of chlorophyll apoprotein translation is an example of a growing list of plastidevents which are regulated at the level of translation. These include regulation of gene expression during Euglena plastid biogenesis (50), regulation of rbcL translation in Amaranth cotyledons (9) and in pea buds (51), and synthesis of the psbA geneproduct in mature Spirodela plants (52). These examples suggest that translational regulation may beimportant for coordination of nuclear and plastidgene expression and thecoordination of chromophore and cofactor biosynthesis during chloroplast biogenesis. REFERENCES 1. Robertson, D.,and Laetsch, W. M. (1974) Plant Physiol. 5 4 , 148-159 2. Mackender, R. 0.(1978) Plant Physiol. 6 2 , 499-505 3. Boffey, S. A., Sellden, G., and Leech, R. M. (1980) Plant Physiol. 65,680-684 4. Smith, H.(1970) Phytochemistry 9,965-975 5. Henningsen, K. W., and Boynton, J. E. (1969) J.Cell Sci. 5,757793 6. Siddell, S. G., and Ellis, R. J. (1975) Biochem. J. 146,675-685 7. Vierling, E., and Alberte, R. S. (1983) J. Cell Biol. 97,1806-1814 8. Nechushtai, R., and Nelson, N. (1985) Plant Mol. Biol. 4 , 377384 9. Berry, J. O., Nikolau, B. J., Carr, J. P., and Klessig, D.F. (1985)

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