Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum PSI-J is required for efficient electron transfer and stable accumulation of photosystem I Andreas Hansson 1 , Katrin Amann 2 , Agnieszka Zygadlo 1 ,Jo ¨ rg Meurer 2 , Henrik V. Scheller 1 and Poul E. Jensen 1 1 Plant Biochemistry Laboratory, Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark 2 Department Biologie I, Botanik, Ludwig-Maximilians-Universita ¨ t-Mu ¨ nchen, Germany The photosystem I (PSI) complex of higher plants con- sists of at least 19 different polypeptides [1–3]. PSI mediates light-driven electron transfer from reduced plastocyanin (Pc) in the thylakoid lumen to oxidized ferredoxin in the stroma. The PSI core in higher plants contains at least 15 different subunits named PSI-A to PSI-L, PSI-N to PSI-P. Two subunits present in cyanobacteria, PSI-M and PSI-X, are missing from plants. In addition to the PSI core, higher plants con- tain a peripheral antenna associated with PSI, also known as light-harvesting complex I (LHCI), which is mainly composed of four different Lhca proteins. The major subunits of PSI, PSI-A and PSI-B, form a heterodimer, which binds the components of the elec- tron-transfer chain: the primary electron donor P700 and the electron acceptors A 0 ,A 1 and F x [1,4,5]. The two remaining electron acceptors, F A and F B , are bound to the PSI-C subunit. PSI-C is located towards the stro- mal side of PSI and, together with PSI-D and PSI-E, provides the docking side for soluble ferredoxin [5,6]. Keywords antenna size; electron transport; photosynthesis; plastocyanin kinetics; thylakoid membrane Correspondence P. E. Jensen, Plant Biochemistry Laboratory, Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark Fax: +45 35 28 33 33 Tel: +45 35 28 33 40 E-mail: peje@life.ku.dk (Received 30 August 2006, revised 21 December 2006, accepted 31 January 2007) doi:10.1111/j.1742-4658.2007.05722.x The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Ho- moplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild- type because of a 13% reduction in chlorophyll content per leaf area caused by an % 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP + photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI- J. Instead, a large fraction of PSI was found to be inactive. Immunoblot- ting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plast- ocyanin-binding domain of PSI. PSI-J is furthermore important for stabil- ity or assembly of the PSI complex. Abbreviations Chl, chlorophyll; Cyt, cytochrome; LHC, light-harvesting complex; Pc, plastocyanin; PS, photosystem. 1734 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS In plants, the three low-molecular-mass subunits, PSI-F, PSI-G and PSI-N, have been implicated in the interaction between PSI and Pc [7–9]. PSI-F contains one transmembrane helix and is exposed to both the lumen and the stroma: its rather large N-terminal domain is situated in the lumen [10], whereas the C-terminus is in contact with PSI-E on the stromal side [6]. The N-terminal part of PSI-F and luminal interhelical loops of PSI-A and PSI-B form a docking site for Pc or cytochrome (Cyt) c 6 [11–15]. In plants, which only use Pc as an electron donor to PSI, a longer N-terminal domain contributes to a helix– loop–helix motif [10], which specifically enables more efficient Pc binding and, as a result, two orders of magnitude faster electron transfer from Pc to P700 [16]. PSI-N is unique to eukaryotic PSI and is entirely located in the thylakoid lumen. However, the recently published structural model of higher-plant PSI based on a crystal structure at 4.4 A ˚ does not reveal the pres- ence of PSI-N [10], and cross-linking experiments have shown little interaction between PSI-N and other small PSI subunits [17]. PSI-J is a hydrophobic low-molecular-mass subunit composed of 44 amino acids with one transmembrane helix that is located close to PSI-F [5,10]. The N-termi- nus of PSI-J is located in the stroma, and the C-termi- nus is located in the lumen [6]. In cyanobacteria, PSI-J binds three chlorophylls (Chls) and is in hydrophobic contact with carotenoids [5], whereas in plants only two Chl molecules are bound (Fig. 1), which has been proposed to be important for energy transfer between LHCI and the PSI core [10]. In cyanobacteria, PSI-J interacts with PSI-F [18]. A psaJ knock-out in Synechocystis PCC 6803 contained only 20% PSI-F subunit compared with wild-type [19]. The corresponding psaJ knock-out in Chlamydomonas contained wild-type levels of PSI-F and PSI, and the cells were able to grow photoautotrophically. A large fraction of the mutant PSI complexes displayed slow kinetics of electron donation from Pc or Cyt c 6 to P700. The absence of PSI-J did not alter the half-lives of the different kinetic phases, but led to the formation of two subpopulations of PSI complexes which differed with respect to electron transfer to P700 + . One popu- lation behaved like wild-type with fully functional PSI-F, and the other population had characteristics similar to a PSI-F-deficient strain [20]. It was conclu- ded that, in 70% of the PSI complexes lacking PSI-J, the N-terminal domain of PSI-F is unable to provide an efficient binding site for either Pc or Cyt c 6 and was explained by a displacement of this domain. Thus, PSI-J does not appear to participate directly in binding of Pc or Cyt c 6 , but plays a role in maintaining a precise recognition site for the N-terminal domain of PSI-F required for fast electron transfer from Pc and Cyt c 6 to PSI [20]. To determine the role of PSI-J in plants, we gener- ated homoplastomic psaJ knock-outs in tobacco. Transplastomic transformants were obtained and ana- lyzed for differences in electron transport and antenna function. In contrast with results obtained with PSI-J- deficient Chlamydomonas, the content of PSI was reduced by 20% and the remaining PSI had a decreased in vitro NADP + -photoreduction activity. A secondary loss of the luminal subunit, PSI-N, was seen when PSI complexes were analysed and kinetic analysis revealed a large fraction of inactive PSI. Thus, we pro- pose a dual function of PSI-J in higher plants; one for assembly of the PSI core complex and the other for integrity and stabilization of a luminal domain invol- ving at least PSI-N and the N-terminal part of PSI-F which is required for efficient electron transfer. Fig. 1. Alignment of PSI-J sequences representing cyanobacteria, algae and higher plants. In total, 44 full-length PSI-J sequences were aligned using CLUSTAL W. In the alignment shown are the sequences from plants [Arabidopsis thaliana (ARATH) and Nicotiana tabacum (TOBAC)], algae [Chlamydomonas reinhardtii (CHLRE) and Porphyra purpurea (PORPU)] and cyanobacteria [Synechcoccus elongatus (SYNEL) and Prochlorococcus marinus (PROMA)]. Amino-acid residues involved in Chl binding [W (Trp), E (Glu) and H (His)] are indicated with green arrows. Note that the histidine residue is only conserved in cyanobacteria, in agreement with the notion that PSI-J of cyanobacteria is involved in binding three Chls, whereas plant PSI-J only binds two. Amino-acid residues making contact with b-carotene [I (Ile) and R (Arg)] are indicated with orange arrows. The underlined residues are completely conserved in plants, algae and cyanobacteria. A. Hansson et al. Knock-out of the J subunit of PSI FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1735 Results Targeted inactivation of the tobacco chloroplast psaJ gene To determine the function of PSI-J in plants, we have taken a reverse genetics approach and constructed a knock-out allele for targeted disruption of the tobacco psaJ (Fig. 2A). The knock-out allele was introduced into the tobacco plastid genome by particle bombard- ment-mediated chloroplast transformation [21]. From 10 bombarded leaf samples, 19 chloroplast transformants were selected and verified by PCR and DNA gel blot analysis (data not shown). Two independent transplastomic lines were subjected to additional rounds of regeneration on spectinomycin- containing medium to obtain homoplastomic tissue. In Fig. 2B, an example of PCR verification of one of the homoplastomic psaJ knock-out lines is shown. Nor- thern blot analysis was also performed to demonstrate that the psaJ gene was disrupted by the insertion of the aadA cassette (Fig. 2C). Finally, PSI particles (PSI holocomplexes) were prepared from wild-type and plants disrupted in the psaJ gene and subjected to immunoblot analysis. An antibody originally raised against electroeluted PSI-I [22] and subsequently found to recognize both PSI-I and PSI-J [17] was used to confirm the absence of PSI-J protein from the mutant (Fig. 2D). Altogether this clearly shows that the psaJ gene has been disrupted causing elimination of the PSI-J protein. Plants devoid of PSI-J are fully viable and fertile but display a clear phenotype When plants lacking PSI-J were transferred to soil, they grew photoautotrophically and were fully fertile (Fig. 3). The original transformed lines were self-polli- nated, and the seeds produced were germinated directly on soil. The resulting offspring displayed the same characteristics as the first generation (results not shown). Tobacco plants lacking PSI-J were slightly smaller than wild-type plants (Fig. 3). This was observed for plants grown in either a growth-chamber or a green- house and suggests that elimination of the PSI-J pro- tein from PSI affects the overall photosynthetic performance. Besides being slightly smaller than wild-type, the psaJ knock-out plants were visibly paler. Pigment WT ΔJ T ΔJ M 4 7 16 17 34 45 55 105 kDa M123 564 947 831 1375 1584 2027/1904 3530 A B C D 3.7 kb 1.9 kb WT WT 68293 70823 PetG TrnW TrnP PsaJ Rpl33 Rps18 250-bp ScaI TrnP (PsaJ) Rpl33 (ScaI/SmaI) (PsaJ) (HindIII/ScaI) AadA ΔJΔJ Fig. 2. (A) Construction of the plastid trans- formation vector. Schematic map of the 2.53-kb chloroplast genomic fragment con- taining the psaJ gene. The aadA cassette is inserted in a ScaI site within the coding sequence of psaJ in the sense orientation. (B) PCR confirmation that the aadA cassette has inserted in the psaJ gene. M, marker; 1, total DNA from transgenic plant as tem- plate; 2, plasmid DNA used to transform the plants as template; 3, total DNA from wild- type tobacco as template. (C) Northern blot showing that there is no wild-type-sized psaJ mRNA (as a loading control the left hand side shows the stained and the right hand side the actual Northern blot). (D) Immunoblot analysis of PSI complexes from wild-type and DpsaJ plants. The panel on the left is the stained gel, and the panel on the right is an immunoblot using an antibody directed against a mixture of PSI-I and PSI-J. The arrow indicates PSI-J. Knock-out of the J subunit of PSI A. Hansson et al. 1736 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS extraction of leaf discs using boiling ethanol and spec- trophotometric quantification showed a 13% reduction in the content of Chl per leaf area compared with wild-type (Table 1). Estimated from the leaf extracts, the Chl a ⁄ b ratio was 2.95 in the psaJ knock-out leaves compared with 3.25 in the wild-type leaves. This differ- ence was caused by a bigger decrease in Chl a (15% less) and a smaller decrease in Chl b (6% less) in the mutant (Table 1). Similar measurements on several independent preparations of thylakoids also revealed a lower Chl a ⁄ b ratio in the mutant, although the abso- lute numbers were different. The reduced Chl a ⁄ b ratio suggests that plants without PSI-J either have less of the core complexes or increased content of the Chl b containing peripheral antenna. To monitor the photosynthetic electron flow through PSI during steady-state photosynthesis in vivo, we esti- mated the redox state of P700 in the light by measuring oxidation of P700 in the leaf as DA at 810 minus 860 nm as described in Experimental procedures. The light dependence of the P700 oxidation ratio (DA ⁄ DA max ) was examined, and, in both the wild-type and DPSI-J plants, P700 oxidation was almost linearly related to increasing light intensity. However, in the DPSI-J plants the redox state of P700 was higher than wild-type at all light intensities (Fig. 4). This means that P700 stays more oxidized in the absence of PSI-J. This usually sug- gests that electron donation from Pc to P700 + is affec- ted. Comparison of the curves suggested that about 20% of the PSI has very inefficient electron donation from Pc in the absence of PSI-J. Table 1. Chl a and b content per leaf area, Chls per PSI reaction centre, PSI activity, and the plastoquinone redox state under different light conditions. Wild-type n DPSI-J n Chl (lg ⁄ cm 2 ) Leaf 19.1 ± 2.1 6 16.6 ± 1.0* 6 Chl a ⁄ b Leaf 3.25 ± 0.3 6 2.95 ± 0.1* 6 Chl a (lg) Leaf 14.6 ± 1.8 6 12.4 ± 0.7* 6 Chl b (lg) Leaf 4.5 ± 0.3 6 4.2 ± 0.3 6 Chl ⁄ P700 Thylakoids 435 ± 17 3 531 ± 32* 3 NADP + photoreduction a Thylakoids 24.8 ± 2.0 3 11.1 ± 1.0*** 3 [lmol NADP + Æs )1 Æ(lmol P700) )1 ] 1–q P Growth chamber ⁄ growth light 0.024 ± 0.003 3 0.04 ± 0.01* 5 1–q P Greenhouse ⁄ cloudy and rainy 0.013 2 0.019 2 1–q P Greenhouse ⁄ sunny, no clouds 0.028 2 0.065 2 a Mean of three independent thylakoid preparations. *P<0.05; ***P<0.001. WT ΔPsaJ Fig. 3. Phenotype of homoplastomic DpsaJ plants grown under growth chamber conditions. Note that the DpsaJ plant is slightly smaller and paler than the wild-type plant. Li g ht intensity (μE) 0 100 200 300 400 oitar noitadixo 007P 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 WT ΔJ Fig. 4. P700 oxidation state in leaves of wild-type and DpsaJ plants. Light response of P700 oxidation ratio (DA ⁄ DA max ) in leaves of wild-type (WT) and DPSI-J plants (DJ). All data points are mean ± SD (n ¼ 3), but in some cases the error bars are covered by the marker. A. Hansson et al. Knock-out of the J subunit of PSI FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1737 The PSII excitation pressure (estimated as 1–q P ) was subsequently measured in vivo in the growth chamber under the light conditions to which the plants were adapted. Under these conditions 1–q P was increased 1.7-fold in the plants lacking PSI-J (Table 1), indica- ting that the PSII excitation pressure was significantly increased as the result of a more reduced plastoqui- none pool. Measuring 1–q P under greenhouse condi- tions on either a cloudy or a sunny day confirmed the higher excitation pressure in plants without PSI-J, especially under conditions where the plants have to cope with higher light intensities (Table 1). This is in agreement with a restriction of electron flow at PSI. The amount of PSI is reduced in the absence of PSI-J To analyze the content of PSI further, the amount of P700 was determined in solubilized thylakoids using flash-induced absorption changes in P700 at 834 nm. The number of Chls per P700 reaction centre was esti- mated to be 435 ± 17 for wild-type and 531 ± 32 for thylakoids from the PSI-J-less plants (Table 1). Similar values were obtained using chemical oxidation and reduction of P700 (data not shown). This clearly indi- cates an % 20% reduction in P700 in plants lacking PSI-J. To investigate this by an independent method and also to analyze whether the absence of PSI-J caused changes in photosynthetic complexes, we performed immunoblot analysis of thylakoid proteins using a variety of antibodies directed against subunits of the PSI, PSII and ATP synthase complexes (Fig. 5). The gels were loaded with proteins corresponding to equal amounts of Chl. This analysis showed that subunits of PSII and the ATP synthase were present in amounts equal or close to the amounts found in wild-type (Fig. 5). In contrast, the amounts of the analysed sub- units of the PSI core were consistently reduced by 15– 25% compared with the wild-type (Fig. 5A). This shows that there are fewer PSI core complexes in the absence of PSI-J and confirms the spectroscopic deter- mination of Chl per P700 above. Together this sug- gests that PSI-J is implicated in stable accumulation of PSI because of a requirement for this subunit either during assembly or subsequently for the stability of the PSI complex. To analyse the effect of the absent PSI-J in more detail, immunoblot analysis of PSI particles purified using sucrose density gradient centrifugation was also performed (Fig. 6). This revealed that most of the sub- units analysed were present in the complex of the mutant in amounts similar to that found in the wild- type. This included the PSI-F subunit, which is known to be located next to PSI-J in the complex [5,10]. Sur- prisingly, the only subunit that was reduced in content was PSI-N, which was reduced to 30–40% of the wild- type level. Fig. 5. Immunoblot analysis of proteins in thylakoids prepared from DpsaJ and wild-type plants. (A) Content of a range of PSI core pro- teins and ATP synthase (CF 1 -b). Thylakoids were prepared from leaves from two to four different wild-type or DpsaJ plants. A dilu- tion series containing protein corresponding to 1.0, 0.5, and 0.25 lg Chl of the wild-type and 1.0–0.5 lg Chl of the mutant was separated by SDS ⁄ PAGE, blotted and analyzed with the antibodies indicated. Wild-type (WT) and DpsaJ dilutions were run side by side, and, for each antibody, the resulting signal was quantified using the LabWorks software as described in Experimental proce- dures. Quantification was performed on two independent prepara- tions of both wild-type and DpsaJ thylakoids. (B) Content of light- harvesting Chl a ⁄ b proteins of PSI. Thylakoid proteins were separ- ated as above and the blots were incubated with antibodies as indi- cated. The Lhca2 antibody also detects Lhcb4 (CP29). (C) Content of light-harvesting Chl a ⁄ b proteins of PSII and PSII core proteins. Thylakoid proteins were separated as above, and the blots were incubated with antibodies as indicated. Knock-out of the J subunit of PSI A. Hansson et al. 1738 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS PSI-J is not involved in binding LHCI The four Lhca proteins, which constitute the major part of the peripheral antenna of PSI (LHCI), were not reduced to the same extent as the core subunits. Lhca1 and Lhca4 were present in near wild-type amounts, and Lhca2 and Lhca3 were reduced by 15–25% compared with wild-type (Fig. 5B). This indi- cates that some of the Lhca proteins are present in rel- ative excess of the PSI core complexes. The antenna properties were further analysed by fluorescence emission measurements at low tempera- ture. Fluorescence emission spectra between 650 and 800 nm during excitation at 435 nm at 77 K using intact leaves of wild-type plants and plants devoid of PSI-J are shown in Fig. 7. The spectra revealed that, in the absence of PSI-J, there is a 2–3 nm blue shift in the far-red emission originating from PSI–LHCI. The blue shift suggests a perturbation of the peripheral antenna, which is because either PSI-J plays a func- tional role in the binding ⁄ function of the LHCI antenna or free Lhca complexes are present in the membrane. However, low-temperature fluorescence emission measurements on PSI–LHCI particles enriched using sucrose density gradient centrifugation as shown in Fig. 8 did not display the 2–3 nm blueshift (data not shown), indicating that the blue shift is caused by excess free Lhca complexes in the thylakoid membrane. This was further supported by estimation of the functional antenna size of PSI using light-induced P700 absorption changes at 810 nm after very gentle solubilization of the thylakoid membrane using digito- nin as described in Experimental procedures. We have WT ΔJ B PSI-D PSI-E PSI-F PSI-K PSI-H PSI-L PSI-J PSI-C PSI-N % of WT)(ecnadnu baevitaleR 0 20 40 60 80 100 Δ J A Fig. 6. Immunoblot analysis of proteins in PSI particles prepared from DpsaJ and wild- type plants. (A) Quantification of the signals obtained in the immunoblot analysis. (B) Representative example of the signals with the various PSI antibodies. Emission wavelen g th (nm) 660 680 700 720 740 760 780 ecnecseroulf evi taleR 0.0 0.5 1.0 1.5 2.0 WT ΔJ Fig. 7. Low-temperature fluorescence emission. Shown are the spectra of intact leaves from a wild-type plant (WT) and a DpsaJ plant (DJ). Leaves from several individual plants of both genotypes were measured, and the mutant consistently showed a 3-nm blue shift in the far-red florescence emission peak. Excitation wave- length was 435 nm, and the spectra were normalized to the peak at 685 nm. A. Hansson et al. Knock-out of the J subunit of PSI FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1739 previously used this method to successfully detect changes in PSI antenna caused by association with LHCII during state transitions [23] or genetic elimin- ation of individual Lhca proteins in Arabidopsis [24]. The functional PSI antenna size was expressed by the t 1 ⁄ 2 value which is defined as the time it takes to oxidize 50% of the P700 in the sample and was esti- mated at three different intensities of actinic light. At all three light intensities, there was no significant dif- ference in t 1 ⁄ 2 in the samples lacking PSI-J compared with the values obtained with wild-type samples (Table 2), suggesting that the PSI antenna size is unaffected by the elimination of PSI-J and further- more ruling out the possibility that PSI-J is strictly required for binding of any of the Lhca antenna proteins. The presence of free Lhca1 and Lhca4 in the thylakoid membrane was verified by gentle solubiliza- tion of the various thylakoid membrane complexes using dodecyl-b-d-maltoside and subsequent separation of the complexes using sucrose density gradient centrif- ugation. After separation, the gradients were harvested in 0.5-mL fractions, and the individual fractions were analysed by gel electrophoresis and immunoblotting using antibodies against the four Lhca proteins and the PSI-F subunit (Fig. 8). This revealed that signifi- cant amounts of free Lhca1 and Lhca4 proteins indeed were found in the fractions where mainly LHCII trim- ers and ⁄ or Lhcb monomers are normally found. How- ever, this analysis also suggested that PSI–LHCI complexes devoid of PSI-J are slightly more sensitive to the detergent treatment, as some free Lhca2 and Lhca3 proteins were also detected. Table 2. Measurements of antenna size using time course of P700 photo-oxidation in solubilized thylakoid preparations from wild-type and DPSI-J plants. lE, l moles photonÆm )2 Æs )1 . lE t 1 ⁄ 2 (ms) Wild-type n DPSI-J n 20 104.5 ± 3.4 3 102.6 ± 12.8 4 33 66.3 ± 4.6 3 61.7 ± 7.7 4 58 38.0 ± 1.2 3 37.5 ± 3.9 4 12345678910111213141516171819202122232425 wt ΔJ PSI-LHCI PSII-core LHCII trimers and monomers wt Lhca1 wt Lhca2 wt Lhca3 wt Lhca4 ΔJ Lhca2 ΔJ Lhca3 ΔJ Lhca4 ΔJ Lhca1 wt PsaF ΔJ PsaF Fig. 8. Analysis of the distribution of Lhca proteins in the thylakoid membrane of DpsaJ (DJ) plants. Shown is the centrifuga- tion tubes after separation of the solubilized membrane complexes in a sucrose density gradient (top panels) and an immunoblot analysis using the four Lhca antibodies and a PSI-F antibody on individual fractions har- vested from the sucrose density gradient fraction (bottom part). Knock-out of the J subunit of PSI A. Hansson et al. 1740 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS PSI-J is important for proper electron transfer On the basis of work with mutants of Chlamydomonas lacking PSI-J, it has been proposed that the function of PSI-J is to maintain PSI-F in the correct orienta- tion, facilitating fast electron transfer from Pc or Cyt c 6 to P700 [20]. A similar role for PSI-J in higher plants is likely, and, in order to analyse this, NADP + photoreduction was determined using thylakoids puri- fied from plants without PSI-J and wild-type plants. In our standard assay with 2 lm Pc, an activity of 24.8 ± 2.0 lmol NADPHÆs )1 Æ(lmol P700) )1 was obtained with thylakoids from wild-type and 11.1 ± 1.0 lmol NADPHÆ s )1 Æ(lmol P700) )1 with thyl- akoids devoid of PSI-J (Table 1). Thus, PSI devoid of PSI-J only has 45% of the NADP + photoreduction activity of the wild-type. This result clearly suggests that PSI-J affects electron transport. As indicated from work with green algae [20] and the in vivo measurement of the P700 redox level in Fig. 4, the most obvious step to be affected is the elec- tron transfer from Pc to P700. To investigate the kinetics of the Pc–P700 interaction, flash-induced P700 absorp- tion transients were determined by following the absorp- tion at 834 nm in the presence of Pc. Flash excitation of PSI results in a very rapid absorption increase at 834 nm caused by photo-oxidation of P700 to P700 + , followed by a slower absorption decrease due to reduction of P700 + by Pc. The reaction between Pc and P700 is a multistep reaction, which can be divided into three major steps: binding of Pc to P700, electron transfer within a complex between Pc and P700, and release of oxidized Pc from the complex between Pc and P700. The absorp- tion decrease at 834 nm can be modelled as the sum of three exponential decays discerned as a fast phase corres- ponding to the electron transfer between preformed Pc– PSI complexes, an intermediate phase corresponding to the bimolecular reaction between Pc in solution and PSI, and a slow phase corresponding to inactive PSI and a contribution from absorption of oxidized Pc at 834 nm [25–27]. For analysis of wild-type and mutants lacking PSI-J, Pc concentrations of 5 an 25 lm were used, and the first 20 ls of the data were ignored. With 5 and 25 lm Pc, the fast reduction of P700 + by Pc bound to PSI before photo-oxidation is negligible. Therefore, good fits to the experimental data could be obtained using a sum of two exponential decays. The results show that there is no difference between wild-type and mutant in the apparent second-order rate constants (Table 3), sug- gesting that PSI-J does not affect the electron transfer from Pc to PSI directly. However, the amplitude of the intermediate phase is 80% in wild-type and only 63% in the PSI-J-less samples, indicating that the absence of PSI-J results in % 20% more inactive PSI compared with wild-type. Thus, the observed decrease in NADP + pho- toreduction can, at least in part, be explained by a larger fraction of inactive PSI in the absence of PSI-J. Discussion PSI-J is a subunit of PSI in almost all photosynthetic organisms studied so far. However, the unicellular cyanobacterium, Gleobacter violaceus PCC 7421, appears to have a PSI without PSI-J [28,29]. The func- tion of PSI-J in higher plants has so far not been investigated. We have successfully generated transgenic Nicotiana tabaccum plants devoid of the J subunit of PSI and been able to investigate the role of PSI-J in higher plants. The PSI-J-less plants were analysed with various biochemical and physiological methods. PSI-J is required for stable accumulation of PSI In the absence of PSI-J, the steady-state accumulation of PSI is reduced by % 20%, as evidenced by the esti- mates of Chl ⁄ P700, the immunoblotting analysis of thylakoid proteins (Fig. 5), and the lower Chl a ⁄ b ratio (Table 1). This suggests that PSI-J is implicated in sta- bility or assembly of the PSI complex in tobacco. This is in contrast with results reported for Chlamydomonas lacking PSI-J, where it was concluded that steady-state accumulation of PSI does not require the PSI-J sub- unit [20]. Differences between higher plants and green algae with respect to PSI stability and function have also been reported after removal of PSI-F, which in Arabidopsis resulted in severe destabilization of PSI and especially loss of stromal subunits such as PSI-C, PSI-D and PSI-E [8]. In contrast, a deletion of PSI-F Table 3. Apparent second-order rate constant (k) for the reduction of P700 + by plastocyanin. The rate constants were obtained from a curve-fitting analysis of flash-induced absorption transients recorded at 834 nm in samples of dodecyl-b- D-maltoside-solubilized thylakoids. Wild-type n DPSI-J n k ( M )1 Æs )1 ) 1.75 · 10 8 ± 2.17 · 10 7 10 1.97 · 10 8 ± 4.76 · 10 7 8 Percentage of amplitudes relative to the total amplitude 80 ± 5 10 63 ± 9 8 A. Hansson et al. Knock-out of the J subunit of PSI FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1741 in Chlamydomonas did not affect the stability of the PSI complex [11,20]. Transgenic Arabidopsis plants without PSI-N, PSI-H, PSI-K and PSI-L compensate for a poorly functioning PSI by making 15–20% more PSI [7,30–32]. Apparently, the plants devoid of PSI-J cannot compensate in a sim- ilar way, which again suggests that PSI-J affects the sta- bility or assembly in a different way from the absence of PSI-N, PSI-H, PSI-K and PSI-L. In some aspects, plants devoid of PSI-J display certain similarities to plants devoid of PSI-G [9,33,34]. In the absence of PSI-G, less PSI core, a relative excess of LHCI, and a less stable PSI is also observed. To distinguish whether it is the stability or the assembly of the PSI complex that is affected needs further investigation. The reduced content of PSI was readily revealed by the appearance of the transgenic tobacco plants, which were slightly smaller and paler than wild-type. Plants devoid of PSI-G or PSI-K have been reported to be reduced in mean size [34], and plants devoid of PSI-G have a 40% reduction in content of PSI [33] and also a slightly lighter pigmentation [34]. Thus, there is good correlation between the amount of PSI, plant size, and pigmentation, although one would not expect a 20% reduction in PSI to affect the growth to the extent seen for the tobacco plants without PSI-J. However, com- bined with a less efficient PSI, as both the in vitro NADP + measurements and the in vivo estimations of the PSII excitation pressure indicate, the observed growth phenotype is explainable. PSI-J is not necessary for binding of the peripheral light-harvesting antenna The two Chls bound to PSI-J in higher plants are sug- gested to be important for the energy transfer between LHCI and the PSI core [10]. However, the functional PSI antenna size is unaffected by the elimination of PSI-J from the PSI complex (Table 2). Thus, PSI-J is not required for binding or the function of the peripheral antenna, or at least the PSI that is formed is unaffected by the missing PSI-J. The measurements of the functional antenna size using P700 oxidation rates do not allow enough time resolution to tell whe- ther the absence of the two Chl molecules bound to PSI-J causes inefficient transfer of excitation energy from the peripheral antenna to the core. In vitro the absence of PSI-J affects the stability of the PSI–LHCI complex. The results of the fractionation of mildly solubilized thylakoid membrane complexes as pre- sented in Fig. 8 indicate that some Lhca proteins, mainly Lhca1 and Lhca4 are present in relative excess compared with the core subunits, as also indicated in the immuno- blot analysis on nonsolubilized thylakoids (Fig. 5) and the 77 K fluorescence emission measurements on detached leaves (Fig. 7). Alternatively, the solubilization with detergent affects the PSI-J-deficient complexes more than the wild-type complexes, and thereby more of the Lhca proteins are released from the complex. PSI-J is required for efficient electron transfer PSI-J affects the electron transport through PSI. Meas- ured as in vitro NADP + photoreduction activity, a 55% decrease in the steady-state electron transport in the absence of PSI-J was observed. The kinetic analysis of the reaction between Pc and P700 did not reveal any significant difference in the second-order rate con- stant between wild-type and PSI-J-deficient plants that can explain the observed decrease in PSI activity. The kinetic parameters of the reaction between Pc and P700 was also found to be unaffected when PSI from DPSI-J and wild-type Chlamydomonas was analysed [20], and it therefore seems that PSI-J does not partici- pate directly in the binding of Pc in either plants or green algae. In Chlamydomonas, the amplitude of the PSI-F-dependent second-order kinetics was 76% and 42% of the total amplitude with wild-type and PSI-J- deficient thylakoid membranes, respectively [20], which correspond to a 45% decrease. This decrease is thought to be caused by an increased proportion of PSI complexes incompetent for fast electron transfer in the absence of PSI-J and has been suggested to be due to a stabilizing effect of PSI-J on PSI-F [20]. Similar to this, we observe a 20% decrease in the amplitude of the second-order component of electron transfer with plant thylakoids devoid of PSI-J. Thus, in plants, there is also an increased proportion of PSI complexes that are incompetent for efficient electron transfer. Interest- ingly, the immunoblotting analysis of PSI particles purified using sucrose density gradient centrifugation after solubilization with dodecyl-b-d-maltoside clearly suggested that binding of the luminal PSI-N to PSI was affected in the absence of PSI-J (Fig. 6). This loss of PSI-N is probably due to increased sensitivity to detergent during preparation of the PSI particles, but, despite this, it strongly suggests a perturbation of the luminal side of PSI involving PSI-F and PSI-N. The absence of PSI-J might affect the conformation of PSI-F, which, in turn, changes the binding of PSI-N. PSI-F provides part of the Pc-binding site in plants [16], and it is known that the depletion of PSI-F by antisense suppression of the corresponding gene leads to a secondary loss of PSI-N [8], indicating an interac- tion between these two subunits. PSI-N has further been shown to be necessary for the efficient interaction Knock-out of the J subunit of PSI A. Hansson et al. 1742 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS with Pc, as the second-order rate constant was reduced by 40% in the absence of PSI-N [7]. The increase in the pool of inactive PSI observed in plants devoid of PSI-J is not caused by the absence of PSI-N because mutants lacking PSI-N clearly have a changed second-order rate constant for Pc–P700 inter- action but not an increased proportion of inactive PSI complexes [7]. Furthermore, the immunoblotting ana- lysis of thylakoid proteins (Fig. 5) clearly indicates that PSI-N is present in amounts similar to the other PSI core subunits. Instead it seems plausible that the chan- ged conformation of PSI-F in the absence of PSI-J renders a fraction of the PSI complexes inactive. The in vivo measurements of the P700 redox level indicate that P700 in the DPSI-J plants constantly stays more oxidized, which is usually caused by a limi- tation of electron-transfer activities on the donor or lu- minal side of PSI. The 20% permanently oxidized PSI estimated from the in vivo experiment is in excellent agreement with the 20% inactive PSI determined with the flash excitation. At the same time, the plastoqui- none pool is more reduced, as indicated by the increased PSII excitation pressure. These observations are consistent with a greater pool of inactive PSI cen- tres in the absence of PSI-J in vivo. However, the 20% increase in the pool of inactive PSI complexes in the absence of PSI-J does not explain the dramatic reduction in PSI activity measured by NADP + photoreduction activity. The kinetic analysis clearly indicates that the second-order rate constant for electron transfer from Pc to P700 is unaffected. However, the release of oxidized Pc has been shown to limit electron transfer between the cytochrome b 6 f complex and PSI in vivo [35], and the absence of PSI-J may affect the k off value, so that oxidized Pc stays lon- ger in the active site and thereby blocks efficient exchange with reduced Pc. Alternatively, the changed conformation of PSI-F in the absence of PSI-J could affect proper functioning of stromal subunits in con- tact with PSI-F, such as PSI-E or PSI-D. These sub- units are involved in docking and efficient electron transfer to ferredoxin [6], and, from the structures, it is known that PSI-E is in contact with the C-terminus of the PSI-F subunit [5]. Changes in binding or amounts of any of the stromal subunits of PSI were not detec- ted in our immunoblot analysis; however, a subtle change in arrangement of the subunits is still possible. In conclusion, PSI-J is needed for stable accumulation of the PSI core complex and proper electron transfer. Despite the location of PSI-J close to the rim of the core complex facing LHCI, it is not needed for correct inter- action with the peripheral antenna complexes. Clearly the luminal side of PSI is perturbed, probably because of destabilization of PSI-F in the absence of PSI-J, resulting in an increased pool of inactive PSI. Experimental procedures Vector construction, chloroplast transformation, and plant material The region of the tobacco chloroplast genome containing 700 bp upstream and downstream of the psaJ reading frame was amplified using PCR. The 1535-bp fragment was ligated into the SacI and BamHI sites of pUC19. The psaJ knock-out allele was created by digestion of this construct with ScaI, and a chimeric aadA gene conferring resistance to aminoglycoside antibiotics [21] was inserted into this ScaI site to disrupt psaJ and to facilitate selection of chloroplast transformants. ScaI causes disruption of the 132-bp psaJ coding region after nucleotide 38. A plasmid clone carrying the aadA gene in the same orientation as psaJ yielded the transformation vector pPsaJ (Fig. 2). Chloroplasts of N. tabaccum cv. Petit Havanna were transformed by particle bombardment of leaves [21]. Selec- tion and culture of transformed material as well as assess- ment of plastome segregation and the homoplastomic state were performed essentially as described by De Santis- Maciossek et al. [36] and Swiatek et al. [37]. Essentially, 10 leaves were used for particle bombardment, and 19 antibi- otic resistant transformants were selected. The material was maintained on agar-solidified MS medium [38] containing 2% sucrose, and grown in 12 h dark ⁄ light cycles at 25 °C and 20 l mol photonsÆm )2 Æs )1 and, under selective condi- tions, 500 lgÆmL )1 spectinomycin. For thylakoid isolation and physiological measurements, wild-type and transformed plants (originating from two independent transplastomic lines) were planted in compost and kept in growth chamber conditions in 8 h light and 120–140 lmol photonsÆm )2 Æs )1 . Isolation of thylakoid membranes and PSI particles from tobacco Leaves from 6–8-week-old plants were used for isolation of thylakoids as described previously [7]. PSI particles were iso- lated from thylakoids after solubilization with dodecyl-b-d- maltoside and sucrose density gradient ultracentrifugation as described in [31]. Chl content and the Chl a ⁄ b ratio were determined in 80% acetone as described previously [39]. The samples were frozen in liquid nitrogen and stored at )80 °C. RNA gel blot analysis Northern blot analysis of total leaf RNA was performed using DNA probes and was carried out as described by Meurer et al. [40]. A 33 P-labelled DNA fragment corres- ponding to the psaJ gene was used as probe. A. Hansson et al. Knock-out of the J subunit of PSI FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1743 [...]... antibodies directed against subunits of the various thylakoid membrane complexes as indicated in the Figure legends An antibody originally raised against electroeluted PSI -I [22] but subsequently found to recognize both PSI -I and PSI-J [17] was used to detect PSI-J Primary antibodies were detected using a chemiluminescent detection system (Immun-Star, Bio-Rad, Herlev, Denmark; Super-Signal, Pierce, Rockford,... plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF Biochemistry 36, 6343–6349 Hippler M, Drepper F, Haehnel W & Rochaix J-D (1998) The N-terminal domain of PsaF: precise recognition site for binding and fast electron transfer from cytochrome c (6) and plastocyanin to photosystem I of Chlamydomonas reinhardtii Proc Natl Acad Sci USA 95, 7339–7344 Hippler M, Rimbault B & Takahashi Y (2002)... Mimuro M (2004) Unique constitution of photosystem I with a novel subunit in the cyanobacterium Gloeobacter violaceus PCC 7421 FEBS Lett 578, 275–279 30 Naver H, Haldrup A & Scheller HV (1999) Cosuppression of photosystem I subunit PSI-H in Arabidopsis thaliana J Biol Chem 274, 10784–10789 31 Jensen PE, Gilpin M, Knoetzel J & Scheller HV (2000) The PSI-K subunit of photosystem I is involved in the interaction... resolved into two exponential decay components using a Levenberg–Marquardt nonlinear regression procedure Knock-out of the J subunit of PSI 9 10 11 12 Acknowledgements We wish to thank Ingrid Duschanek and Elli Gerick for excellent technical assistance, and Steen Malmmose for assistance with growing plants The Danish National Research Foundation, the Danish Veterinary and Agricultural Research Council (23-03-0105)... (2000) Downregulation of the PSI-F subunit of Photosystem I in 14 16 17 18 19 20 21 Arabidopsis thaliana The PSI-F subunit is essential for photoautotrophic growth and antenna function J Biol Chem 275, 31211–31218 Zygadlo A, Jensen PE, Leister D & Scheller HV (2005) Photosystem I lacking the PSI-G subunit has higher affinity for plastocyanin and is less stable Biochim Biophys Acta 1708, 154–163 Ben-Shem... absence of the PSI-G subunit J Biol Chem 277, 2798–2803 1746 34 Varotto C, Pesaresi P, Jahns P, Lessnick A, Tizzano M, Schiavon F, Salamini F & Leister D (2002) Single and double knockouts of the genes for photosystem I subunits G, K, and H of Arabidopsis Effects on photosystem I composition, photosynthetic electron flow, and state transitions Plant Physiol 129, 616–624 35 Finazzi G, Sommer F & Hippler... at 700 nm The thylakoids were solubilized with 0.2% Triton X-100, and the measurements were repeated 3–5 times on several independent thylakoid preparations For spectroscopic determination of the amount of P700, the maximal flash-induced P700 absorption was determined by supplying a series of saturating flashes as outlined below (under Kinetic measurements) and using an e at 834 nm for P700 of 5 mm)1... (2004) Light-harvesting complex II binds to several small subunits of photosystem I J Biol Chem 279, 3180–3187 24 Klimmek F, Ganeteg U, Ihalainen JA, van Roon H, Jensen PE, Dekker JP, Scheller HV & Jansson S (2005) The structure of higher plant LHCI In vivo characterization and structural interdependence of the Lhca proteins Biochemistry 44, 3065–3073 25 Bottin H & Mathis P (1985) Interaction of plastocyanin... Chlorophylls and caroteinoids: pigments of photosynthetic biomembranes Methods Enzymol 148, 350–382 40 Meurer J, Meierhoff K & Westhoff P (1996) Isolation of high-chlorophyll-fluorescence mutants of Arabidopsis thaliana and their characterisation by spectroscopy, immunoblotting and Northern hybridization Planta 198, 385–396 41 Klughammer C & Schreiber U (1994) An improved method,using saturating light pulses, for. . .Knock-out of the J subunit of PSI A Hansson et al Chl content per leaf area Total leaf Chls were extracted by boiling leaf disks in 95% ethanol for 30 min After cooling to room temperature and volume adjustment, the Chl content and Chl a ⁄ b ratio was determined in 95% ethanol as described [39] Immunoblotting Immunoblotting analysis was performed essentially as described previously [31] using antibodies . explained by a larger fraction of inactive PSI in the absence of PSI-J. Discussion PSI-J is a subunit of PSI in almost all photosynthetic organisms studied. Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum PSI-J is required for efficient electron transfer and stable accumulation