Plastocyanin Is Indispensable for Photosynthetic Electron Flow in Arabidopsis thaliana *

Plastocyanin is a soluble copper-containing protein present in the thylakoid lumen, which transfers electrons to photosystem I. In the chloroplast of the flower-ing plant Arabidopsis thaliana , a cytochrome c 6 -like protein is present, which was recently suggested to function as an alternative electron carrier to plastocyanin. We show that Arabidopsis plants mutated in both of the two plastocyanin-coding genes and with a functional cytochrome c 6 cannot grow photoautotrophically be- cause of a complete block in light-driven electron transport. Even increased dosage of the gene encoding the cytochrome c 6 -like protein cannot complement the dou- ble mutant phenotype. This demonstrates that in Arabidopsis only plastocyanin can donate electrons to photosystem I in vivo .

Plants use light energy to drive electron and proton transport across the thylakoid membrane, resulting in the synthesis of NADPH and ATP and involving photosystem I (PSI) 1 and II (PSII), cytochrome b 6 /f, and the plastid ATPase. Plastocyanin transfers electrons from cytochrome b 6 /f to PSI (1), and in cyanobacteria and some algae, plastocyanin can be replaced by the heme protein cytochrome c 6 (2,3). The familiar idea that in higher plants only plastocyanin operates as a mobile electron donor to PSI has recently been challenged. Arabidopsis thaliana lines in which both of the plastocyanin-coding genes, PetE1 and PetE2, had been silenced by RNA interference (RNAi) were viable, whereas plastocyanin-RNAi lines that in addition lacked a cytochrome c 6 -like protein did not survive (4). From these data, it was concluded that in higher plants neither plastocyanin nor cytochrome c 6 is essential for plant growth and development but that both proteins can transport electrons from cytochrome b 6 /f to PSI.
Plastocyanin-null mutants have been identified and characterized in several cyanobacterial and algal species (2,(5)(6)(7). In these organisms, cyanobacterial cytochrome c 6 can replace plastocyanin in plastocyanin-null genotypes (5) as well as in wild-type cultures grown under conditions that result in copper deficiency (8). Accordingly, Chlamydomonas cells mutated for plastocyanin only perform photosynthesis when cytochrome c 6 expression is induced by making the cells deficient for copper (3). In this work, we show that Arabidopsis plants mutated in both of the two plastocyanin-coding genes and with a functional cytochrome c 6 cannot grow photoautotrophically because of a complete blockade in light-driven electron transport. This demonstrates that in Arabidopsis only plastocyanin can donate electrons to photosystem I in vivo.

Plant Propagation and Mutant Isolation-The En-mutagenized
A. thaliana (ecotype Columbia 0) population and growth of plants under short and long day conditions have been described previously (9,10). pete1-1.1 pete2-1.1 plants were propagated on solid MS medium (11) containing 2% sucrose. Transposon insertions within PetE1 and PetE2 were identified by screening the En-tagged population using genespecific primers in combination with En-specific primers followed by hybridization with a gene-specific probe as described previously (12). Positive lines were confirmed by sequencing PCR-amplified insertion sites. The stable frameshift mutants pete1-1.1 and pete2-1.1 were identified according to Varotto et al. (13), and the double mutant pete1-1.1 pete2-1.1 was generated by crossing the single mutants.
Complementation of the pete1 pete2 Double Mutant-The PetE2 and Atc6 cDNAs were introduced by recombination cloning (Gateway, Invitrogen) into the plant expression vector pJAN33, placing them under the transcriptional control of the Cauliflower Mosaic Virus 35 S promoter. In addition, a genomic fragment containing PetE2 and 1000 and 500 bp of DNA 5Ј and 3Ј to the gene, respectively, were introduced by recombination cloning into the vector pP001VS-GW. Flowers of pete1-1.1/pete1-1.1 PetE2/pete2-1.1 plants were transformed according to Clough and Bent (14). Plants were transferred to the greenhouse, and seeds were collected after 3 weeks. At least 200 independent transgenic plants were selected on the basis of their resistance to kanamycin (pJAN33) or to the herbicide Basta (pP001VS-GW). The presence of the respective transgenes in double homozygous mutant plants was confirmed by PCR using primers that specifically allowed the amplification of the transgenes and of the endogenous PetE2 and pete2-1.1 alleles. Successful complementation of the double mutation was confirmed by measurements of growth and of chlorophyll fluorescence.
Immunoblot and Chlorophyll Fluorescence Analyses-Total proteins were isolated from 6-week-old plants as described previously (15). For denaturing PAGE analysis, 40 g of total proteins determined by using the Bio-Rad protein assay based on the Bradford dye-binding procedure (16) were loaded for each genotype. Decreasing amounts of wild-type proteins (28 and 16 g) were loaded in parallel lanes (0.7ϫ WT and 0.4ϫ WT). For immunoblot analyses, proteins were transferred to Immobilon-P membranes (Millipore, Eschborn, Germany) and incubated with antibodies specific for the large subunit of Rubisco (polyclonal chicken anti-RbcL raised against a peptide target conserved in all of the known Type I RbcL proteins (Agrisera, Vä nnä s, Sweden)), plastocyanin (polyclonal antibody raised against the mature form of the spinach plastocyanin expressed in E. coli, obtained from Ralph Bernd Klösgen * This work was supported by the European Community's Human Potential Programme (HPRN-CT-2002-00248 (PSICO)), by the Deutsche Forschungsgemeinschaft (Grant DL 1265/8), and by the Bundesministerium fü r Bildung und Forschung (Förderkennzeichen 03117519). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to the work. ** To whom correspondence should be addressed. Tel.: 49-221-5062415; Fax: 49-221-5062413; E-mail: leister@mpiz-koeln.mpg.de. 1 The abbreviations used are: PSI, photosystem I; PSII, photosystem II; RNAi, RNA interference; F V /F M , maximum quantum yield; ⌽ II , effective quantum yield; 1 Ϫ qP, the fraction of Q A (the primary electron acceptor of PSII) present in the reduced state; WT, wild type; MIPS, Munich Information for Protein Sequences. Germany) as described previously (12). Pulses (800 ms) of white light (6000 mol photons⅐m Ϫ2 ⅐s Ϫ1 ) were used to determine the maximum fluorescence (F M ) and the ratio (F M Ϫ F o )/F M ϭ F V /F M . A 20-min illumination with actinic light (FL101/E; Walz, Effeltrich, Germany) at a rate of 80 mol photons⅐m Ϫ2 ⅐s Ϫ1 was used to drive electron transport between the two photosystems before measuring the effective quantum yield of PSII (⌽ II ) and photochemical quenching (qP ϭ (F MЈ Ϫ F S )/(F MЈ Ϫ F oЈ )). Fluorescence induction kinetics were measured with a home-made fluorometer (17). Actinic light was provided by an array of green light emitting diodes (HLMP CM15, 520 nm, Hewlett-Packard). Light was concentrated on a leaf spot of ϳ20 mm 2 , and fluorescence was detected on the opposite side of the sample. Emission was recorded at discrete times with a maximum resolution time of 30 s.

RESULTS AND DISCUSSION
In A. thaliana, we have isolated transposon insertion mutants for both plastocyanin-coding genes PetE1 (MIPS accession number At1g76100) and PetE2 (MIPS accession number At1g20340). Starting from the unstable insertion mutants, stable null alleles of both genes, pete1-1.1 and pete2-1.1, were generated by selecting germinal revertants with frameshift mutations (Fig. 1A). The double mutant pete1-1.1 pete2-1.1 was obtained by crossing single mutants and genotyping F2 progenies. In pete1-1.1 pete2-1.1 plants, no plastocyanin was detectable by immunoblot analysis, whereas wild-type amounts of the cytochrome c 6 polypeptide were found (Fig. 1B).
In both pete1-1.1 and pete2-1.1 plants, growth was affected (Fig. 1C). The plastocyanin double mutants did not survive when grown on soil but could be propagated in axenic culture on medium supplemented with sucrose. However, heterotrophically grown double mutants had a substantially reduced growth rate and displayed a high chlorophyll fluorescence phenotype upon illumination with UV light (Fig. 1D). This last observation indi- cated that photosynthetic electron flow was blocked.
Parameters of chlorophyll fluorescence induction were determined to characterize the electron flow in the plastocyanin double null mutant. Although the maximum (F V /F M ) quantum yield was decreased by ϳ35% in pete1-1.1 pete2-1.1 plants compared with the wild type (F V /F M : 0.54 Ϯ 0.04 versus 0.81 Ϯ 0.01), the effective quantum yield (⌽ II ) was close to zero in the double mutant with respect to the wild type (⌽ II : 0.04 Ϯ 0.01 versus 0.74 Ϯ 0.02), again pointing to a severe perturbation in electron flow. In addition, the fraction of Q A (the primary electron acceptor of PSII) present in the reduced state was drastically increased (1 Ϫ qP: 0.94 Ϯ 0.01 versus 0.06 Ϯ 0.02), implying that electron flow through PSII is still occurring while the block exists at a later electron transfer step.
Consistent with these data is the comparison of the fluorescence induction curves measured in the pete1-1.1 pete2-1.1 mutant and wild-type plants (Fig. 2). Whereas the PSII photochemical rate measured in the pete1-1.1 pete2-1.1 line was close to that of the WT (data not shown), the area above the fluorescence induction curve (a parameter proportional to the size of the PSII acceptors; Refs. 18 and 19) was largely reduced in this mutant. This finding suggests that the size of PSII electron acceptor pool was largely decreased by the mutation. The area was nevertheless larger than the one measured in the WT in the presence of DCMU (to block the reoxidation of Q A ), confirming that the effect of the mutation was localized downstream of PSII. As shown in Fig. 2, the mutant was blocked in a high fluorescence state, whereas in the WT, the fluorescence yield started to decrease after approximately 10 s of continuous illumination. This decrease is at least partly attributable to the activation of the Benson Calvin cycle (20). Its absence in the mutant gives a further indication that electron transfer is blocked. Moreover, the mutant did not display the plateau observed in the WT in the 20 -100-ms time range during continuous illumination. This plateau has been recently ascribed to the reduction of PSI acceptors (see Ref. 17 for discussion). Its absence is consistent with a block of electron flow located downstream of the cytochrome b 6 /f. Taken together, these results strongly strengthen the interpretation of the non-photoautotrophic mutant phenotype as the consequence of a complete block in light-driven electron transport, which seems to occur as expected at the level of the PSI donor side.
To test whether increased doses of cytochrome c 6 can replace plastocyanin in the pete1 pete2 double mutant, the Arabidopsis cDNA for cytochrome c 6 was fused to the 35 S promoter of cauliflower mosaic virus and introduced into the pete1 pete2 line. In all of the 35 S::Atc6 pete1 pete2 individuals analyzed, Atc6 mRNA was detected at high levels (Fig. 1E). However, unlike the case of the plastocyanin-overexpressing 35 S::AtPetE2 construct that served as a positive control, 35 S::Atc6 could neither reverse nor reduce the effects of the double mutation, indicating that cytochrome c 6 cannot replace plastocyanin in vivo. Also, cytochrome c 6 expressed from T-DNA constructs containing the Atc6 gene under the transcriptional control of its natural promoter had no effect on the phenotype of the pete1 pete2 double mutant.
The data concerning the photosynthetic behavior of the pete1 pete2 double mutant are in conflict with the findings of Gupta et al. (4). Their oxygen evolution reconstruction assays indicate that Arabidopsis cytochrome c 6 can function along with plastocyanin in photosynthetic electron transport (4). But does Arabidopsis cytochrome c 6 actually donate electrons to PSI in vivo? Unlike the case in Chlamydomonas, cytochrome c 6 expression in A. thaliana is constitutive and is not up-regulated by plastocyanin depletion due to copper deficiency (4). Moreover, cytochrome c 6 of higher plants compared with that of prokaryotes or algae may have acquired novel functions as suggested from phylogenetic analyses (Fig. 3). The plastocyanin sequences from Arabidopsis and several other plant species branch from algal sequences, indicating a continuous evolution from the cyanobacterial endosymbiont to the plastocyanin of modern chloroplasts. On the contrary, the cytochrome c 6 -like sequences of higher plants form a distinct clade, which is relatively distantly related to both algal and cyanobacterial proteins. This indicates that higher plant cytochrome c 6 has diversified in sequence perhaps in association with the acquisition of function(s) different from transferring electrons to PSI.
The RNAi-based silencing of the genes for plastocyanin as well as the combination of plastocyanin-RNAi and Atc6 knockout was performed in the Wassilewskija ecotype of A. thaliana (4), whereas the pete1 pete2 double mutant (this study) was generated in the Columbia 0 background. However, it seems improbable that intraspecific variation in photosynthetic functions could account for the difference in response. A second possibility can explain the contrasting results. Residual plastocyanin expression can take place during RNAi because of leakiness in gene silencing (21). Thus, the plastocyanin-RNAi lines may have retained enough plastocyanin to allow a residual level of photosynthesis.
If cytochrome c 6 cannot replace plastocyanin, at least in Columbia 0, what might its function be under normal physiological conditions? The protein might play a regulatory role rather than quantitatively transferring electrons to PSI, or in light of the results of recent proteomics analyses of the thylakoid lumen (22,23), cytochrome c 6 could be involved in one of the diverse functions of the thylakoid lumen, including crosstalking with immunophilins with which it can interact in the yeast two-hybrid experiments (4).