Cosuppression of Photosystem I Subunit PSI-H in Arabidopsis thaliana
1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês
10.1074/jbc.274.16.10784
ISSN1083-351X
AutoresHelle Naver, Anna Haldrup, Henrik Vibe Scheller,
Tópico(s)Photoreceptor and optogenetics research
ResumoPSI-H is an intrinsic membrane protein of 10 kDa that is a subunit of photosystem I (PSI). PSI-H is one of the three PSI subunits found only in eukaryotes. The function of PSI-H was characterized in Arabidopsis plants transformed with apsaH cDNA in sense orientation. Cosuppressed plants containing less than 3% PSI-H are smaller than wild type when grown on sterile media but are similar to wild type under optimal conditions. PSI complexes lacking PSI-H contain 50% PSI-L, whereas other PSI subunits accumulate in wild type amounts. PSI devoid of PSI-H has only 61% NADP+ photoreduction activity compared with wild type and is highly unstable in the presence of urea as determined from flash-induced absorbance changes at 834 nm. Our data show that PSI-H is required for stable accumulation of PSI and efficient electron transfer in the complex. The plants lacking PSI-H compensate for the less efficient PSI with a 15% increase in the P700/chlorophyll ratio, and this compensation is sufficient to prevent overreduction of the plastoquinone pool as evidenced by normal photochemical quenching of fluorescence. Nonphotochemical quenching is approximately 60% of the wild type value, suggesting that the proton gradient across the thylakoid membrane is decreased in the absence of PSI-H. PSI-H is an intrinsic membrane protein of 10 kDa that is a subunit of photosystem I (PSI). PSI-H is one of the three PSI subunits found only in eukaryotes. The function of PSI-H was characterized in Arabidopsis plants transformed with apsaH cDNA in sense orientation. Cosuppressed plants containing less than 3% PSI-H are smaller than wild type when grown on sterile media but are similar to wild type under optimal conditions. PSI complexes lacking PSI-H contain 50% PSI-L, whereas other PSI subunits accumulate in wild type amounts. PSI devoid of PSI-H has only 61% NADP+ photoreduction activity compared with wild type and is highly unstable in the presence of urea as determined from flash-induced absorbance changes at 834 nm. Our data show that PSI-H is required for stable accumulation of PSI and efficient electron transfer in the complex. The plants lacking PSI-H compensate for the less efficient PSI with a 15% increase in the P700/chlorophyll ratio, and this compensation is sufficient to prevent overreduction of the plastoquinone pool as evidenced by normal photochemical quenching of fluorescence. Nonphotochemical quenching is approximately 60% of the wild type value, suggesting that the proton gradient across the thylakoid membrane is decreased in the absence of PSI-H. Photosystem I (PSI) 1The abbreviations used are: PS, photosystem; LHC, light harvesting complex; Chl, chlorophyll.1The abbreviations used are: PS, photosystem; LHC, light harvesting complex; Chl, chlorophyll. is a pigment-protein complex that mediates the light-driven electron transport across the thylakoid membrane from the soluble electron donor, plastocyanin, to the soluble electron acceptor, ferredoxin. PSI from plants contains 13 different subunits of which three are only found in plants, namely PSI-G, PSI-H, and PSI-N. The remaining 10 subunits are shared between cyanobacteria and plants. In addition to the 13 subunits of PSI in a narrow sense, plants contain light harvesting complex I (LHCI), which is composed of four different polypeptides, Lhca1–4, that are specifically associated with PSI (1Jansson S. Biochim. Biophys. Acta. 1994; 1184: 1-19Crossref PubMed Scopus (585) Google Scholar,2Scheller H.V. Naver H. Møller B.L. Physiol. Plant. 1997; 100: 842-851Crossref Google Scholar). The PSI-A/B heterodimer coordinates the reaction center P700 (a chlorophyll (Chl) a dimer) and the electron acceptors A0 (Chl a), A1 (phylloquinone), and FX (a [4Fe-4S] iron-sulfur cluster). The terminal electron acceptors FA and FB are [4Fe-4S] clusters bound to the stromal PSI-C subunit (3Høj P.B. Svendsen I. Scheller H.V. Møller B.L. J. Biol. Chem. 1987; 262: 12676-12684Abstract Full Text PDF PubMed Google Scholar, 4Schubert W.-D. Klukas O. Krauss N. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (224) Google Scholar).The 10 common subunits are highly conserved from cyanobacteria to plants apart from the presence of extended N- termini of PSI-D, -E, -F, and - L from plants. The role of specific subunits in PSI has mostly been investigated by gene knock-out studies in cyanobacteria and algae. However, despite the sequence similarities, PSI subunits of cyanobacteria and plants show important functional differences. For example, the plant-specific N terminus of PSI-F plays a role in supporting plastocyanin-mediated donation of electrons to P700+ (5Farah J. Rappaport F. Choquet Y. Joilot P. Rochaix J.D. EMBO J. 1995; 14: 4976-4984Crossref PubMed Scopus (108) Google Scholar, 6Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar). In cyanobacterial PSI, this electron transfer mostly follows a simple second order reaction, whereas a stable plastocyanin-PSI complex is formed in plants before electron transfer (6Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar). PSI-L is essential for formation of PSI trimers in cyanobacteria (7Chitnis V.P. Chitnis P.R. FEBS Lett. 1993; 336: 330-334Crossref PubMed Scopus (189) Google Scholar), but plant PSI complexes are not assembled in trimers, and the function of PSI-L in plants is thus far unsolved. Finally, the N-terminal extension of the PSI-D subunit is important for the stable binding of PSI-C (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar). PSI-C is anchored to the PSI-A/B heterodimer directly through a domain of eight amino acid residues (9Naver H. Scott M.P. Golbeck J.H. Møller B.L. Scheller H.V. J. Biol. Chem. 1996; 271: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Naver H. Scott M.P. Golbeck J.H. Olsen C.E. Scheller H.V. J. Biol. Chem. 1998; 273: 18778-18783Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and indirectly via PSI-D (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar, 11Li N. Zhao J.D. Warren P.V. Warden J.T. Bryant D.A. Golbeck J.H. Biochemistry. 1991; 30: 7863-7872Crossref PubMed Scopus (143) Google Scholar, 12Diaz-Quintana A. Leibl W. Bottin H. Setif P. Biochemistry. 1998; 37: 3429-3439Crossref PubMed Scopus (75) Google Scholar). Treatment with chaotropic agents selectively dissociates the extrinsic subunits PSI-C, -D, and -E, but a much harsher and more prolonged treatment is required to dissociate these subunits from plant PSI compared with cyanobacterial PSI. (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar, 9Naver H. Scott M.P. Golbeck J.H. Møller B.L. Scheller H.V. J. Biol. Chem. 1996; 271: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Naver H. Scott M.P. Golbeck J.H. Olsen C.E. Scheller H.V. J. Biol. Chem. 1998; 273: 18778-18783Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,13Golbeck J.H. Parrett K.G. Mehari T. Jones K.L. Brand J.J. FEBS Lett. 1988; 228: 268-272Crossref Scopus (71) Google Scholar).The role of the three plant-specific subunits is less understood than the role of the 10 common subunits. PSI-N is a luminal protein, and very recent data have shown a function of this subunit in the interaction with plastocyanin (14Haldrup A. Naver H. Scheller H.V. Plant J. 1999; (in press): 17Google Scholar). The PSI-H protein is membrane intrinsic and has a molecular mass of about 10 kDa. PSI-H can be cross-linked with PSI-D, PSI-I, and PSI-L, and the three membrane intrinsic subunits are positioned peripherally in the model of the plant PSI complex with PSI-L and -I located on the outside (15Andersen B. Koch B. Scheller H.V. Physiol. Plant. 1992; 84: 154-161Crossref Scopus (53) Google Scholar, 16Jansson S. Andersen B. Scheller H.V. Plant Physiol. (Bethesda). 1996; 112: 409-420Crossref PubMed Scopus (143) Google Scholar). The properties of PSI-H combined with the relatively high stability of plant PSI have led us to suggest that the role of the N terminus of PSI-D in stability is mediated through an interaction with the PSI-H subunit (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar). Because light-harvesting chlorophylla/b-binding proteins are only present in plants and not in cyanobacteria, an alternative suggestion for the function of PSI-H has been an involvement in the interaction with LHCI (17Scheller H.V. Møller B.L. Physiol. Plant. 1990; 78: 484-494Crossref Scopus (53) Google Scholar). However, the topological studies indicate that such a function is less likely because PSI-H appears not to be in close contact with any LHCI protein (16Jansson S. Andersen B. Scheller H.V. Plant Physiol. (Bethesda). 1996; 112: 409-420Crossref PubMed Scopus (143) Google Scholar).To investigate the role of PSI-H we transformed Arabidopsisplants with a psaH cDNA in sense orientation under the control of a constitutive promoter. Cosuppressed transformants with undetectable levels of PSI-H were obtained. The down-regulated plants were analyzed with several methods both at the biochemical and leaf level. We conclude that PSI-H is essential for efficient electron flow in the PSI complex. Furthermore, PSI-H is required for interaction with PSI-L, stabilization of FX, and the overall stability of the PSI complex.DISCUSSIONThe cosuppression strategy in Arabidopsis was successful and yielded plants without detectable PSI-H. This results has enabled us to investigate the function of the PSI-H subunitin vivo as well as in vitro. Although the approach was successful, the frequency of substantial down-regulation in the transformed plants was low, and no plants lacking PSI-H were obtained in the T1 generation. It has previously been reported that down-regulation of LHCII in an antisense approach was not observed, although the mRNA level was reduced to extremely low levels (28Flachmann R. Kühlbrandt W. Plant Cell. 1995; 7: 149-160Crossref PubMed Scopus (51) Google Scholar). On the other hand, down-regulation of the PSI-N subunit was very efficient in a similar cosuppression and antisense approach (14Haldrup A. Naver H. Scheller H.V. Plant J. 1999; (in press): 17Google Scholar), and the successful down-regulation of the LHCI subunit Lhca4 by an antisense approach was recently reported (29Zhang H. Goodman H.M. Jansson S. Plant Physiol. (Bethesda). 1997; 115: 1525-1531Crossref PubMed Scopus (47) Google Scholar). Possibly, the difficulty in obtaining down-regulation of PSI-H is related to the presence of two expressed copies of psaH inArabidopsis. In contrast, PSI-N is expressed from a single gene. Gene knock-out by homologous recombination is an excellent way of studying the role of PSI subunits in cyanobacteria. However, in plants this technique is not yet straightforward. The present results show that PSI subunits can be efficiently down-regulated in plants by cosuppression. Thus, this approach can be very useful in dissecting the role of individual components of PSI.During growth on sterile medium, plants without PSI-H showed pronounced stunting of growth and yellowing of leaves (Fig. 3). Surprisingly, however, no difference in visual appearance of the plants was seen when pants were grown in soil. Only plants grown in soil were used for biochemical and physiological studies.Forward Electron Transport Is Decreased in Plants Lacking PSI-HSteady state electron transport is clearly decreased in PSI lacking PSI-H. The decrease does not appear to be due to heterogeneity of PSI in the absence of PSI-H because there is no difference at low ferredoxin concentration. The interaction between ferredoxin and PSI is kinetically complex (12Diaz-Quintana A. Leibl W. Bottin H. Setif P. Biochemistry. 1998; 37: 3429-3439Crossref PubMed Scopus (75) Google Scholar, 30Setif P.Q.Y. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar). Ferredoxin forms a complex with PSI in both the reduced and oxidized form. Intracomplex electron transfer is heterogeneous and takes place with several different time constants. At ferredoxin concentrations below the dissociation constant for the PSI-ferredoxin complex, the limiting factor for electron transfer is the second order rate constant. Thus, this rate constant appears to be unaltered in PSI lacking PSI-H. At higher ferredoxin concentration, intracomplex electron transfer may become limiting, and the lower rate in the absence of PSI-H suggests that intracomplex electron transfer is affected. This may be due to different rate constants of transfer or to different relative contributions of the different time constants. The situation in PSI lacking PSI-H may resemble the situation in cyanobacterial PSI where the rate of ferredoxin reduction is much lower than in plant PSI, at least in a large fraction of complexes (30Setif P.Q.Y. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar). In principle, the difference at high ferredoxin concentration could also be due to another limiting step in electron transfer. However, we find this unlikely. Forward electron transfer from P700 to FA/FB is very fast compared with the rates of NADP+ reduction, and the interaction between plastocyanin and PSI appears to be largely governed by PSI-F and PSI-N. Nevertheless, further detailed investigations of different steps of electron transport will be necessary to determine the precise mechanism by which PSI-H affects electron transport. A likely explanation is that lack of PSI-H perturbs the binding of PSI-D and PSI-C, causing a less productive binding of ferredoxin.Plants Lacking PSI-H Accumulate More PSIThe plants devoid of PSI-H have compensated by synthesizing more PSI as evidenced by the 15% lower Chl/P700 ratio. This compensation is sufficient under optimal conditions, where lack of PSI-H had little impact on plant growth. The lower efficiency of PSI might be expected to lead to overreduction of the electron transport components connecting PSI and PSII. However, again the compensation seems sufficient because no significant increase in qP was observed. Furthermore, the size of the functional PSI antenna is identical in plants with and without PSI-H, at least in state 1 (25Naver H. Haldrup A. Gilpin M. Scheller H.V. Garab G. Photosynthesis: Mechanisms and Effects. I. Kluwer Academic Publishers, Dordrecht1998: 631-634Google Scholar). Plants lacking PSI-H show less nonphotochemical quenching of fluorescence, which may indicate a smaller transthylakoidal proton gradient. A smaller proton gradient could result from less overall electron transport. However, because the plants do not exhibit differences in growth, we do not think that this is likely. Alternatively, the less efficient reduction of ferredoxin may lead to altered redox levels in the stroma with a resulting decrease in cyclic electron transport and therefore a decrease in proton pumping. Finally, it may be imagined that lack of PSI-H lead to altered permeability of the membrane for protons.The lower scope for PSI activity and the lower nonphotochemical quenching in the absence of PSI-H may have little significance under optimal and constant growth conditions. However, under photoinhibitory conditions it may be predicted that plants lacking PSI-H would suffer more severely from overreduction of plastoquinone. Other stress condition such as low light intensity or conditions where the demand for ATP is increased may also be expected to lead to more severely affected plants.PSI-H Stabilizes PSIA substantial decrease in stability of PSI was observed in thylakoids from plants without PSI-H. The relative instability resembles the situation in cyanobacteria, where urea has a much faster and stronger effect on PSI than in plants. We have hypothesized that PSI-H interacts with the N-terminal extension of PSI-D which is important for the high stability of plant PSI (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar). The lower stability of the PSI complex lacking PSI-H is therefore in good agreement with the hypothesis. Future experiments with in vitro reconstitution in the presence or absence of PSI-H should allow us to test the hypothesis further and investigate the interaction of PSI-H with PSI-D and other subunits in detail. The instability of FX and earlier acceptors was a surprising result because PSI-H is unlikely to be directly involved in coordinating electron acceptors. Possibly, lack of PSI-H leads to a general instability of the PSI complex in the presence of urea and a progressive disintegration of the entire complex. Although the instability of PSI was easily observed in the in vitro experiments, no severe disintegration of the PSI complex appears to take place during normal growth because all electron acceptors were functional in the thylakoids lacking PSI-H. However, the partial lack of PSI-L suggests a lower stability of the complex also under in vivo conditions. Possibly, more severe disintegration of PSI could occur under certain stress conditions, e.g. heat stress. Because PSI-L content is decreased, it can be speculated whether the role of PSI-H in electron transport is mediated through PSI-L. We find this unlikely because PSI-L is relatively far removed from the site of interaction of the soluble electron transfer proteins (4Schubert W.-D. Klukas O. Krauss N. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (224) Google Scholar). However, experiments with plants lacking only PSI-L will be required to address this issue.ConclusionIn summary, PSI-H has been shown to be not important for LHCI interaction with PSI (25Naver H. Haldrup A. Gilpin M. Scheller H.V. Garab G. Photosynthesis: Mechanisms and Effects. I. Kluwer Academic Publishers, Dordrecht1998: 631-634Google Scholar) but to be essential for efficient electron transfer of PSI and for stability of the PSI complex. The cyanobacterial PSI complex is dissociated eight times faster in the monomeric than in the trimeric form as shown by urea treatment of monomers and trimers (4Schubert W.-D. Klukas O. Krauss N. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (224) Google Scholar, 31Luneberg J. Fromme P. Jekow P. Schlodder E. FEBS Lett. 1994; 338: 197-202Crossref PubMed Scopus (59) Google Scholar). Plant PSI is a monomer that is more stable upon urea treatment than the trimeric cyanobacterial PSI (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar). The peripheral antenna in cyanobacteria consists of extrinsic phycobiliproteins. In contrast, plants have adopted a different antenna consisting of membrane intrinsic Chl a/b-binding proteins, i.e. LHCI and LHCII. It can be speculated that the trimeric structure of PSI was abandoned as LHCI became associated with PSI in plants, and this resulted in a need for stabilizing the now monomeric PSI complex. PSI-H may have evolved simultaneously, fulfilling the role as a stabilizing factor. Photosystem I (PSI) 1The abbreviations used are: PS, photosystem; LHC, light harvesting complex; Chl, chlorophyll.1The abbreviations used are: PS, photosystem; LHC, light harvesting complex; Chl, chlorophyll. is a pigment-protein complex that mediates the light-driven electron transport across the thylakoid membrane from the soluble electron donor, plastocyanin, to the soluble electron acceptor, ferredoxin. PSI from plants contains 13 different subunits of which three are only found in plants, namely PSI-G, PSI-H, and PSI-N. The remaining 10 subunits are shared between cyanobacteria and plants. In addition to the 13 subunits of PSI in a narrow sense, plants contain light harvesting complex I (LHCI), which is composed of four different polypeptides, Lhca1–4, that are specifically associated with PSI (1Jansson S. Biochim. Biophys. Acta. 1994; 1184: 1-19Crossref PubMed Scopus (585) Google Scholar,2Scheller H.V. Naver H. Møller B.L. Physiol. Plant. 1997; 100: 842-851Crossref Google Scholar). The PSI-A/B heterodimer coordinates the reaction center P700 (a chlorophyll (Chl) a dimer) and the electron acceptors A0 (Chl a), A1 (phylloquinone), and FX (a [4Fe-4S] iron-sulfur cluster). The terminal electron acceptors FA and FB are [4Fe-4S] clusters bound to the stromal PSI-C subunit (3Høj P.B. Svendsen I. Scheller H.V. Møller B.L. J. Biol. Chem. 1987; 262: 12676-12684Abstract Full Text PDF PubMed Google Scholar, 4Schubert W.-D. Klukas O. Krauss N. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (224) Google Scholar). The 10 common subunits are highly conserved from cyanobacteria to plants apart from the presence of extended N- termini of PSI-D, -E, -F, and - L from plants. The role of specific subunits in PSI has mostly been investigated by gene knock-out studies in cyanobacteria and algae. However, despite the sequence similarities, PSI subunits of cyanobacteria and plants show important functional differences. For example, the plant-specific N terminus of PSI-F plays a role in supporting plastocyanin-mediated donation of electrons to P700+ (5Farah J. Rappaport F. Choquet Y. Joilot P. Rochaix J.D. EMBO J. 1995; 14: 4976-4984Crossref PubMed Scopus (108) Google Scholar, 6Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar). In cyanobacterial PSI, this electron transfer mostly follows a simple second order reaction, whereas a stable plastocyanin-PSI complex is formed in plants before electron transfer (6Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar). PSI-L is essential for formation of PSI trimers in cyanobacteria (7Chitnis V.P. Chitnis P.R. FEBS Lett. 1993; 336: 330-334Crossref PubMed Scopus (189) Google Scholar), but plant PSI complexes are not assembled in trimers, and the function of PSI-L in plants is thus far unsolved. Finally, the N-terminal extension of the PSI-D subunit is important for the stable binding of PSI-C (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar). PSI-C is anchored to the PSI-A/B heterodimer directly through a domain of eight amino acid residues (9Naver H. Scott M.P. Golbeck J.H. Møller B.L. Scheller H.V. J. Biol. Chem. 1996; 271: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Naver H. Scott M.P. Golbeck J.H. Olsen C.E. Scheller H.V. J. Biol. Chem. 1998; 273: 18778-18783Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and indirectly via PSI-D (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar, 11Li N. Zhao J.D. Warren P.V. Warden J.T. Bryant D.A. Golbeck J.H. Biochemistry. 1991; 30: 7863-7872Crossref PubMed Scopus (143) Google Scholar, 12Diaz-Quintana A. Leibl W. Bottin H. Setif P. Biochemistry. 1998; 37: 3429-3439Crossref PubMed Scopus (75) Google Scholar). Treatment with chaotropic agents selectively dissociates the extrinsic subunits PSI-C, -D, and -E, but a much harsher and more prolonged treatment is required to dissociate these subunits from plant PSI compared with cyanobacterial PSI. (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar, 9Naver H. Scott M.P. Golbeck J.H. Møller B.L. Scheller H.V. J. Biol. Chem. 1996; 271: 8996-9001Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Naver H. Scott M.P. Golbeck J.H. Olsen C.E. Scheller H.V. J. Biol. Chem. 1998; 273: 18778-18783Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,13Golbeck J.H. Parrett K.G. Mehari T. Jones K.L. Brand J.J. FEBS Lett. 1988; 228: 268-272Crossref Scopus (71) Google Scholar). The role of the three plant-specific subunits is less understood than the role of the 10 common subunits. PSI-N is a luminal protein, and very recent data have shown a function of this subunit in the interaction with plastocyanin (14Haldrup A. Naver H. Scheller H.V. Plant J. 1999; (in press): 17Google Scholar). The PSI-H protein is membrane intrinsic and has a molecular mass of about 10 kDa. PSI-H can be cross-linked with PSI-D, PSI-I, and PSI-L, and the three membrane intrinsic subunits are positioned peripherally in the model of the plant PSI complex with PSI-L and -I located on the outside (15Andersen B. Koch B. Scheller H.V. Physiol. Plant. 1992; 84: 154-161Crossref Scopus (53) Google Scholar, 16Jansson S. Andersen B. Scheller H.V. Plant Physiol. (Bethesda). 1996; 112: 409-420Crossref PubMed Scopus (143) Google Scholar). The properties of PSI-H combined with the relatively high stability of plant PSI have led us to suggest that the role of the N terminus of PSI-D in stability is mediated through an interaction with the PSI-H subunit (8Naver H. Scott M.P. Andersen B. Møller B.L. Scheller H.V. Physiol. Plant. 1995; 95: 19-26Crossref Scopus (25) Google Scholar). Because light-harvesting chlorophylla/b-binding proteins are only present in plants and not in cyanobacteria, an alternative suggestion for the function of PSI-H has been an involvement in the interaction with LHCI (17Scheller H.V. Møller B.L. Physiol. Plant. 1990; 78: 484-494Crossref Scopus (53) Google Scholar). However, the topological studies indicate that such a function is less likely because PSI-H appears not to be in close contact with any LHCI protein (16Jansson S. Andersen B. Scheller H.V. Plant Physiol. (Bethesda). 1996; 112: 409-420Crossref PubMed Scopus (143) Google Scholar). To investigate the role of PSI-H we transformed Arabidopsisplants with a psaH cDNA in sense orientation under the control of a constitutive promoter. Cosuppressed transformants with undetectable levels of PSI-H were obtained. The down-regulated plants were analyzed with several methods both at the biochemical and leaf level. We conclude that PSI-H is essential for efficient electron flow in the PSI complex. Furthermore, PSI-H is required for interaction with PSI-L, stabilization of FX, and the overall stability of the PSI complex. DISCUSSIONThe cosuppression strategy in Arabidopsis was successful and yielded plants without detectable PSI-H. This results has enabled us to investigate the function of the PSI-H subunitin vivo as well as in vitro. Although the approach was successful, the frequency of substantial down-regulation in the transformed plants was low, and no plants lacking PSI-H were obtained in the T1 generation. It has previously been reported that down-regulation of LHCII in an antisense approach was not observed, although the mRNA level was reduced to extremely low levels (28Flachmann R. Kühlbrandt W. Plant Cell. 1995; 7: 149-160Crossref PubMed Scopus (51) Google Scholar). On the other hand, down-regulation of the PSI-N subunit was very efficient in a similar cosuppression and antisense approach (14Haldrup A. Naver H. Scheller H.V. Plant J. 1999; (in press): 17Google Scholar), and the successful down-regulation of the LHCI subunit Lhca4 by an antisense approach was recently reported (29Zhang H. Goodman H.M. Jansson S. Plant Physiol. (Bethesda). 1997; 115: 1525-1531Crossref PubMed Scopus (47) Google Scholar). Possibly, the difficulty in obtaining down-regulation of PSI-H is related to the presence of two expressed copies of psaH inArabidopsis. In contrast, PSI-N is expressed from a single gene. Gene knock-out by homologous recombination is an excellent way of studying the role of PSI subunits in cyanobacteria. However, in plants this technique is not yet straightforward. The present results show that PSI subunits can be efficiently down-regulated in plants by cosuppression. Thus, this approach can be very useful in dissecting the role of individual components of PSI.During growth on sterile medium, plants without PSI-H showed pronounced stunting of growth and yellowing of leaves (Fig. 3). Surprisingly, however, no difference in visual appearance of the plants was seen when pants were grown in soil. Only plants grown in soil were used for biochemical and physiological studies.Forward Electron Transport Is Decreased in Plants Lacking PSI-HSteady state electron transport is clearly decreased in PSI lacking PSI-H. The decrease does not appear to be due to heterogeneity of PSI in the absence of PSI-H because there is no difference at low ferredoxin concentration. The interaction between ferredoxin and PSI is kinetically complex (12Diaz-Quintana A. Leibl W. Bottin H. Setif P. Biochemistry. 1998; 37: 3429-3439Crossref PubMed Scopus (75) Google Scholar, 30Setif P.Q.Y. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar). Ferredoxin forms a complex with PSI in both the reduced and oxidized form. Intracomplex electron transfer is heterogeneous and takes place with several different time constants. At ferredoxin concentrations below the dissociation constant for the PSI-ferredoxin complex, the limiting factor for electron transfer is the second order rate constant. Thus, this rate constant appears to be unaltered in PSI lacking PSI-H. At higher ferredoxin concentration, intracomplex electron transfer may become limiting, and the lower rate in the absence of PSI-H suggests that intracomplex electron transfer is affected. This may be due to different rate constants of transfer or to different relative contributions of th
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