The Role of Plastids in the Thylakoid Proteins Studied ... · further data are required to settle...

Plant Physiol. (1994) 105: 1355-1364

The Role of Plastids in the Expression of Nuclear Genes for Thylakoid Proteins Studied with Chimeric P-Glucuronidase

Gene Fusions'

Cordelia Bolle, Sudhir Sopory, Thomas Lübberstedt, Ralf Bernd Klosgen, Reinhold G. Herrmann, and Ralf Oelmüller*

Botanisches lnstitut der Ludwig-Maximilians-Universitat, Menzingerstrasse 67, 80638 München, Germany

We have analyzed plastid and nuclear gene expression in tobacco seedlings using the carotenoid biosynthesis inhibitor norflurazon. mRNA levels for three nuclear-encoded chlorophyll- binding proteins of photosystem I and photosystem II (CAB I and II and the CP 24 apoprotein) are no longer detedable in photobleached seedlings, whereas those for other components of the thylakoid membrane (the 33- and 23-kD polypeptides and Rieske Fe/S polypeptide) accumulate to some extent. Transgenic tobacco seedlings with promoter fusions from genes for thylakoid membrane proteins exhibit a similar expression behavior: a CAB-8- glucuronidase (CUS) gene fusion is not expressed in herbicide- treated seedlings, whereas PC-, FNR-, PSAF-, and ATPC-promoter fusions are expressed, although at reduced levels. All identified segments in nuclear promoters analyzed that have been shown to respond to light also respond to photodamage to the plastids. Thus, the regulatory signal pathways either merge prior to gene regulation or interad with closely neighboring cis elements. These results indicate that plastids control nuclear gene expression via different and gene-specific ris-regulatory elements and that CAB gene expression i s different from the expression of the other genes tested. Finally, a plastid-directing import sequence from the maize Waxy gene is capable of directing the CUS protein into the photodamaged organelle. Therefore, plastid import seems to be functional in photobleached organelles.

Mayfield and Taylor (1984) first demonstrated that the expression of nuclear genes for plastid proteins depends on the developmental stage of the plastids (reviewed by Oel- müller, 1989; Taylor, 1989). Based on the analysis of pleiotropic mutants in which plastid biogenesis is inhibited at early stages of development (Mayfield and Taylor, 1984; Batschauer et al., 1986; Mayfield et al., 1986a; McHale et al., 1990) and of seedlings in which plastids are destroyed by photooxidation because of the absence of carotenoids (Sie- fermann-Harms, 1987; cf. Mayfield et al., 1986b), a plastid- derived signal has been postulated that specifically influences the expression of nuclear genes for plastid proteins. Organelle control of nuclear gene expression is probably part of a more general intracellular communication system, which may also include peroxisomes (Feierabend and Schubert, 1978; Feier-

' This work was supported by the Deutsche Forschungsgemein- schaft (SFB 184). S.S. was supported by the Alexander-von-Hum- boldt-Stiftung, Bonn, Germany.

* Corresponding author; fax 49-89-1782-274.

abend and Kemmerich, 1983) and mitochondria (King and Attardi, 1990). In yeast, for instance, heme, synthesized in mitochondria, controls the transcription of the nuclear-encoded iso-1-Cyt c gene (Guarente and Mason, 1983; Forsburg and Guarente, 1989; Liao and Butow, 1993).

Although the nature of the plastid-derived signal(s) is still unknown, various physiological studies, performed to char- acterize its action and relation to discrete metabolic pathways, have suggested that the signal is not directly related to photosynthesis, since the plastids of severa1 white mutants (Palomares et al., 1993) or of etiolated (Oelmüller et al., 1986a) and FR-grown (Oelmiiller et al., 1986b) seedlings, in which the photoconversion of Pchlide to Chlide is inhibited, can trigger nuclear gene expression. The signal also seems to be required continuously, disappears rapidly after impair- ment of the plastid compartment (Batschauer et al., 1986; Oelmüller et al., 1986b), and influences nuclear gene transcription (Batschauer et al., 1986; Taylor et al., 1986; Burgess and Taylor, 1988; Emst and Schefbeck, 1988; Sagar et al., 1988; Stockhaus et al., 1989). Growth of rye seedlings under permissive temperatures (32OC), which prevent the assembly of plastid ribosomes (Feierabend and Schrader-Reichhardt, 1976; Feierabend, 1978), and studies with inhibitors of the plastid translation machinery suggest that nuclear gene expression is not dependent on plastid protein synthesis (Oelmiiller et al., 1986b; cf. Bunger and Feierabend, 1980; Biekmann and Feierabend, 1985) but requires plastid transcription (Rapp and Mullet, 1991).

Although these results are consistent with the hypothesis that cytoplasmic synthesis of plastid polypeptides may be controlled by plastid-synthesized RNA (Bradbeer et al., 1979), further data are required to settle the argument that organel- lar nucleic acids are in fact directly involved in the intracellular communication. It needs to be established to what extent the inhibition of plastid transcription affects the biogenesis of the organelle in general, which, in tum, results in the downregulation of nuclear gene expression. Analyses of the expression of chimeric promoter/GUS gene fusions in trans-

Abbreviations: ATPC, gene for subunit y of the ATP synthase; CAB, Chl a/b-binding protein; CaMV, cauliflower mosaic virus; CP 24, apoprotein of the CP 24 complex; D, darkness; FNR, Fd-NADP+- oxidoreductase; FR, far-red light; GUS, P-glucuronidase; PC, plastocyanin; NF, norflurazon; NiR, nitrite reductase; NR, nitrate reductase; PSAF, gene for subunit I11 of PSI reaction center; WL, white light.

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1356 Bolle et al. Plant Physiol. Vol. 105, 1994

genic plants (Stockhaus et al., 1989; Liibberstedt et al., 1994b) and in isolated protoplasts (Harkins et al., 1990) suggest that the action of the signal is restricted to a particular tissue, or even to the cell, in which the signal is released from the organelle.

For most of the investigations conceming this topic, mem- bers of the RBCS and CAB gene families were used. Damage to plastids causes a reduction of their expression in maize (Mayfield and Taylor, 1984; Taylor et al., 1986, 1987), mustard (Oelmiiller and Mohr, 1986), rice (Emst and Schefbeck, 1988), barley (Rapp and Mullet, 1991), tobacco (McHale et al., 1990), and Arabidopsis (Susek et al., 1993), although the extent of the response of the two gene families differs significantly. In general, the CAB genes were found to be more sensitive than the RBCS genes. Batschauer et al. (1986) established conditions in barley under which transcription of the CAB gene family was sensitive to treatments acting on plastids, whereas no effect was noted for the transcription of the RBCS and NADPH-Pchlide oxidoreductase genes. This led to the hypothesis that intermediates of the Chl biosynthesis pathway exhibit a specific inhibitory effect on CAB gene expression (Batschauer et al., 1986), similar to the situation in Chlamydomonas (Johanningmeier and Howell, 1984; Johan- ningmeier, 1988). Because of the differences between CAB and RBCS genes, and because of different response pattems of various other nuclear genes for plastid proteins (Burgess and Taylor, 1987), it could not be ruled out convincingly that more than one signal or even gene-specific signals are involved in this process. More recently, a nuclear-encoded gene for a 28-kD chloroplast RNA-binding protein was found to be expressed at normal rates in spite of severely impaired organelles (Tonkyn et al., 1992). Obviously, nuclear genes for plastid proteins can respond quite differently to the developmental stage of the plastids.

An additional complexity derives from the observation that transcript levels of nuclear genes coding for cytoplasmic enzymes with functions related to plastids, such as NR, also fail to be expressed in tissues with impaired plastids (Bomer et al., 1986; Oelmiiller et al., 1988; Oelmiiller and Briggs, 1989). However, this might be caused by a completely different regulatory network. Expression of the N I A genes (encoding NR) in tobacco, for example, is influenced by nitrogen metabolites from the organelle (Caboche and Rouze, 1990; Vincentz et al., 1993), which have no effect on other nuclear- encoded genes for plastid proteins tested (R. Oelmüller, unpublished results).

More recently, Susek et al. (1993) have identified at least three Arabidopsis loci necessary for coupling the expression of some nuclear genes to the functional state of the chloroplast. The homozygous recessive mutations allow nuclear gene expression in the absence of chloroplast development and may interfere with the switch from dark-grown to light- grown development. The authors used a chimeric CAB promoter construct, which was responsive to photooxidative damage to the chloroplasts. Also, studies with other promoter constructs suggested that defined cis-regulatory elements in the 5’ flanking region of nuclear genes are the major target site for the action of the postulated plastid signal(s) (cf. Harkins et al., 1990). Thus, the question arises whether plastid-specific responsive elements can be identified that are

specifica.lly involved in the interorganelle commurication and whether they are different from those determining the quan- titative or light-regulated expression.

We took advantage of DNA fragments for specific plastid- encoded genes from spinach (Herrmann et al., 1985, 1991), of a variety of homologous cDNA probes for nuclear genes encoding thylakoid membrane proteins from tobacco (Palo- mares et al., 1991) and of transgenic tobacco seeds with chimeric promoter fusions from nuclear genes for plastid proteins from spinach (Bichler and Herrmann, 1990; Oel- miiller et al., 1992, 1993; Flieger et al., 1993b; Lii‘jberstedt et al., 1994.a, 1994b) to analyze this topic in greater detail. Our data confirm that CAB gene expression, but also that of another Chl-binding protein, the CP 24 apoprotc?in, is more sensitive to plastid damage compared to the expression of nuclear genes that do not code for Chl-binding proteins and that the signal is perceived by gene-specific and quite divergent cis-regulatory elements in the respective promoters. Separate regions for light responsiveness and plastid-dependent expression could not be identified.

MATERIALS AND METHODS‘

Tobacco seedlings (Nicotiana tabacum Samsun. NN) were grown on one-half-strength Murashige-Skoog mcrdium (Mu- rashige and Skoog, 1962), supplemented with SUC (3%, w/v), casein hydrolysate (0.05%, w/v), and amm.onium succinate (I5 mM) in the presence or absence of NF (5 PM; Zoecon Corp., Palo Alto, CA) in temperature-controlled (25 f 0.5OC:) growth chambers. The light qualities and quantities used for the individual experiments are specified below.

Most of the methods used have been describetl, including the determination of enzyme activities (Oelmiiller et al., 1986b; Liibberstedt et al., 1994a), the isolation and radiola- beling of gene-specific fragments and hybridiza tion conditions, RNA isolation, dot blot and northem analysis, quanti- fication of hybridization signals (Flieger et al., 1993a; Oelmiiller et al., 1993), and westem analysis with monospe- cific antisera raised against spinach thylakoid membrane proteins (Herrmann et al., 1985, 1991). The light ijources and filters have been described by Palomares et al. (1991).

GUS .activities were determined by a standard protocol described by Liibberstedt et al. (1994a). Since thc accumulation of the fluorescent reaction product increaijes linearly with regard to time (t,,,,, tested = 6 h) as long as the product formation is not limited by the amount of substrate, but the background fluorescence is constant, low GUS activities can easily be detected. The background fluorescence (no transgenic tobacco or extracts without substrate) leads to calculated values of 0.09 k 0.03 nmol mg-’ protein min-!. In a11 in- stances, :fluorescente in “active” extracts can easily be detected even with a UV hand lamp after longer incubaticns.

RESULTS

The Effect of NF on Plastid Development and Gene Expression

Since photodamage of plastids not only affwts carbon metabolism but also nitrate assimilation (Bomer c?t al., 1986; Oelmiiller, 1989; Oelmiiller and Briggs, 1989; Seith et al.,



Role of Plastids for Nuclear Gene Expression 1357

1991; Vincentz et al., 1993), tobacco seedlings were grown on one-half-strength Murashige-Skoog medium, supplemented with Suc and NH4+ succinate in the presence or absence of NF, a herbicide known to inhibit phytoene desaturase (Sandmann et al., 1989; Chamovitz et al., 1991). This antimetabolite causes an inhibition of plastid development in high light intensities (Bartels and McCullough, 1972). After 12 d in WL (30 W m-’), herbicide-treated seedlings contained no immunologically detectable Chl-binding proteins (CAB 11, CP 47, subunit I of PSI reaction center) and only trace amounts of the 23- and 33-kD polypeptides of the oxygen- evolving system, of the Rieske Fe/S polypeptide, PC, and the subunits CY and p of the chloroplast ATP synthase (data not shown). The activities of the stromal NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase and ribulose- 1,5-bisphosphate carboxylase were at least 20-fold reduced (1.4 and 4.6%, respectively, of the corresponding water controls) and that of NiR was approximately 10-fold lower (9.4% of the water control), but the activities of the cytoplasmic NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were slightly increased (1 13 and 126%, respectively). The only exception is the cytoplasmic NR activity, which is not detectable in photobleached seedlings (less than 1 %), consistent with previous reports from other species (Borner et al., 1986; Rajasekhar and Oel- miiller, 1987; Oelmiiller et al., 1988). Quantitation of dot blot signals from RNA hybridized to probes specific for plastid genes demonstrate that the steady-state transcript levels of the rDNA, psbA, psbE, petA, and atpA genes are approximately 20-fold lower compared to the water controls and comparable to the levels found in tobacco seeds, whereas etiolated seedlings contain almost identical or slightly reduced

120

” rDNA psbA psbE petA atpA

Figure 1. Relative amounts (WL control = 100‘70) of RNA of four plastid genes and rDNA in seeds and 12-d-old tobacco seedlings that were kept in either WL or D in the presence or absence of NF. FR + WL: NF-treated seedlings were germinated in FR for 4 d before transfer to photodamaging WL for another 8 d. Results are based on four independent RNA extractions and dot blot assays; SD values are between 3 and 10%. After hybridization, the dots were excised, and the radioactivity was quantitated by liquid scin- tillation counting.

mRNA levels as WL-grown seedlings (Fig. 1). However, if NF-treated tobacco seedlings were germinated in FR for 4 d before transfer to photodamaging WL for another 8 d, the psbA transcript level increased strongly, but no accumulation was observed for the other four transcript levels tested (Fig. 1). The increase is caused by the accumulation of psbA transcripts during the photostress, since 4-d-old FR-grown seedlings contain only 22 f 4% of the transcripts detectable in 12-d-old control seedlings. This suggests that plastid development, including plastid gene expression, is prevented completely if the photodamage takes place from the very beginning. When the plastid transcription machinery has been established (obviously within the first 4 d after sowing), it seems to be relatively stable and highly selective in tran- scribing plastid genes.

Herbicide Treatment Affects the Expression of Nuclear Genes for the Chl-Binding Proteins and Those for Other Thylakoid Proteins

Northem analyses with cDNAs for six thylakoid membrane proteins demonstrate that the transcript levels of three polypeptides associated with Chls (CAB I, CAB 11, and the CP 24 apoprotein; genes CAB I, CAB 11, CP 24, respectively) are below or at the limits of detectability in herbicide-treated seedlings, whereas those for the Rieske Fe/S polypeptide (PETC), as well as for the 33-kD (PSBO) and 23-kD (PSBP) polypeptides, were only moderately reduced (Fig. 2A). Fur- thermore, transcripts for three enzymes of the nitrate assimilation pathway, NR, NiR, and Gln synthase (genes NIA, NIR, GS, respectively) respond quite differently to the herbicide treatment; whereas the NIA transcript level fails to accumulate in photobleached seedlings, NIR transcripts accumulate to some extent (Fig. 2A), and the GS transcript level does not respond to the treatment (see “Discussion”).

Comparison of the steady-state mRNA levels for thylakoid membrane proteins with results obtained for the expression of chimeric promoter/GUS gene fusions in transgenic tobacco seedlings suggests that the observed differences seem to reflect differences in nuclear gene transcription and are caused by different response pattems of cis-regulatory elements in their promoters: the spinach CAB promoter does not direct GUS gene expression in herbicide-treated seedlings, whereas the PC, FNR, PSAF, and ATPC promoter fusions are clearly expressed, albeit with substantially reduced rates as compared to water controls (Fig. 2B). Finally, an approximately 2-kb-long DNA fragment with the 5’ flanking region of the Arabidopsis NIA-1 gene fails to direct GUS gene expression in transgenic tobacco (average value of 24 independent primary transformants: 0.09 f 0.01 nmol mg-’ protein min-’, which is identical with the background; cf. “Materials and Methods”).

To demonstrate that these results are caused by different sensitivities of the expression of the trans-genes toward photooxidative damage, seedlings harboring the above-men- tioned constructs were grown on NF in dim FR (0.5 W m-’) for 5 d, were then illuminated with high intensity WL (30 W m-’) for O (control), 1, 4, 12, and 24 h, and transferred back to FR until the 12th d. Figure 3 demonstrates that 1 h of WL




-NT +NF -NF +NF -NF + NF

CAB I

CP24

PETC

PSBO

PSBV

NIANIR

OS

m

B

PC ATPC FNR PSAF

Figure 2. A, Northern analysis of nuclear-encoded transcripts forplastid proteins and for the NR in 12-d-old tobacco seedlings thatwere grown on either water (—NF) or NF (+NF, 5 jjmol). CAB II (I),Gene for the light-harvesting Chl a/b-binding proteins of PSII (I); CP24, gene for the apoprotein of the CP 24 complex; PETC, gene forthe Rieske-Fe/5-protein of the Cyt b/f complex; P5BO(P), genes forthe 33-kD (23-kD) polypeptide of the oxygen-evolving complex;NIA (NIR), gene for NR (NIR); CS, gene for Gin synthase. B, GUSactivity in 12-d-old transgenic tobacco seedlings harboring chimericpromoter/GUS gene fusions. The following spinach promoter frag-ments were used: CAB, -377/+71; PC, -259/+60; ATPC, -992/+ 173); FNR, -3200/+231; P5AF, -220/+162. Based on the analysisof offsping from at least 10 independent primary transformants.Bars represent SD values. For each construct, the activity of thewater control was taken as 100, and the other values were ex-pressed relative to it. The absolute levels for the water controls are(nmol mg'1 min~'): CAB, 175 ± 52; PC, 75 ± 28; ATPC, 2.89 ± 0.22;FNR, 38 ± 14; PSAF, 58 ± 19.

suffices to prevent accumulation of the GUS activity in "CAB"seedlings, whereas 12 and 24 h of WL are required for a 25and 35%, respectively, reduction of the GUS activity in "PC"seedlings. No inhibitory effect, even after 24 h in WL, wasobserved for seedlings bearing the other promoter fragments(ATPD, FNR, and PSAF; data not shown). This demonstratesthat the CAB promoter exhibits the highest sensitivity towardphotodamage to the plastids. Figure 2A suggests that thisregulation is not restricted to CAB II transcripts but seemsalso to be true for transcripts for other, nuclear-encoded Chl-binding proteins. Comparison of these results with thoseobtained for psbA (Fig. 1) demonstrates that transcripts forChl-binding proteins of nuclear and plastid origin can exhibitan opposite regulation in herbicide-treated seedlings (cf. Figs.1 and 2A).

Short Promoter Fragments Seem to Be Involved in thePerception of the Plastid-Derived Signal

Four promoter segments, the -220/-119- and -118/-29-bp regions from the FNR promoter fused to the spinach CAB-1 minimal promoter (-77/+71), designated FNR-1 and FNR-2, respectively, and the -2S9/-79- and -168/-79-bpregions from the PC promoter fused to the 35S RNA CaMVminimal promoter (—90/+3), designated PC-1 and PC-2, re-spectively, which are sufficient to confer light-regulatedexpression to heterologous TATA boxes (Liibberstedt et al.,1994a, 1994b), respond to photodamage of plastids (Fig. 4A).Promoter fragments, which are active in plants but fail torespond to light, such as the trimer of the G-box sequencefrom the RBCS promoter from spinach (Liibberstedt et al.,1994a) also fail to respond to photodamage to the plashds(Fig. 4A). These results suggest either that the cis elements inthese promoter fragments, which are responsible for the light-regulated and plastid-dependent expression, are located inthe vicinity of each other or that both signal pathways mergeprior to gene regulation. In addition, since two adjacent FNRpromoter fragments are capable of directing plastid-depend-ent gene expression to the CAB-1 minimal promoter, morethan one cis element can be involved in the perception ofphotooxidative damage to the chloroplasts. None of thesegments shows obvious sequence similarities.

The suggestion that at least one major target site for theplastid-derived signal(s) is a cis element in the vicinity of therespective transcription start sites is further supported byresults obtained with 5' promoter deletions. The — 377/+71-

120

1 h 4 h 12h 24 h

Figure 3. GUS activities in 12-d-old, FR-grown (5 W m"2) tobaccoseedlings that were grown in the presence of NF (5 ̂ mol). Seedlingscontain either a CA6 (-377/+71) or a PC (-259/+60) promoterfragment. After sowing (5 d), the FR treatment was interrupted byphotodamaging WL (30 W m"2); the duration of this treatment isindicated in the abscissa. The GUS activity in control plants (nophotodamage = 0 h) was taken as 100%, and the others areexpressed relative to it. For details and statistics, see legend toFigure 2. Except for the CAB/CUS gene fusion, for which theabsolute activity in FR-grown seedlings was 49 ± 6 nmol mg~'min"', the enzyme activity directed by the PC construct wasnot significantly different from the value given in the legend toFigure 2. www.plantphysiol.orgon March 23, 2019 - Published by Downloaded from

Copyright © 1994 American Society of Plant Biologists. All rights reserved.



A 120

e 80 1 O 0

0 60

Y W

40

20

o FNR-1 FNR-2 PC-1 PC-2 G-box

B 120

1 O 0

e 80

g 8 60 o) 2 3 CJ

40

20

O

FNR PC PSAF ATPC RBCS

C

+ + + m o m - . . . . . . " e m m o

C y N ; ? E q ; PC PSA F

Figure 4. The effect of N F (5 pmol) on the accumulation of GUS activities in 12-d-old tobacco seedlings. A, Promoter fragments fused to minimal promoters. FNR-1 (-2), The -220/-119-bp (-1 18/ -28-bp) promoter fragment from the FNR gene was fused to the inactive -70/+71 -bp CAB-1 gene promoter from spinach (Oel- müller et al., 1993). PC-1 (-2), The -259/-79-bp (-169/-79-bp) PC promoter region was fused to the CaMV minimal promoter (-90/ +3; Lübberstedt et al., 1994b). G-box, A trimer of the spinach RBCS-1 C-box sequence (Lübberstedt et al., 1994a). The absolute GUS activities (in nmol mg-' protein min-') are: FNR-1, 5.4; FNR-2, 1.9; PC-1, 13.6; PC-2, 2.9; G-Box, 2.2. B, Short promoter fragments fused to the CUS gene: FNR, -118/+231 bp; PC, -169/+60 bp;

For details and statistics see legend to Figure 2. The absolute GUS activities (in nmol mg-' protein min-') are: FNR, 19.6; PC, 4.7; PSAF, 11.4; ATPC, 2.8; RBCS, 11.3. C, Ratio of the GUS activities of water control seedlings and NF-treated seedlings kept in high intensity WL. Every point represents t h e ratio from t h e offspring of an independent primary transformant. The promoter fragments are indicated on the abscissa.

PSAF, -1 78/+162 bp; ATPC, -1 72/+173 bp; RBCS, -298/+80 bp.

bp CAB (Fig. 2B), -118/+231-bp FNR, -168/+60-bp PC, -178/+162-bp PSAF, -172/+173-bpATPC, and -298/+98- bp RBCS-1 fragments are sufficient to respond to both stimuli (Fig. 4B, data for the light-response have been reported by Liibberstedt et al., 1994a), whereas further 5' deletions to

-88/+162 bp (PSAF), -47/+173 bp (ATPC), and -77/+98 bp (RBCS) result in the complete loss of the respective promoter activities (for more detailed information about the promoters, see Flieger et al., 1993b; Oelmiiller et al., 1993; Lübberstedt et al., 1994a, 1994b). However, Figure 4C demonstrates that regions located further upstream are also involved in the perception of the developmental stage of the plastids. The -168/+60-bp PC and -178/+162-bp PSAF promoter regions cause an approximately 2-fold reduction of the GUS leve1 in herbicide-treated seedlings, whereas an approximately 4-fold (PSAF) and even 8- to 10-fold (PC) reduction is observed with longer fragments.

-77/+71 bp (CAB), -28/+231 bp (FNR), -50/+60 bp (PC),

The lmportance of Chloroplasts in the Expression of the Chimeric CAB Gene Fusion

Histological expression studies demonstrate that the GUS activities under the control of the PC, ATPC, FNR, and PSAF promoters in etiolated seedlings are almost exclusively detectable in the vascular tissue (for details, see Flieger et al., 1993b; Liibberstedt et al., 1994b; R. Oelmüller, unpublished data) presumably because this expression is caused by cell- type-specific factors that operate independently of light (Flie- ger et al., 1993b; Liibberstedt et al., 1994b). However, the CAB promoter/GUS gene fusion was never expressed in any tissue of etiolated seedlings (Lübberstedt et al., 1994a), sug- gesting that functional plastids are also required for its expression in the vascular tissue. To test this we have transformed the white plastome mutant SRlV35 (Palomares et al., 1993) with the CAB and PC gene promoter constructs. As expected, the CAB fusion was not expressed, but the PC gene fusion was, although at low levels (approximately 20% compared to activity in the SRl wild-type, cf. Lübberstedt et al., 1994b). In vivo GUS staining revealed that this activity is only detectable in the vascular tissue of the white mutant, consistent with the expression pattem observed in etiolated seedlings (data not shown). The plastome mutation is unsta- ble, and green spots are frequently detectable in different tissues of the mutant. This change is not only accompanied by a significant increase in the expression of the chimeric PC gene fusion but also by the expression of the CAB gene fusion, irrespective of the tissue (vascular tissue, stem, or leaf region with mesophyll cell) in which the change occurred (Table I). This supports the conclusion that the activity of the CAB promoter depends strictly on the stage of the plastids and suggests that its regulation is different from that of other thylakoid membrane proteins.

Furthermore, the CAB-GUS fusion is not expressed in the style of the ovary, whereas very high GUS levels were detected for the other chimeric gene fusions tested (Fig. 5 ) . However, microscopy revealed that this tissue contains fully developed chloroplasts that lack properly developed grana thylakoids, an indication for a reduction of PSII (G. Wanner and R. Oelmüller, personal communication). We are currently




Table I. CUS activities in white and green sections of the SR1V35mutant

Construct Tissue GUS activity

nmol min ' mg ' protein

CAB (-377/+71) White leafGreen leafWhite stemGreen stemWhite styleGreen style

PC (-259/+60) White leafGreen leafWhite stemGreen stemWhite styleGreen style

0.09.40.03.60.00.0

18.134.2

2.28.91.1

13.1

trying to test whether the decreased expression of the CABfusion is correlated with the development and/or assemblyof PSII.

Photodamage to the Plastids Does Not Affect the OverallProtein Import into Plastids

A possible explanation for the reduced transcription ofnuclear genes for thylakoid membrane proteins in herbicide-treated seedlings is that precursor polypeptides synthesized

Figure 5. GUS staining in the stigmatoid tissue of the style of theovary. The transgenic tobacco plant was transformed with the — 118/+231-bp FNR promoter fragment, fused to the GUS gene. Compa-rable expression patterns were observed for chimeric GUS genefusions with promoter/leader segments from the spinach PSAD,PS/AF, PC, and ATPD genes (data not shown). E + C, Epidermis andcuticula; P, parenchyma; ST, stigmatoid tissue.

10 15 20 25

Fraction number

30 35 40

Figure 6. Cell fractionation of crude organelle extracts from 12-d-old NF-treated potato seedlings harboring chimeric GUS genefusions with (Waxy-GUS) and without (GUS) the plastid-directingWaxy transit peptide on a Sue gradient. For details, see Klosgen etal. (1986, 1989). After fractionation, the GUS activity was deter-mined and compared with a cytoplasmic and plastid marker enzyme(the NAD- versus NADP- glyceraldehyde-3-phosphate dehydrogen-ase, GDH). Note that the optical test is sensitive enough to detectthe residual (approximately 1%) NADP-glyceraldehyde-3-phos-phate dehydrogenase activity in herbicide-treated seedlings.

in the cytoplasm cannot be imported into the impaired or-ganelles because essential components might have been de-stroyed by photooxidation (Oelmuller, 1989). The precursorpolypeptides could then exert an inhibitory effect on theexpression of their own genes in the nucleus, for instance viaa feedback mechanism. We took advantage of chimeric GUSgene fusions, which contain the plastid-directing Waxy transitpeptide of maize and the constitutively expressed 35S RNACaMV promoter (Klosgen et al., 1986, 1989; Klosgen andWeil, 1991). Since this promoter does not respond signifi-cantly to herbicide treatments (data not shown), it also directsexpression in photobleached seedlings. Thus, the GUS levelin transgenic potato seedlings with the chimeric 35S RNACaMV/Waxy/GUS gene fusion is only slightly reduced inherbicide-treated seedlings (12% compared to the water con-trol) and comparable to the levels obtained for seedlingsharboring a control construct without the import sequence(35S RNA CaMV/GUS, data not shown). Cell fractionationin Sue gradients demonstrates that the GUS activity derivingfrom the Waxy gene fusion is located in the same fractionsthat contain the rudimentary plastids, as indicated by usingthe residual activity of the marker enzyme NADP+-depend-ent glyceraldehyde-3-phosphate dehydrogenase (Fig. 6).Thus, the GUS protein with the Waxy import sequenceappears to be quantitatively imported into the rudimentaryplastid structures (see 'Discussion').

DISCUSSION

Our data demonstrate that nuclear genes for plastid pro-teins exhibit different sensitivities against photodamage of www.plantphysiol.orgon March 23, 2019 - Published by Downloaded from

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plastids and that the stage of the plastid is perceived via gene-specific and quite divergent cis elements in the respective promoters. At least some of these elements are located relatively close to the transcription start site. It is also evident that plastid control for the expression of the C A B gene is different from that for other nuclear genes (Table I; Fig. 5). This raises the question of whether more than one signal originating from the organelle may exist, as has been suggested for the phytochrome-induced signal pathway (Neu- haus et al., 1993).

Although the nature of the postulated plastid signal(s) remains elusive, a particularly interesting observation is that a11 identified promoter fragments that are involved in light perception (Liibberstedt et al., 1994a) seem to be also involved in the perception of the plastid signal. This does not exclude the fact that additional elements that were not identified by our approach are also involved in the regulated expression of these genes. However, since the chosen spinach promoter fragments respond to both signal pathways (and probably also pathways from other stimuli) in tobacco, it is likely that they merge prior to gene regulation and act at the same cis elements and through the same truns factors in a given promoter or that distinct cis elements are located very close to each other or overlap. In addition, more than one promoter region can be involved in the perception of both signals (Fig. 4, A and C; cf. Liibberstedt et al., 1994a). Since these fragments contain no obvious sequence similarities, we suggest that they are target sites for different regulatory proteins.

In an attempt to link nuclear gene expression to regulatory pathways, various DNA fragments have been identified in the genes studied that specifically interact with protein factors in gel shift assays (Oelmiiller et al., 1992, 1993; Flieger et al., 1993b; Liibberstedt et al., 1994b). We prepared extracts from NF-treated and control plants, the white plastome mutant SRlV35 (cf. Palomares et al., 1993), which exhibits strongly reduced expression levels of nuclear genes for thylakoid proteins (R. Palomares and R. Oelmüller, unpublished observations), and cauliflower inflorescences that do not express ‘photosynthetic” genes and used them for gel retardation assays. None of the fragments tested showed any significant and reproducible difference in the retardation pattern. In some cases, even stronger complexes were formed with extracts from white tissues (R. Oelmiiller, unpublished data).

Regulation of the Expression of Plastid- and Nuclear- Encoded Cenes for Chl-Binding Proteins

Photobleached seedlings fail to accumulate Chl-binding proteins of plastid and nuclear origin. Nevertheless, the psbA transcripts can accumulate even under conditions when the RNAs for the nuclear-encoded components disappear. Thus, signal pathways that control the expression of the plastid and nuclear genes for Chl-binding proteins appear to operate differently in the two organelles. Figure 2 indicates that the expression of the three analyzed nuclear genes for Chl- binding proteins is controlled by a similar or common regulatory pathway(s), which probably operates via cis elements in their corresponding promoters. In addition, the response

pattem seems to be conserved in spinach and tobacco (Fig. 2B).

Comparison of the upstream sequences of a number of CAB gene promoters revealed highly conserved regions even from different species (cf. Dunsmuir, 1985; Oelmiiller et al., 1993). This suggests that the C A B multigene family derives from a single ancestor and that the regulatory elements have been multiplied along with the structural gene. This raises the question of why promoters from other genes can respond to the same stimuli, since they originated and evolved independently of each other and from those of the CAB genes. A possible explanation could be that the regulatory elements have been under an evolutionary pressure that streamlines the expression of genes for functionally related proteins. On the other hand, signal pathways and responsive elements may have co-evolved; not only cis elements but also steps in the transduction chains could have been modified during this process. Some of these pathways (e.g. those for the light response or response to the circadian rhythm) could derive from prokaryotic roots, since similar response pattems are established already at this level. However, the intracellular communication system between organelles has probably been developed de novo after the establishment of eukar- yotes.

Tissue-Specific Expression of the Chimeric CUS Cene Fusions

Our data indicate that the expression of the chimeric CAB gene fusion is under a different control from the organelle than those of other photosynthetic genes. The absence of the expression of the CABIGUS gene fusion in the style of the tobacco ovary is consistent with the idea that a CAB-specific plastid-derived signal is not released from the organelle in this tissue. Since the other chimeric gene fusions tested are highly expressed in the interior stigmatoid tissue of the style, analysis of this plastid type might be helpful for a better understanding of the interorganelle communication. On the other hand, since some thylakoid membranes are detectable in the inner tissue of the style, Chl-binding proteins should be present. The most likely interpretation is that the CAB genes have been expressed during earlier stages of development and are no longer expressed in the mature style.

Photodamage in Carotenoid-Free Seedlings

The effect of the photodamage in carotenoid-free seedlings is probably more complicated than anticipated to date. Whereas membrane components and various soluble enzymes are usually at the limits of detectability, others such as NiR activity are clearly detectable, albeit at severely reduced rates (cf. Harpster et al., 1984; Mayfield et al., 1986a; Mohr et al., 1992). The thylakoid membrane is the primary target site for the photodamage (Siefermann-Harms, 1987), where excited triplet Chls react with ground-state oxygen. This results in the generation of singlet oxygen (Feierabend and Winkelhiisener, 1982). This component reacts primarily with unsaturated fatty acids of membrane lipids, aromatic amino acids, and purines, but the half-life of singlet oxygen is long enough to interact also with components in the plastid




stroma. Obviously, some of the components are quite sensitive to this insult, but others are only moderately affected. For instance, the psbA transcript level after 4 d in FR is relatively low (22 * 4% of the level detectable after an additional growth of the seedlings under photooxidative conditions for 8 d, cf. Fig. 1). Thus, the almost normal accumulation of psbA transcripts in herbicide-treated seedlings suggests that the plastid transcription machinery, once established, is relatively resistant to photodamage (Fig. 1). Figure 6 demonstrates that transport into (and perhaps also out of) the rudimentary organelles is functional. This excludes the possibility of a general feedback mechanism on nuclear gene expression, if cytoplasmically synthesized precursor polypeptides cannot be imported into the photodamaged organelle. Furthermore, these results are consistent with various previous observations that showed that the outer membrane seems to be unimpaired in photobleached cells (Bach- mann et al., 1967; Reiss et al., 1983; Mayfield et al., 198613). Since the Waxy organelle-directing import sequence derives from an amyloplast polypeptide (Klosgen et al., 1986), we cannot exclude that chloroplast-specific mechanisms controlling the import of chloroplast proteins are absent or impaired in photobleached organelles. This seems to be unlikely since, for instance, plastid RNA polymerases contain nuclear-encoded subunits, which must be present in and imported into the photobleached organelle.

The accumulation of psbA transcripts in photobleached seedlings demonstrates that essential components for transcription are present and functional in photobleached organelles (Sagar and Briggs, 1990; Tonkyn et al., 1992). Under the chosen conditions, psbA transcripts can only accumulate in tobacco seedlings if the carotenoid-free plastids were allowed to develop for a certain period under protective light. They are not detectable if the development of the seed proplastids into chloroplasts in the light or etioplasts in the dark is continuously blocked by photodamaging light. This indicates (a) that the establishment of the plastid transcription machinery occurs at early stages of plastid biogenesis, (b) that it is relatively stable toward photodestruction, and (c) that it shows a high selectivity for the transcription of plastid genes under permissive conditions. Furthermore, since the effect of the plastid signal(s) can be detected as early as the organelle starts to develop from a proplastid to a chloroplast (green tissue) or etioplast (etiolated tissue), and since this development ceases completely if plastid transcription is inhibited (Oelmiiller, 1989), it is not surprising that plastid transcription is a prerequisite for the release of this signal (cf. Rapp and Mullet, 1991).

The quite different effect of the herbicide treatment on the accumulation of the NIA, NIR, and GS transcript levels is consistent with the idea that the reduction of nitrate by NR is rate limiting for the nitrogen assimilation pathway and that a crucial step in this regulation is the control of NIA gene expression. The transcript level for the second enzyme of this pathway, NiR, is less sensitive to the photodamage, which ensures reduction of the toxic nitrite even under photostress. The relatively high GS mRNA level in photobleached seedlings could be caused by the hybridization of the probe to mRNA species for the cytoplasmic and plastidic isoforms, because isolated cDNAs for both genes exhibit a high degree

of sequence similarities (cf. Elmlinger et al., 1994). It is conceivable that only the transcript level for the plastidic enzyme iresponds to the photodamage. Finally, the responsive elemente of the Arabidopsis NIA-1 gene are probably not exclusively located within the first 2 kb upstream of the gene, since chimeric GUS gene fusions with this prom oter/leader fragment are not expressed in transgenic tobacco.

ACKNOWLEDCMENT

The authors thank Prof. W. Riidiger (Botanical Institute, Munich) for critically reading the manuscript.

Received January 18, 1994; accepted April5, 1994. Copyright Clearance Center: 0032-0889/94/105/1355/ 10.

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