ANTIOXIDANTS & REDOX SIGNALINGVolume 2, Number 3, 2000Mary Ann Liebert, Inc.

Original Research Communication

Redox Modulation of Chloroplast DNA Replication in Chlamydomonas reinhardtii

KEN W.K. LAU, JIANPING REN, and MADELINE WU

ABSTRACT

We constructed a plasmid probe containing DNA sequences unique for chloroplast (Cp) genome and nucleargenome of Chlamydomonas reinhardtii. Using this probe and quantitative Southern blot analyses, we determinedthe content ratio of Cp DNA/nuclear DNA in total DNA isolated from cells grown in different conditions. Algalcells grown photoheterotrophically with acetate as an added carbon source contain the highest amount of Cp DNAcompared with cells grown in other conditions tested. We investigated the effect of nitrogen limitation, 5-fluo-rodeoxyuridine treatment, cadmium exposure, photoautotrophic growth, and heterotrophic growth in darkness.The change in the Cp/nuclear DNA ratio in cells shifted from one growth condition to another depended on celldivision; Cp DNA content in undivided cells remained constant. Therefore, the reduction of Cp DNA content wasattributed by under replication rather than selective degradation of Cp DNA. Cells with low Cp DNA content of-ten contained less reduced glutathione, suggesting the possible effect of redox status. Low Cp DNA content wasdetected in cells treated with inhibitors that block electron flow of photosystems and in mutants with PS I de-fective phenotype. On the basis of these data, we propose that in C. reinhardtii, Cp DNA replication be modu-lated by redox status. Antiox. Redox Signal. 2, 529–535.

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INTRODUCTION

IN EUKARYOTIC CELLS, energy metabolism takesplace in mitochondria and chloroplasts (Cp).

Both organelles contain their own genomes inmultiple copy. The mitochondria genome hasbeen studied extensively, as have the copy-number responses to developmental signalsand functional needs; however, the factor(s) af-fecting copy number are not clear (Shadel andClayton, 1997). Cp is the best-studied memberof the plastid family, which consists of severalrelated organelles unique for plant cells. Mul-tiple copy genome was detected in many typesof plastids, with the number varying from a

few copies in a proplastid to a few hundredcopies in a Cp. The dramatic increase in Cpnumber and Cp genome copy number that ac-companies leaf development in higher plantshas been documented (Bendich 1987), but theunderlying mechanism as well as the necessityfor the change remains to be investigated.

In this study, we analyzed the Cp DNA copynumber change in the well-studied eukaryoticgreen alga Chlamydomonas reinhardtii. This uni-cellular alga is particularly suitable for thisstudy for the following reasons. The cell con-tains a solitary Cp, so that the effect of Cp num-ber on Cp DNA content can be eliminated.Treatment with 5-fluorodeoxyuridine (FdUrd)

Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, HongKong, SAR, PRC.

and mutant studies indicated that Cp DNA ispresent in excess of the minimal functionalneed (Harris, 1989). The alga uses acetate forheterotrophic growth and allows a wide rangeof growth condition manipulation.

MATERIALS AND METHODS

Algal strains and culture conditions

The selection of C. reinhardtii 950 from C. rein-hardtii cc 178 (CW-15 mt2) was reported inTang et al. (1995). Other algal strains and mu-tants were obtained from the ChlamydomonasGenetics Center, Department of Bontany, DukeUniversity (Durham, N.C.). The high salt (HS),nitrogen-free high salt (HS-N), high saltmedium containing acetate (HSA), Tris-ac-etate-phosphate (TAP), culture media wereprepared according to Harris (1989). TP wasmodified from TAP by using Tris HCl insteadof acetic acid to adjust the medium pH. For cul-tures used in Cd treatment, glycerophosphatewas used to provide phosphate source. CdCl2was added to the final concentration of 50 mMor 100 mM. The treatment time for Cd and otherinhibitors was 48 hr. Algal cultures were keptat 21°C, on a shaker in a 12-hr light/12-hr darkcycle under 60 mE/m2 sec light intensity in aculture chamber. Chemicals of reagent gradewere purchased from Sigma (St. Louis, MO).

General molecular biology procedures

Quick isolation of total DNA from cells andthe separation of Cp DNA from nuclear DNAwere performed according to Tang et al. (1995).Construction of pBHg was described in Hsiehet al. (1991). Plasmid pCS2.1 was a generous giftfrom Dr Michel Goldschmidt-Clamont (Gold-schmidt-Clamont, 1986). The 900-bp Eco RIfragment in pCS2.1 was purified (Sambrook etal., 1989) and inserted into the Eco RI site ofpBHg to generate pCN6. Plasmid DNA isola-tion, restriction enzyme digestion, and South-ern blotting analysis were carried out accord-ing to Sambrook et al. (1989). Optimal range tomeasure hybridization bands was determinedby loading different amounts of DNA in the geland calibrating the band intensity with knownstandards. The relative intensity of hybridiza-

tion bands was measured by using a Phospho-Imager scanner (Molecular Dynamics).

Determination of reduced glutathione content

The washed cells were resuspended in 10%sulfosalic acid and ground in liquid nitrogen.After removing debris by centrifugation, theclear supernatant was collected for protein andglutathione (GSH) measurements. Protein con-centration was determined by using the com-mercial protein assay kit (Bio-Rad). Measure-ment of GSH was performed according toRauser (1991) using a C18 reverse-phase col-umn and Waters 600 high-performance liquidchromatography (HPLC) system.

RESULTS

Measurement of the Cp DNA/nuclear DNA content ratio by quantitative Southern blot analysis

Plasmid pCN6 contains 224-bp Cp-genome-specific and 900-bp nuclear-genome-specificsequence. In Figure 1A, the assignment of Cand N1, N2 bands to Cp and nuclear genome,respectively, was based on its hybridizationpattern to each component purified by CsClgradient. The algal cell contains two copies ofthe rbcS gene (Goldschmidt-Clamont, 1986).The theoretical Cp DNA/nuclear DNA ratiofor cells containing one copy of Cp DNAshould be 224/900 3 2 5 0.1244. The experi-mental Cp DNA/nuclear DNA ratio was de-termined by C/N1 1 N2. Figure 1B shows thestatistical analysis of the Cp DNA/nuclearDNA ratio in cells grown in several conditions.The Cp DNA/nuclear DNA ratio showed thatC. reinhardtii 950 in photoheterotrophic growthin HSA contained 98 6 2 copies of Cp DNA percell. Treatment with 1 mM FdUrd for 8 days re-duced the Cp DNA content to 10 6 2 copies percell. The average Cp DNA copy number forcells grown in HS medium was 38 6 4 per cell.After transferring cells to nitrogen-depleted HS(HS-N) for 24 hr, the Cp DNA content was fur-ther reduced to 17 6 4 copy per cell. The effectof FdUrd treatment and nitrogen limitation toCp DNA content was reported in cytologicalstudies using DNA-specific fluorochrome, 49,6-

LAU ET AL.530

diamidino-2-phenylindole (DAPI) (Matagneand Hermesse, 1981). Our results were in gen-eral agreement with theirs.

The effect of acetate and cadmium on Cp DNA content

One unexpected finding was that cells grewphotoautotrophically in HS medium that con-

tained less Cp DNA. The result indicated thatthe increased demand for photosynthetic out-put did not increase the copy number and/orthe presence of acetate increased the Cp DNAcontent. Figure 1B also shows the effect of cad-mium (Cd) on Cp DNA content. The presenceof 100 mM Cd reduced the cellular Cp DNAcontent from 98 6 2 to 25 6 3. This treatmentcondition did not inhibit growth, as deter-mined by cell counting and chlorophyll mea-surement. To evaluate whether these effectswere unique for the particular strain and cul-ture medium, we studied the response in sev-eral strains in different culture media. Table 1shows the results obtained from C. reinhardtiicc-125 mt1. To test the effect of acetate, wegrew cells in equivalent culture media with orwithout acetate, e.g., HSA versus HS, TAP ver-sus TP. In separate experiments, we observeda similar pattern and extent of changes in C.reinhardtii cc-124 mt2 and the cell wall-con-taining strains, C. reinhardtii 89 and C. rein-hardtii 90 (data not shown). The results indi-cated that the effect of acetate or Cd wasindependent of strain, mating type, or culturemedia. In control condition of photohetero-trophic growth, the Cp DNA copy number ofall tested strains showed no significant differ-ence.

Under-replication of Cp DNA attributes to thereduction of Cp DNA content

The reduction of Cp DNA content could re-sult from selective degradation and/or underreplication of Cp DNA. To elucidate the possi-ble cause, log-phase cells were transferred fromone growth condition to another. Cell number

REDOX AFFECTS CHLOROPLAST DNA CONTENT IN CHLAMYDOMONAS 531

FIG. 1. (A) Southern blot used for quantitative analy-sis. N1, N2, and C mark the positions of two nuclear DNAand one Cp DNA hybridization bands, respectively. Po-sitions of the size markers in kb are included. The Eco RIdigest of purified Cp DNA was loaded in lane 1. Lanes2, 3, and 4 contain the Eco RI digest of the total DNA iso-lated from strains 950, 124, and 125, respectively. All cellswere grown photoheterotrophically in HSA. (B) The ef-fect of different growth conditions on the Cp DNA/nu-clear DNA ratio in C. reinhardtii 950. For each treatment,duplicate tests of at least five independently isolated DNAsamples were carried out. Data are presented as the meanand standard deviation; all treatments show significantdifference from that of cells grown in HSA (*p , 0.05).

TABLE 1. THE EFFECT OF ACETATE AND CD

ON CP DNA CONTENT OF C. REINHARDTII

Culture Cp DNA contentmedia Added ingredient (copies/cell)

HS None 42 6 5HSA None 96 6 3HSA 50 mM CdCl2 39 6 4HSA 100 mM CdCl2 26 6 3TP None 43 6 6TAP None 97 6 4TAP 50 mM CdCl2 46 6 5TAP 100 mM CdCl2 28 6 3

The algal strain used was C. reinhardtii (cc-125 mt1).

and Cp/nuclear DNA ratio was monitored pe-riodically after the transfer. In cultures trans-ferred from photoheterotrophic growth in HSAto photoautotrophic growth in HS, cell numberdid not increase significantly within 3 days,and the Cp DNA/nuclear DNA ratio remainedconstant. Within 5 days, cell number increasedabout two-fold and the Cp DNA content re-duced from 98 6 2 to 56 6 4 copy per cell(Table 2). Similar types of results were obtainedfrom culture transferred from photohetero-trophic growth in HSA to heterotrophic growthin HSA in darkness and from photohetero-trophic growth in HSA without Cd to HSA con-taining Cd (data not shown). The results sug-gested that the under-replication of Cp DNAwas the major cause for the reduction of CpDNA content. The constant Cp/nuclear DNAratio in undivided cells indicated that Cp DNAwas not selectively degraded under the condi-tions we investigated. When we reversed thedirection of transfer, the increase of cellular CpDNA content preceded the cell division (datanot shown). The results again supported thecontribution of Cp DNA replication to thechanges of Cp DNA content.

Influence of redox status to Cp DNA content

Using this measurement method, we also ob-served that cells survived in extreme condi-tions contained less Cp DNA. We searched forcommon factor(s) among these conditions.Upon exposure to Cd, algal cells synthesizedphytochelatins to sequester this heavy metal(Howe and Merchant, 1992). Concomitant withthe synthesis of phytochelatins that use GSH assubstrate, a decrease in the cellular GSH poolwas predicted. We actually detected the re-duction of cellular GSH content from 3.17 60.34 to 2.08 6 0.21 nmol/mg protein upontreatment with 100 mM Cd for 48 hr (Table 3).After measuring the GSH content in cells sub-jected to other treatments that affected the CpDNA content, we detected a general correlationbetween low GSH level and low Cp DNA con-tent. GSH is a major reducing cofactor for oxy-gen detoxification and contributes significantlyfor the maintenance of cellular redox homeo-stasis. In this connection, we carried out the fol-lowing experiments.

Effect of photosynthetic inhibitors on Cp DNA content

In photosynthetic cells, photosystem (PS) I isthe main source of the reducing equivalents.Paraquat blocks PS I electron flow and can pro-vide information on the possible involvementof PS I (Hess, 1980). We tested the effect ofParaquat and other photosynthesis inhibitorson Cp DNA content (Table 4). When cells weregrown photoheterotrophically in HSA, pres-ence of 0.3 mM Paraquat in the culture mediumfor 48 hr did not affect cell division but reducedthe Cp DNA content from 98 6 2 to 61 6 8copies per cell. Paraquat could form superox-

LAU ET AL.532

TABLE 2. CHANGES OF CELL DENSITY AND CP DNACONTENT IN ALGAL CULTURE TRANSFERRED FROM

PHOTOHETEROTROPHIC GROWTH IN HSA TOPHOTOAUTOTROPHIC GROWTH IN HS

Days after Cell density Cp DNA contenttransfer (106 cells/ml) (copies/cell)

0 2.96 6 0.12 98 6 21 3.11 6 0.09 96 6 33 3.69 6 0.13 89 6 55 5.63 6 0.21 56 6 47 6.68 6 0.27 47 6 5

The algal strain used for this study was C. reinhardtii950.

TABLE 3. CP DNA AND GSH CONTENT IN C. REINHARDTIIGROWN IN DIFFERENT CULTURE CONDITIONS

GSH content Cp DNA contentCulture condition (nmol/mg protein) (copies/cell)

Control 3.17 6 0.34 98 6 2Treated with 100 mM Cd 2.08 6 0.21 25 6 3Deplete of nitrogen 2.33 6 0.08 28 6 5Treated with methylamine 3.26 6 0.21 118 6 15

The strain used for this study was C. reinhardtii 950. Control was photo-heterotrophic growth in HSA. The treatment time was 48 hr.

ide that damages the lipid components. To de-termine whether Paraquat reduced Cp DNAcontent through its inhibition of photosyntheticelectron transport or its effect on the lipid component, we tested the effect of 3-(39,49-dichlorophenyl)-1, 1-dimethylurea (DCMU).DCMU blocks electron transport between theprimary acceptor of PS II and plastoquinone.Noncyclic photosynthetic electron transportgenerates ATP and NADPH by operating PSIand PSII in series; it is affected by DCMU. Thetreatment with DCMU at 1026 M did not affectcell division but reduced the Cp DNA contentto 78 6 5 copies per cell. We also tested the ef-fect of the uncoupler of photophosphorylation,methylamine, and the inhibitor of water mole-cule splitting, hydroxylamine. Both inhibitorsdid not reduce Cp DNA content over the testedconcentration range of 1028 M to 1023 M for 48hr. These results suggested that Cp DNA repli-cation is more sensitive to the redox status thanthe energy status.

Mutants with defective PS I contain less Cp DNA

To assay the in vivo effect of PS I, we mea-sured the Cp DNA content in PS I-defectivemutants with different lesions. C. reinhardtii cc-742 contains mutation in the Cp genome. Thetwo nuclear mutants tested were C. reinhardtii

cc-1062 F23 mt1 that was mapped at the AC212 locus and C. reinhardtii cc-1042 which lacksP700, Cp1, and polypeptide 2 (Harris, 1989).We detected low Cp DNA and GSH content inall mutants. The results are summarized inTable 5. Therefore, the limited supply of re-ducing equivalents within the cell is correlatedwith the low Cp DNA content.

DISCUSSION

In C. reinhardtii, the change in the DAPI stain-ing pattern of Cp nucleoids has been studiedduring the mating process (Kuroiwa et al., 1982)and other developmental stages (Coleman,1984). Due to the dynamic morphologicalchanges of Cp nucleoids (Ehara et al., 1990), andthe strong interference of nuclear DNA, quan-titative measurement of Cp DNA content byDAPI staining is difficult. Separation of CpDNA from nuclear DNA and mitochondriaDNA for the purification of each componenthas been successful, but this method is againdifficult for quantitative detection of each com-ponent. The current method is more suitablefor quantitative estimate of Cp DNA content,particularly for cells grown in a wide range ofstress conditions.

We demonstrated that in C. reinhardtii Cp

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TABLE 4. THE EFFECTS OF PHOTOSYNTHESIS INHIBITORS

ON CP DNA CONTENT IN C. REINHARDTII

Cp DNA contentInhibitor Function and target site (copies/cell)

Control None 98 6 2Paraquat Blocks the electron flow of PS I 61 6 8DCMU Electron flow between PS II and PS I 78 6 5Methylamine Uncoupler of photophosphorylation 118 6 15Hydroxylamine Inhibits water molecule splitting 116 6 13

The algal strain used for this study was C. reinhardtii 950.

TABLE 5. CP DNA AND GSH CONTENT IN DIFFERENT MUTANT STRAINS OF C. REINHARDTII

PS I Location Cp DNA content GSH contentStrains phenotype of mutation (copies/cell) (nmol/mg protein)

950 Wild type None 98 6 21 3.17 6 0.34cc-106 Defective Nuclear 35 6 81 2.28 6 0.17cc-1042 Defective Nuclear 32 6 61 2.35 6 0.23cc-742 Defective Cp 68 6 13 2.66 6 0.15

DNA replication is more sensitive than nuclearDNA replication to changing redox status. Inall experiments, we tried to collect data fromsynchronous culture by inoculating the testingculture with synchronous cells and monitoringthe synchrony by periodic cell counting. We de-tected in some treatment conditions cells thatdid not divide synchronously, particularly inheterotrophic growth. Therefore, the measure-ment is not the absolute value. Our datashowed that cells with limited supply of re-ducing equivalents support a lower level of CpDNA replication and produced progeny cellswith reduced Cp DNA content. The Cp DNAreplication system appears to be responsive tothe availability of reducing equivalents that isthe difference between production and demand.The cells can generate reducing equivalentsfrom either biological oxidation of organic car-bon source(s) and/or photosynthetic electrontransports. The demand for reducing equiva-lents involves various assimilation pathwaysand stress responses. This hypothesis is consis-tent with the following observations; the reduc-tion of Cp DNA content in Cd-treated cells andthe enhancing effect of acetate to Cp DNA con-tent for many strains under photoheterotrophicgrowth. It also explains the reduction of CpDNA content in cells subjected to nitrogen lim-itation. When carbon is available, nitrogen lim-itation leads to increased content of fatty acidsand carbohydrates (Behren et al., 1994) by trig-gering biosynthetic pathways that demand ad-ditional reducing equivalents.

The redox status affect signal transduction(Staal et al., 1994), apoptosis (Sato et al., 1995),transcription (Storz et al., 1990; Ziegelhofferand Kiley, 1995), translation (Danon and May-field, 1994), post-translational regulation (Mal-ter and Hong, 1991), and protein activity(Fontecave et al., 1989; Rainwater et al., 1995;Ross et al., 1995). A few examples are cited here.This is the first report to implicate redox mod-ulation of DNA replication. Further studies arerequired to determine whether this is a uniquefeature for Cp DNA of this alga or a commonphenomenon for the replication of other DNA.Redox regulation is usually mediated by thiol-disulfide balance to modulate protein confor-mation change and/or protein affinity withDNA or RNA. The regulatory role of iron–sul-

fur (Fe-S) proteins in redox sensing has beenreported. The redox state of the Fe-S center ofthe E. coli global transcription factor, FNR, af-fects DNA-binding activity and regulates geneexpression in response to oxygen deprivation(Khoroshilova et al., 1995). In a superoxidestress pathway, the Fe-S center of the SoxR pro-tein is involved in sensing the redox state byoxidation-reduction (Hidalgo et al., 1998). Pre-viously, we have reported the sequence-spe-cific interaction of a Fe-S protein with thecloned Cp DNA replication origin in vitro andin vivo (Hsieh et al., 1991; Wu et al., 1989, 1993).The role of this protein–DNA interaction in theredox modulation of Cp DNA replication re-quires further investigation.

ACKNOWLEDGMENTS

We thank grant support from Research GrantCouncil of Hong Kong and Biotechnology Re-search Institute, HKUST.

ABBREVIATIONS

Cd, cadmium; Cp, Chloroplast; DAPI, 49,6-diamidino-2-phenylindole; DCMU, 3-(39,49-dichlorophenyl)-1,1-dimethylurea; FdUrd, 5-fluorodeoxyuridine; Fe-S, iron-sulfur; GSH,glutathione; HAS, high salt medium-contain-ing acetate; HPLC, high-performance liquidchromatography; HS, high salt; HS-N, nitro-gen-free high salt; N2, nitrogen; PS, photosys-tem; TAP, tris acetate phosphate.

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Address reprint requests to:Dr. Madeline Wu

Department of BiologyThe Hong Kong University of Science

and TechnologyClear Water Bay

Kowloon, Hong Kong SAR, PRC

E-mail: bomwu@ust.hk

Received for publication December 23, 1999; ac-cepted March 26, 2000.

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