Control of cab gene expression in synchronized Chlamydomonas reinhardtii cells

J. Photo&em.

Photobid.

139

B: Biol., II (1991) 139-150

Control of cab gene expression in synchronized Chlamydomonas reinhardtii cells Fred Jasper, Beate

Quednau,

Mouika

Korteujauu

and Udo Johauuingmeiert Ruhr-Vniversitiit W-4630 Bochum

Bochum, (F’.R.G.)

Lehrstuhl _fTir Biochemie

akr Pjlanam,

Vniversitiitstr.

150,

(Received February 26, 1991; accepted April 30, 1991)

Keywords. Light-regulation, nuclear gene expression, transcriptional control, chlorophyll intermediates.

Abstract In light-dark synchronized Chlamydomonas reinhurdtii cultures transcripts of at least two members of the cab gene family coding for chlorophyll a/b binding proteins are highly abundant in the light, but almost undetectable in the dark. “Run-on” transcription assays in isolated nuclei were used to show that the rapid increase in cab mRNA levels during the light phase is primarily due to regulation at the transcriptional level. Functionally unrelated inhibitors such as dipyridyl and cycloheximide as well as anaerobic conditions block chlorophyll synthesis, presumably by interfering with the conversion of magnesium protoporphyrin monomethyl ester to protochlorophyllide. Under .these conditions, cab mRNA does not accumulate and nuclei isolated from inhibitortreated cells do not support cob gene transcription. Inhibitors such as dioxoheptanoic acid and diphenyl ether herbicides block earlier steps within the chlorophyll synthesis pathway without substantial effects on cab mRNA accumulation and transcription. A possible control of transcription by intermediates of the chlorophyll biosynthesis pathway is discussed.

1. Introduction

Chloroplast proteins are encoded by both the nuclear and the plastid genome. Gene expression in both compartments has to be coordinated in order to produce a functional chloroplast. Such coordination is especially important during chloroplast development and requires the exchange of regulatory signals between the organelle and the nucleus. One such signal appears to be released by the chloroplast and affects the accumulation of specific nuclear transcripts [ 11. The nature of this signal (or signals) is unknown and is subject to intensive investigations. ‘Author to whom correspondence should be addressed.

loll-1344/91/$3.50

0 1991 - Elsevier Sequoia, Lausanne

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Chlamydomonas cultures synchronized by alternating light-dark cycles provide a model system to study the interaction between the chloroplast and nuclear compartment. During the light phase transcripts encoding chloroplast proteins increase to maximal levels. It has been shown that relatively high levels of plastid-encoded transcripts such as the psbA, psbD or rbcL mRNAs persist throughout the dark period [ 2-4 1, whereas certain nuclear transcripts coding for chloroplast proteins are almost undetectable in the dark, but highly abundant in the light [5-S]. An example for a highly variable nuclear transcript is the cab mRNA, which codes for the light-harvesting chlorophylla/b-binding protein (LHCP). One member of the cab gene family in Chlamydomonas, designated cabII-I, has been characterized [ 91. Its transcription product exhibits a wave-like accumulation pattern in the light phase [ 5, 61. Unlike in higher plants, cab mRNA accumulation in Chlamydomonus requires continuous or intermittent illumination conditions, and blue rather than red light appears to be effective [6, 81. In addition, there is indirect evidence that chlorophyll precursors are involved in the regulation of light-responsive genes [6, 71. Experiments carried out so far in Chlamydomanas have focused on the determination of steady state RNA levels, which can be the result of a transcriptional and/or post-transcriptional regulation. In order to characterize factors that are involved in cab mRNA regulation it is necessary to determine whether they affect RNA stability or RNA transcription rates. One way to differentiate between these two control levels is the in vitro transcription of already initiated RNAs in isolated nuclei. We have used this approach to ask (i) whether the rapid increase in cab mRNA levels during the light phase is due to increased transcription rates and (ii) whether the failure to accumulate cab transcripts in the presence of certain inhibitors blocking chlorophyll accumulation is due to a transcriptional or post-transcriptional control. In addition, the effect of diphenyl ether herbicides on chlorophyll synthesis and cab mRNA levels has been investigated. 2. Materials

and methods

2.1. Algal strains and culture conditions Chlumydomonas reinhardtii cell wall-less strain CW 15 + (Sammhmg von Algenkulturen, Giittingen) was synchronized according to the method of Surzycki [ 10 1. Cells were grown photoautotrophically at 2 1 “C under alternating 12 h light-l 2 h dark cycles in TMP medium. Inhibitors were added to the cultures at the beginning of the third or fourth light period (LO) [6]. At this time cell densities varied in the range (l-2) X lo6 cells ml-‘. Anaerobic conditions were obtained by bubbling N2 through the culture. Chlorophyll was measured as described earlier [7]. Cycloheximide, 2,2’dipyridyl and 4,6dioxoheptanoic acid (Succinylaceton) were purchased from methyl 5-[ 2-chIoro-4-(trifluoromeSigma. Acinuorphen methyl (AFM; thyl)phenoxy]-2-nitrobenzoate) and chlorophthalim (Cim; N-(4-chlorophenyl)3,4,5,6-tetrahydrophthalimide) were gifts from Dr. R. ScaIla and Dr. P. Boger.

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2.2. RNA extraction For determination of steady-state transcript levels, isolation of RNA was carried out as described earlier [ 71. 10 pg of total RNA were separated on 1,5% formaldehyde-agarose gels as described by Maniatis et al. [ 111, transferred onto nitrocellulose filters (BA 85, Schleicher & Schiill) and hybridized to 32P-labelledDNA probes. Hybridization was carried out overnight in 5 X SSC (1 X SSC: 0.15 M NaCl, 0.015 M sodium citrate), 5 X Denhardt’s solution [ 171, 100 pg denatured herring sperm DNA ml-’ and 50% formamide. Northern blots were washed at 55 “C in 0.1 X SSC, 0.1% sodium dodecyl sulphate (SDS). 2.3. Nuclei isolation Isolation of nuclei essentially followed the procedure described by Keller et al. [ 121,but includes an additional Percoll gradient purification step [ 181. About (l-2) x 1O9nuclei obtained after lysis with Nonidet P-40 were pelleted, washed with solution I (25 mM HEPES-NaOH, pH 7.5, 20 mM KCL, 20 mM MgC12,0.6 M sucrose, 10% glycerol, 5 mM dithiothreitol), resuspended in 1 ml Percoll buffer (0.5 M sorbitol, 5 mM MgCla, 20 mM Tris-HCl, pH 7.5) and layered onto two 4 ml layers of 20% and 80% (by volume) BF Percoll in Percoll buffer. BF Percoll contained 1% (by weight) and 1% (by weight) Ficoll in Percoll. The gradients were centrifuged at 5000g for 30 min. The grey band in the 80% layer was removed and diluted with Percoll buffer and the nuclei were sedimented at 650g. The pellet was resuspended in 2.5% Ficoll, 0.5 M sorbitol, 0.008% spermidine, 5mM MgC12, 10 mM Tris-HCl, pH 7.5, 50% glycerol and 1 mM dithiothreitol and stored frozen at - 80 “C. Isolated nuclei were inspected visually by fluorescence microscopy using mithramycin [ 131. Some aggregation was observed, but size and shape of organelles appeared intact when compared with whole cells. Starch and residual pigments were removed after the Percoll gradient. 2.4. In vitro RNA synth,esk For in vitro RNA synthesis [ 121, about 2.5 X lo8 frozen nuclei were pelleted and resuspended in 0.2 ml transcription buffer containing 1.25% Ficoll, 0.25% sorbitol, 0.004% spermidine, 0.6 mM dithiothreitol, 12 mM MgCIB, 45 m&I Tris-HCl, pH 7.9, 30% glycerol, 150 mM NH&l, 0.16 mM each ATP, GTP and CTP, 100 u RNase inhibitor (from human placenta, Boehringer), and 100 &i [cy-“PJUTP (800 Ci mm01 -I, Amersham). Incubation time was 60 mm at 30 “C. Incorporation proceeded almost linearly during this time and was inhibited by approximately 50% in the presence of either 2 or 20 pg ml-’ of (Y-amanitin (data not shown). Transcription was terminated by the addition of SDS to 1%. RNA was extracted three times with equal volumes of phenol:chloroform:isoamylalcohol(25:24:1) and once with chloroform. RNA was precipitated three times with ethanol in the presence of ammonium acetate and finally resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. In order to characterize the newly formed RNA it was separated on formaldehyde-agarose gels and exposed to X-ray 8h-n.A con-

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tinuous size distribution of labelled RNAs from 100 to 3000 nucleotides was observed (data not shown). 2.5. Plasrnid !INA The following recombinant plasmids were used: pHS 16 contains a 300 bp insert in pBR 322 specific for the cab&l gene (formerly designated’ cabl [6]) encoding a chlorophyll-a/b-binding protein [5]. Plasmid pPI62 contains the complete cab&1 gene and unknown flanking sequences [9]. A 2.4 kb HindIII-EcoRI fragment with exons 2-4 and 3’-flanking sequences of the cab&l gene [ 91 was subcloned into Bluescribe M 13 (Vector Cloning Systems, San Diego, CA) resulting in plasmid pBQ 2.4. Plasmid Ba 2.35 carries nuclear ribosomal DNA in pBR 313 [ 141 and was obtained from J.-D. Rochaix (University of Geneva). Plasmid DNA was purified in CsCl gradients according to the method of Garger et al. [ 151. DNA was cut with restriction enzymes according to the supplier’s instructions. Fragments were separated in 1.2% agarose gels, blotted onto nitrocellulose filters (BA 85, Schleicher & Schiill) and hybridized to 32P-labelled RNA. For dot blot analysis, plasmid DNA was denatured and immobilized on nitrocellulose using a multiple filtration device as described by Mason and Williams [ 16 1. To ensure that filter-bound DNA is in excess, serial dilutions of plasmid DNAs were prepared and applied to filters. 2.6. Hybridization conditions and autoradiography Southern and dot blots were pre-hybridized overnight and hybridized for 48 h at 42 “C with the same number of counts per minute ((2-8) X 10”) in 3 ml hybridization buffer containing 50% deionized formamide, 5 X SSC, 50 mM sodium phosphate, pH 6.5 5 X Denhardt’s solution [17], 100 pg tRNA ml-‘, 50 pg (poly(A) ml-‘, 0.2% SDS. Filters were washed for 15 min at room temperature in 2 x SSC, 0.1% SDS, for 15 min at room temperature in 0.1 X SSC, 0.1% SDS, and for 50 min in 0.1 X SSC, 0.1% SDS at 60 “C. Air-dried filters were wrapped in plastic foil and exposed to pre-flashed X-ray fllm at - 80 “C using Du Pont Lightning Plus intensifying screens. Relative optical density of dots on autoradiograms was quantitated using a laser densitometer (Ultroscan XL and Gelscan XL software, PharmaciaLKB).

3. Results 3.1. cab mJ&YA levels in light- and dark-grown cells DNA sequences specific for the cub&1 member of the LHCP gene family are represented by clone pHS 16, which detects an mRNA species of 1.2 kb on Northern blots [5] and single DNA fragments on Southern blots with total DNA cleaved with different restriction enzymes [9]. Plasmid pBQ 2.4 carries, together with 3’-flanking sequences, exons 2, 3 and 4 of cabI&I. A DNA fragment contained within pBQ2.4 has been shown to detect several

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DNA bands on genomic Southern blots [9], indicating the existence of a small cab gene family. Using pBQ 2.4 as a probe for Northern blot hybridizations reveals two distinct bands (Fig. l), that with the lower mobility being also detected by pHS16. Since the sequences of other cab genes are as yet unknown, both transcripts represent the products of at least two cab genes, or more, if other cub genes produce mRNAs of similar lengths. As can be seen from Pig. 1, both transcripts detected by pBQ 2.4 are highly abundant in L8 RNA preparations, but not at the beginning of the light phase (LO). Equal intensities of ribosomal RNA signals indicate that the same amounts of intact LO and L8 RNA were loaded into each slot. 3.2. In vitro trunsxiption of cub genes in nuclei of light- and durkgrown. cells We have prepared nuclei according to the method described by Keller et al. [ 121 and further purified them in a Percoll gradient [ 181. RNA was synthesized in nuclei isolated from cells just before entering the light phase (LO) and after 8 h in light (L8). In vitro transcripts were hybridized to blots with plasmid DNA from clones pHS 16 (Fig. 2, lane l), pP1 62 (Iane 2), pBR 322 (Iane 3) and to h DNA (lane 4). The cubII-1 gene represented by pHS 16 DNA with a 300 bp insert derived from the 3’-untranslated region is preferentially transcribed in the light. Dot blot analysis indicate very low cubII-I transcription rates at LO (data not shown). When plasmid pP1 62 is cut with EcoRI and HindIII, six fragments are produced (Fig.2, lane 2). The 2.4 kb fragment (the fourth band from the top, cloned in pBQ2.4) contains most of the cubII-I sequences and hybridizes Dark (LO)

Light (L8)

LO L8

123412341234 Fig. 1. Steady-state cab mRNA and nuclear rRNA levels in synchronized cells just before entering the light phase (LO) and after 8 h in light (LB). Total cellular RNA (10 cLg> was separated on formaldehyde-agarose gels and blotted onto nitrocellulose. Blots were hybridized to labelled pBQ2.4 and Ba2.35, detecting two cab mRNA species and rRNA respectively. Fig. with gels RNA

2. Hybridization of in vitro labelled RNA to different plasmids. Plasmids were cleaved restriction endonuclesses, and the resulting DNA fragments were separated on agarose and stained with ethidium bromide (middle panel). Southern blots were hybridized to transcribed from nuclei isolated from synchronized cells just before leaving the dark phase (Lo) and after 8 h in the light (L8). Lane 1, pHS16, linearized with BamHI; lane 2, pP162, cut with Hind111and EcoRI; lane 3, pBR322, linearized with HindIII; lane 4, A DNA, cut with Hind111(23.1, 9.4, 6.6, 4.4, 2.3 and 2.0 kb size markers, indicated from top to bottom by horizontal lines). Arrows point to DNA fragments containing cab gene sequences.

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strongly to labelled RNA from L8 nuclei. Two other fragments (bands 2 and 6) with unknown sequences give strong signals with RNA from LO as well as L8 nuclei and can serve as an internal control for the presence of active transcription in LO nuclei. RNA sequences hybridizing to pBR 322 or A DNA are not detected.

3.3. cab mRIVA levels in the presence of inhibitors Dipyridyl (DP), cycloheximide (CHI) and anaerobic conditions are assumed to induce porphyrin accumulation [7] by inhibiting a late step in the chlorophyll synthesis pathway. DP and CHI completely inhibit accumulation

hs in light 1234567 con

AFM

Cim

cab

2

‘4

0” h g 1 5

12 10 6

g ! 012345676

rRNA @I

hs in light

Fig. 3. Steady state levels of cub mRNAs in cells treated with inhibitors. Total RNA was isolated from synchronized cells kept in light (L) or dark (D) for 7 h after the beghming of the normal light phase, or kept in the light for 7 h under anaerobic conditions (AN), in the presence of 10 pg CHI ml-‘, 1 n-M DP or 0.05 mM DA. Inhibitors were added at the beginning of the normal light phase. cub mRNAs were detected with plasmid pBQ2.4 (cab). The lower panel (rRNA) shows an ethidium bromide stained RNA gel demonstrating intactness and equivalent RNA msss in each lane. Fig. 4. (a) Effect of diphenyl ether herbicides AFM, (10 nM) and Cim, (10 @I) on the accumulation of cab mRNAs as compared with a control (con) culture without inhibitors. Total RNA wss isolated every hour and Northern blots (10 pg RNA lane-‘) were hybridized to pBQ2.4 (b). Inhibition of chlorophyll (chl) accumulation in the absence (a> and presence of AFM (0) and Cim (i).

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of both cub mRNA species. Anaerobiosis also drastically reduces cub mRNA levels, but a low level comparable with that in dark-incubated cells can still be detected (Fig. 3). On the contrary, dioxoheptanoic acid (DA) blocks porphyrin synthesis [29] and has no substantial effect on cab transcript levels. Low concentrations of cab transcripts are present in the prolonged dark phase. A wave-like increase in cub mRNA abundance has been observed when light-dark-entrained cells are kept in the dark during the normal light phase, its amplitude reaching between 5% and 10% of the light value (unpublished observations). A variety of diphenyl ether herbicides have been shown to inhibit chlorophyll synthesis by interfering with the protoporphyrinogen oxidase converting protoporphorinogen to protoporphyrin IX [ 30,311. fro inhibitors of this type have been used to block chlorophyll accumulation in Chlamydomonas cells. Acifluorphen methyl (AFM) and chlorophthalixn (Cim) were added to synchronized cultures at the beginning of the light phase. As compared with the control culture, chlorophyll accumulation was effectively blocked for up to 7 h in the presence of the inhibitors (Fig. 4 (b)). From these cultures total cellular RNA was extracted every hour, separated on denaturing agarose gels, and blotted onto nitrocellulose lilters, and cub mRNA was detected with pBQ2.4. As can be seen from Fig. 4(a), the diphenyl ether herbicides do not significantly interfere with the accumulation of both cab mRNA species. For a certain period of time Chlum~domonas cells appear to be resistant to light-induced peroxidation triggered by protoporphyrin IX accumulation. However, more than 8 h of light in the presence of diphenyl ether herbicides leads to a sudden and drastic reduction in chlorophyll content, indicating that the antioxidative system is no longer able to protect the cells from photo-oxidative damage. 3.4. In vitro transcription of cob genes in nuclei of inhibitor-treated cells Figure 5 illustrates the effect of inhibitors on the transcriptional activity of nuclei isolated from control and inhibitor-treated cells. pBQ 2.4 DNA was spotted onto nitrocellulose filters and hybridized to labelled RNA extracted from nuclei of L7 cells, of cells kept in the dark during the normal light phase (D), or of cells incubated with DA, nitrogen for anaerobic conditions (AN), CHI or DP in the light for 7 h. Light and, to a lesser extent, DA support active transcription of cab genes. Prolonged darkness, anaerobiosis, DP and CHI do not support detectable synthesis of cab mRNA sequences. These data reflect the abundance of cab mRNAs (Fig. 3) under these different conditions. For a control, synthesis of ribosomal RNA was monitored and found to be severely affected by prolonged darkness (Fig. 5(B)). Under anaerobic conditions and in the presence of DP rRNA synthesis is reduced by 30%-40% as compared with the light control. CHI, on the contrary, slightly increases rRNA synthesis.

146

Rig. 5. Effect of different inhibitor treatments on the transcription rates of (A) cab and (B) rRNA genes. Inhibitors were added to cell cultures as described in Fig. 3. Nuclei were isolated and labelled transcripts hybridized to (A) pBQ2.4 and (B) Ba2.35 DNA. Twofold serial dilutions of each sample were spotted onto filters; the top dot in each series contains 4 pg DNA. Relative optical density of dots on autoradiograms was determined as described in Section 2. Concentration of newly synthesized transcripts in nuclei from light (L) grown cells was set to 100%.

4. Discussion It was estimated that the cab gene family in Chlamydomcmasreinhardtii consists of 3-7 members [9]. The recombinant clone pHSl6 identifies the cab&l gene, whereas clone pBQ2.4, containing most of the cab&l gene sequence, also detects an additional cab mRNA with a slightly different mobility (Fig. 1) under stringent hybridization conditions. Accumulation of this transcript is also light dependent. As a prerequisite for the identification of factors regulating transcript accumulation it is important to determine the level of control. In Chlamydomonas, it has not been unambigously demonstrated yet whether the observed increase in cbcbmRNA levels in the light phase is the result of a transcriptional or post-transcriptional control. In experiments with toluenepermeabilized Chlamydomonas cells incorporating [cx-~~P]UTP into RNA it has been shown that the extent of in viva transcription of the cab&l gene is relatively high in the dark and low in the light [19]. Dallman et al. note that this unexpected result could be due to the treatment of cells with toluene, which might affect transcription of different genes in different ways. For our experiments we have chosen to perform “rim-on” transcription assays in isolated nuclei. This method has been successfully utilized to demonstrate differential transcription of CX-and p-tubulin genes before and after cell deflagellation in Chlamydcnnonasreinhurdtii [ 12 J. As is shown here, nuclei are more active in producing cab mRNA when isolated from cells in the light phase than from cells harvested at the end of the dark period (Fig. 2). Although this result does not eliminate the possibility that additional mechanisms affect mRNA stability or processing, a primary control point has been disclosed at the level of transcription.

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In higher plants, cab mRNA accumulation is not only the result of lightinduced enhancement of transcription but is also governed by tissue-specific factors, state of plastid differentiation, circadian rhythms, phytohormones and chloroplast signals [ 1, 20-261. There have been early indications that intermediates of the chlorophyll biosynthesis pathway affect transcription of cab genes in Chlamyd@monus [27, 281. More recent data obtained with inhibitors of chlorophyll synthesis and mutants of Chlamyd&nonus suggested that the accumulation of certain porphyrin compounds may act as negative regulators of cab&l mRNA accumulation [ 6, 7’1. In order to test further aspects of this working hypothesis, the effect of various inhibitors on the accumulation and transcription rates of both cab mRNA species detected by pBQ2.4 has been determined. Accumulation is inhibited by DP, anaerobiosis and CHI, but not by DA (Pig. 3). Nuclei isolated from cells growing anaerobically or in the presence of DP or CHI do not support transcription of cab genes (Pig. 5). It has to be noted, however, that anaerobiosis and DP, but not CHI, also reduce transcription of nuclear rRNA genes to some extent (Pig. 5). These inhibitor treatments have been assumed to induce porphyrin accumulation [7] by interfering with the magnesium protoporphyrin monomethyl ester (Mg-PME) oxidative cyclase complex, which catalyses the formation of the chlorophyll isocyclic ring. On the contrary, DA as an inhibitor of 5aminolevulinate dehydratase [29 1 also blocks chlorophyll synthesis in Chlamydomonus. A slight reduction in cub transcript levels (Pig. 3) is accompanied by an approximately 40% decrease in transcription rate (Pig. 5). This inhibitor, like haemin and levulinic acid, blocks an early step in the pigment biosynthesis pathway and thus does not lead to accumulation of porphyrin compounds. The effect of two diphenyl ether herbicides on chlorophyll synthesis and cub mRNA accumulation in synchronized Chlamydomonas cells has been investigated (Pig. 4). They are known to inhibit the protoporphyrinogen oxidase and promote the accumulation of protoporphyrin IX in algae and higher plants [30-321. AFM and Cim as representatives of these herbicides inhibit chlorophyll synthesis in Chlamydunwnus without inflicting major photo-oxidative damage within the time frame of this experiment. As in the case of DA, accumulation of cab mRNAs is not substantially affected, indicating that this porphyrin compound is not an efficient suppressor of cub gene expression. These results seem to conflict with data obtained with the Chiizmydomonas mutant br,-1 [ 331, which accumulates protoporphyrin IX but no cab mRNA in the light [6]. However, the mode of protoporphyrin IX accumulation appears to be different in the mutant and in inhibitor-treated cells. One would expect the accumulation of protoporphyrinogen in diphenylether-treated cells. Instead, protoporphyrin IX accumulates. This is explained by a non-enzymatic oxidation of protoporphyrinogen, which diffuses out of its site of synthesis [30, 311 within the chloroplast. The mutant appears to be blocked at a step between protoporphyrin IX and magnesium protoporphyrin IX [ 331, which is the branching point of the iron and magnesium porphyrin pathways.

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According to the hypothesis that porphyrin compounds regulate cab mRNA accumulation, it must be concluded from these experiments that chlorophyll intermediates act at the level of transcription by some unknown mechanism. However, a close link between chlorophyll synthesis and cab mRNA accumulation has not been proven yet. Preliminary experiments aimed at the identification of porphyrin compounds induced by various inhibitors confirm that DP induces Mg-PME and Cim protoporphyrin IX accumulation. As for the identification of porphyrin compounds accumulating under anaerobic conditions and in the presence of CHI, no conclusive results have been obtained yet. The inhibitory effect of CHI on cab mRNA accumulation could also be interpreted in a different way. We have observed that in synchronized Chlamg&_nnonascultures CHI also blocks the accumulation of the lightdependent r&S1 mRNA [Sl. It has recently been shown that CHI inhibits cab and rbcS gene transcription in wheat, pea and transgenic tobacco [34]. A labile protein factor has been suggested to play a role in light activation of these genes. It remains to be established whether this also applies to the cab and rbcSI genes in Chlumgdomcmas.

Acknowledgments This work was supported by Deutsche Forschungsgemeinschaft. We thank Dr. P. Biiger and Dr. R. Scalla for providing us with diphenyl ether herbicides and Dr. J.-D. Rochaix for plasmid Ba2. 35.

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