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• W2022337592 abstract "Photosystem II is a large pigment-protein complex catalyzing water oxidation and initiating electron transfer processes across the thylakoid membrane. In addition to large protein subunits, many of which bind redox cofactors, photosystem II particles contain a number of low molecular weight polypeptides whose function is only poorly defined. Here we have investigated the function of one of the smallest polypeptides in photosystem II, PsbJ. Using a reverse genetics approach, we have inactivated the psbJ gene in the tobacco chloroplast genome. We show that, although the PsbJ polypeptide is not principally required for functional photosynthetic electron transport, plants lacking PsbJ are unable to grow photoautotrophically. We provide evidence that this is due to the accumulation of incompletely assembled water-splitting complexes, which in turn causes drastically reduced photosynthetic performance and extreme hypersensitivity to light. Our results suggest a role of PsbJ for the stable assembly of the water-splitting complex of photosystem II and, in addition, support a control of photosystem I accumulation through photosystem II activity. Photosystem II is a large pigment-protein complex catalyzing water oxidation and initiating electron transfer processes across the thylakoid membrane. In addition to large protein subunits, many of which bind redox cofactors, photosystem II particles contain a number of low molecular weight polypeptides whose function is only poorly defined. Here we have investigated the function of one of the smallest polypeptides in photosystem II, PsbJ. Using a reverse genetics approach, we have inactivated the psbJ gene in the tobacco chloroplast genome. We show that, although the PsbJ polypeptide is not principally required for functional photosynthetic electron transport, plants lacking PsbJ are unable to grow photoautotrophically. We provide evidence that this is due to the accumulation of incompletely assembled water-splitting complexes, which in turn causes drastically reduced photosynthetic performance and extreme hypersensitivity to light. Our results suggest a role of PsbJ for the stable assembly of the water-splitting complex of photosystem II and, in addition, support a control of photosystem I accumulation through photosystem II activity. Photosystem II (PSII) 1The abbreviations used are: PSIIphotosystem IIPSIphotosystem ITricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineis a large cofactor-protein complex consisting of at least 17 protein subunits (Ref. 1.Zouni A. Witt H.-T. Kern J. Fromme P. Krauß N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1762) Google Scholar; for review, see e.g. Ref. 2.Barber J. Nield J. Morris E.P. Zheleva D. Hankamer B. Physiol. Plant. 1997; 100: 817-827Crossref Google Scholar). The PSII reaction center is formed by a heterodimer of two pigment-binding proteins, D1 and D2, which, in photosynthetic eukaryotes, are encoded by the chloroplast psbA and psbD genes, respectively. The photochemical reaction carried out by the reaction center converts the energy of a photon into a separation of charge and, in this way, initiates electron flow. Around the reaction center, the outer parts of PSII are assembled, the inner and outer antennae funneling absorbed light energy to the catalytic core and the oxygen-evolving complex splitting water into protons, electrons, and dioxygen (reviewed in Refs. 3.Wollman F.-A. Minai L. Nechushtai R. Biochim. Biophys. Acta. 1999; 1411: 21-85Crossref PubMed Scopus (216) Google Scholar and 4.Choquet Y. Vallon O. Biochimie (Paris). 2000; 82: 615-634Crossref PubMed Scopus (98) Google Scholar). photosystem II photosystem I N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine In addition to the well-studied large protein subunits, purified PSII particles contain a number of low molecular weight polypeptides, many of which are encoded by the plastid genome of photosynthetically active eukaryotes (5.Takahashi Y. J. Plant Res. 1998; 111: 101-111Crossref Google Scholar). Most of these small subunits do not bind redox cofactors and, hence, are unlikely to participate directly in electron transfer reactions. It is generally assumed that they rather function as photosystem-assembling or stabilizing factors. However, in many cases, molecular evidence supporting such a structural role is largely lacking. The successful development of transformation technologies forChlamydomonas (6.Boynton J.E. Gillham N.W. Harris E.H. Hosler J.P. Johnson A.M. Jones A.R. Randolph-Anderson B.L. Robertson D. Klein T.M. Shark K.B. Sanford J.C. Science. 1988; 240: 1534-1538Crossref PubMed Scopus (712) Google Scholar) and tobacco chloroplasts (7.Svab Z. Hajdukiewicz P. Maliga P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8526-8530Crossref PubMed Scopus (506) Google Scholar) has paved the way to functional characterizations of plastid genome-encoded genes by reverse genetics. Linked to a selectable marker gene, mutant alleles can be introduced into plastids by chloroplast transformation, where they replace the endogenous wild-type allele by homologous recombination. During the past decade, reverse genetics has become a powerful tool in plastid functional genomics (reviewed in Ref. 8.Rochaix J.-D. Trends Plant Sci. 1997; 2: 419-425Abstract Full Text PDF Google Scholar). Here, we have taken a reverse genetics approach to define the function of one of the smallest polypeptides in PSII, PsbJ. Using chloroplast transformation, we have generated tobacco plants lacking the PsbJ polypeptide. Physiological and biochemical analyses revealed that the PsbJ-deficient mutant plants accumulate incompletely assembled oxygen-evolving complexes, have reduced levels of PSI and are extremely sensitive to light. Sterile tobacco plants (Nicotiana tabacum cv. Petit Havana) were grown on agar-solidified MS medium containing 30 g/liter sucrose (9.Murashige T. Skoog F. Physiol. Plant. 1962; 15: 493-497Crossref Scopus (53928) Google Scholar). Homoplasmic transplastomic lines were rooted and propagated on the same medium. Photoautotrophic growth was tested on MS medium without sucrose. For protein isolation and physiological measurements, wild type and transformed plants were grown on sucrose-containing medium under low (2 μmol quanta m−2 s−1) and standard light (100 μmol quanta m−2 s−1), respectively. Leaves referred to as “young leaves” were harvested at the age of only a few days in order to avoid interference with photooxidative damage in the mutant chloroplasts. Leaves referred to as “mature leaves” were several weeks old and, in the case of the ΔpsbJ mutant, not yet photobleached. The region of the tobacco plastid genome containing the psbEoperon was isolated as a 2383-bp SalI/SpeI fragment corresponding to nucleotide positions 65,313–67,695 (according to Ref. 10.Wakasugi T. Sugita M. Tsudzuki T. Sugiura M. Plant Mol. Biol. Rep. 1998; 16: 231-241Crossref Scopus (87) Google Scholar). The fragment was cloned into a pBluescript KS vector (Stratagene, La Jolla, CA) cut with SalI andSpeI. To remove the HincII site from the remaining polylinker, the plasmid was linearized with SalI and the recessed ends were filled in using the Klenow fragment of DNA polymerase I followed by religation. The psbE operon was subsequently excised by digestion with HincII andClaI (restriction sites correspond to nucleotide positions 66,341 and 67,167) and replaced by a similarly cut PCR product carrying the psbJ deletion. The deletion of the psbJcoding region was introduced into a PCR product by amplification with primers PT5′ (5′-GACGATCTCACAAAGATGAA-3′) and PΔJ1 (5′-TTTTGTTGACGACTCAATTCATTACCCCACTTCCCTCCA-3′). The 3′ portion of oligonucleotide PΔJ1 binds to the sequence immediately upstream of the psbJ initiator codon and contains in its 5′ portion the nucleotide sequence downstream of the psbJ termination codon up to the HincII site at 66,341. Oligonucleotide PT5′ binds upstream of the ClaI site at 67,167. A chimericaadA gene conferring resistance to aminoglycoside antibiotics (11.Svab Z. Maliga P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 913-917Crossref PubMed Scopus (621) Google Scholar) was cloned into the unique EcoRV site (position 66,054) to facilitate selection of chloroplast transformants. A plasmid clone carrying the aadA gene in the same orientation as the psbE operon yielded the final transformation vector pΔpsbJ (see Fig. 2). Young leaves from sterile tobacco plants were bombarded with plasmid pΔpsbJ-coated 1.1-μm tungsten particles using a biolistic gun (PDS1000He; Bio-Rad). Primary spectinomycin-resistant lines were selected on RMOP regeneration medium containing 500 mg/liter spectinomycin (7.Svab Z. Hajdukiewicz P. Maliga P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8526-8530Crossref PubMed Scopus (506) Google Scholar, 11.Svab Z. Maliga P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 913-917Crossref PubMed Scopus (621) Google Scholar). Plastid transformants were identified by PCR amplification according to standard protocols and using the primer pair P10 (5′-AACCTCCTATAGACTAGGC-3′; complementary to the psbA3′-untranslated region of the chimeric aadA gene) and P11 (5′-AGCGAAATGTAGTGCTTACG-3′; derived from the 3′ portion of theaadA coding region). Four independent transplastomic lines were subjected to four additional rounds of regeneration on RMOP/spectinomycin to obtain homoplasmic tissue. Total plant DNA was isolated by a rapid miniprep procedure (12.Doyle J.J. Doyle J.L. Focus. 1990; 12: 13-15Google Scholar). Total cellular RNA was extracted using the TRIzol reagent (Invitrogen). DNA samples were digested withEcl136II/DraI, separated on 1.2% agarose gels, and blotted onto Hybond N nylon membranes (Amersham Biosciences). Total cellular RNA was electrophoresed on formaldehyde-containing 1.3% agarose gels and transferred onto Hybond N+ membranes. For hybridization, [α-32P]dATP-labeled probes were generated by random priming (Multiprime DNA labeling system; Amersham Biosciences). A radiolabeled PCR product covering part of thepsbE operon (obtained by amplification with primer pair P7652 (5′-CCGAATGAGCTAAGAGAATCTT-3′)/P7355 (5′-GACTATAGATCGAACCTATCC-3′)) was used as probe for the restriction fragment length polymorphism analysis and for detection ofpsbE operon-specific transcripts. Hybridizations were carried out at 65–68 °C in rapid hybridization buffer (Amersham Biosciences). An NdeI restriction fragment corresponding to nucleotide positions 41,487–42,385 in the tobacco chloroplast genome (10.Wakasugi T. Sugita M. Tsudzuki T. Sugiura M. Plant Mol. Biol. Rep. 1998; 16: 231-241Crossref Scopus (87) Google Scholar) was used as a specific probe for detection of transcripts from the plastid psaA/B operon. To control for equal loading, blots were stripped and rehybridized to a cytoplasmic 18S rRNA probe (amplified with primer pair P5′18SNT (5′-GTATATTTAAGTTGTTGCAGT-3′)/P3′18SNT (5′-AAACTTTGATTTCTCATAAGG-3′)). Transcript quantitation was performed with a PhosphorImager using the Quantity One® software (Bio-Rad). Thylakoid proteins from wild-type and mutant plants were isolated from total leaf material following published procedures (14.Machold O. Simpson D.J. Moller B.L. Carlsberg Res. Commun. 1979; 44: 235-254Crossref Scopus (135) Google Scholar). PSII-enriched membranes (BBY) were prepared according to a protocol originally developed for spinach (15.Johnson G.N. Boussac A. Rutherford A.W. Biochim. Biophys. Acta. 1994; 1184: 85-92Crossref Scopus (87) Google Scholar). Following protein quantitation, equal amounts of thylakoid proteins were separated on Tricine-SDS-polyacrylamide gels (16.Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10478) Google Scholar) and stained with silver according to standard protocols (17.Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley & Sons, Inc., New York1995Google Scholar). For Western blot analyses, electrophoretically separated proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences) using the Trans-Blot Cell (Bio-Rad) and a standard transfer buffer (192 mm glycine, 25 mm Tris, pH 8.3). Immunoblot detection was performed using the enhanced chemiluminescence system (ECL; Amersham Biosciences). Determination of PSII activity was performed on dark-adapted leaves from wild-type and mutant plants grown under low light (2 μmol quanta m−2s−1) and standard light (100 μmol quanta m−2 s−1) conditions, respectively. PSII-dependent chlorophyll fluorescence was recorded at 650-nm wavelength with a pulsed amplitude modulation fluorimeter (Walz, Effeltrich, Germany; Ref. 18.Schreiber U. Schliwa U. Bilger W. Photosynth. Res. 1986; 10: 51-62Crossref PubMed Scopus (2381) Google Scholar) under illumination of intact leaf tissue with white actinic light (flux density 50 μmol quanta m−2 s−1 and 100 μmol quanta m−2 s−1; pulse frequency of measuring light, 1.6 kHz). For complete reduction of QA, leaves were exposed to pulses of saturating light (1 s; flux density 6000 μmol quanta m−2 s−1). The redox state of the PSI reaction center chlorophyll P700 was monitored by following the changes in absorbance of dark-adapted leaves from wild-type and mutant plants at a 830-nm wavelength (19.Harbinson J. Hedley C.L. Plant Physiol. 1993; 103: 649-660Crossref PubMed Scopus (65) Google Scholar, 20.Schreiber U. Klughammer C. Neubauer C. Z. Naturforsch. 1988; 43: 686-698Crossref Scopus (239) Google Scholar). Absorbance measurements were performed using the pulsed amplitude modulation fluorimeter with a modified emitter/detector unit. Far red light with a peak wavelength of 730 nm was used to selectively excite PSI. To obtain complete rereduction of PSI, leaves were exposed to a strong white light pulse (5, 50, or 200 ms; 6000 μmol quanta m−2 s−1). Oxygen evolution of young leaves from plants grown at 2 μmol quanta m−2 s−1 was measured with a Clark O2 electrode (Hansatech) at room temperature under saturating CO2 levels to minimize competing O2-consuming reactions. Oxygen evolution was monitored at flux densities of 30 μmol quanta m−2 s−1. Five independent measurements were performed to calculate average O2 evolution values. Thermoluminescence was measured on leaf segments with a home-built apparatus. Thermoluminescence was excited with single turnover flashes at 0 °C. The flashes were spaced with a 1-s dark interval. Samples were then heated with a heating rate of 20 °C/min to 60 °C, and the light emission was recorded. Graphical and numerical data analyses were performed according to Ducruet and Miranda (21.Ducruet J.M. Miranda T. Photosynth. Res. 1992; 33: 15-27Crossref PubMed Scopus (69) Google Scholar). ThepsbJ gene is part of the plastid psbE operon, which comprises the four PSII genes psbE, psbF,psbL, and psbJ. This operon structure is conserved in all photosynthetically active multicellular plant species investigated to date but is not found in the unicellular green algaChlamydomonas reinhardtii (22.Mor T.S. Ohad I. Hirschberg J. Pakrasi H.B. Mol. Gen. Genet. 1995; 246: 600-604Crossref PubMed Scopus (21) Google Scholar). The psbE operon is unique in that its tetracistronic primary transcript does not undergo processing into monocistronic or oligocistronic units (23.Haley J. Bogorad L. Plant Cell. 1990; 2: 323-333PubMed Google Scholar). Consequently, translation must initiate efficiently on all four cistrons of the polycistronic mRNA. The resulting translation products are relatively small subunits of the PSII complex. ThepsbE and psbF genes specify the cytochromeb559 α and β subunits, which are essential for PSII assembly (24.Stewart D.H. Brudvig G.W. Biochim. Biophys. Acta. 1998; 1367: 63-87Crossref PubMed Scopus (216) Google Scholar, 25.Pakrasi H.B. De Ciechi P. Whitmarsh J. EMBO J. 1991; 10: 1619-1627Crossref PubMed Scopus (76) Google Scholar, 26.Morais F. Barber J. Nixon P. J. Biol. Chem. 1998; 273: 29315-29320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The function of the other two polypeptides encoded by the psbE operon, PsbL and PsbJ, is much less clear. PsbL has recently been implicated in the stabilization of the PSII core complex and the dimeric form of PSII (27.Kitamura K. Ozawa S. Shiina T. Toyoshima Y. FEBS Lett. 1994; 354: 113-116Crossref PubMed Scopus (24) Google Scholar, 28.Zheleva D. Sharma J. Panico M. Morris H.R. Barber J. J. Biol. Chem. 1998; 273: 16122-16127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The PsbJ protein has been detected immunologically in thylakoid membranes of cyanobacteria (29.Lind L.K. Shukla V.K. Nyhus K.J. Pakrasi H.B. J. Biol. Chem. 1993; 268: 1575-1579Abstract Full Text PDF PubMed Google Scholar, 30.Regel R.E. Ivleva N.B. Zer H. Meurer J. Shestakov S.V. Herrmann R.G. Pakrasi H.B. Ohad I. J. Biol. Chem. 2001; 276: 41473-41478Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and in purified PSII particles by MALDI-TOF mass spectrometry (1.Zouni A. Witt H.-T. Kern J. Fromme P. Krauß N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1762) Google Scholar). Genetic analyses in cyanobacteria also support an association of PsbJ with PSII and, in addition, have established that, in Synechocystis, PsbJ is not essential for photoautotrophic growth (29.Lind L.K. Shukla V.K. Nyhus K.J. Pakrasi H.B. J. Biol. Chem. 1993; 268: 1575-1579Abstract Full Text PDF PubMed Google Scholar). The psbJ gene of photosynthetic eukaryotes specifies a hydrophobic polypeptide of only 40 amino acids (theoretical molecular mass: 4.1 kDa) which is evolutionarily highly conserved (Fig. 1). In order to define the function of the PsbJ polypeptide in PSII of higher plants, we have constructed a chloroplast transformation vector carrying a psbJ null allele (Fig. 2). The psbJcoding region was deleted from a cloned fragment of the tobacco plastid DNA using PCR-based mutagenesis techniques. A chimeric selectable marker gene aadA was inserted into the intergenic spacer in between the psbE operon and the petA gene (Fig. 2). Earlier work had established that this spacer is a suitable site for the insertion of plastid transgenes (31.Bock R. Kössel H. Maliga P. EMBO J. 1994; 13: 4623-4628Crossref PubMed Scopus (169) Google Scholar, 32.Bock R. Hermann M. Kössel H. EMBO J. 1996; 15: 5052-5059Crossref PubMed Scopus (133) Google Scholar). Biolistic bombardment of sterile tobacco leaves with plasmid pΔpsbJ-coated tungsten particles was followed by selection of spectinomycin-resistant cell lines. Successful chloroplast transformation was verified by tests for double resistance on medium containing two aminoglycoside antibiotics, spectinomycin and streptomycin (11.Svab Z. Maliga P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 913-917Crossref PubMed Scopus (621) Google Scholar), and further confirmed by PCR assays usingaadA gene-specific primers (32.Bock R. Hermann M. Kössel H. EMBO J. 1996; 15: 5052-5059Crossref PubMed Scopus (133) Google Scholar). Chloroplast transformants were purified to homoplasmy by passing them through additional regeneration cycles under antibiotic selection. Homoplasmy of the transplastomic lines (i.e. absence of any residual copies of the wild-type chloroplast genome) was confirmed by restriction fragment length polymorphism analysis (Fig. 3). Since there is no absolute linkage between the introducedpsbJ deletion and the selectable marker gene aadAin our transformation vector (Fig. 2), two different types of chloroplast transformants are obtained: (i) transformants carrying only the aadA but not the psbJ deletion and (ii) transformants having incorporated both the aadA gene and thepsbJ null allele by homologous recombination upstream ofpsbJ (Fig. 2). Southern blot analyses confirmed the isolation of transplastomic lines from each of the two types (Fig. 3). As expected, lines belonging to the first type were phenotypically indistinguishable from the wild type (as shown earlier; Ref. 31.Bock R. Kössel H. Maliga P. EMBO J. 1994; 13: 4623-4628Crossref PubMed Scopus (169) Google Scholar), whereas lines of the second type all exhibited the same mutant phenotype (subsequently referred to as ΔpsbJ plants; see below). Since the psbJ gene is part of an operon that is transcribed as a tetracistronic mRNA, it was important to verify that the deletion introduced into psbJ did not affect transcription of the psbE operon or stability of its mRNAs. We therefore comparatively analyzed accumulation of psbE operon transcripts in wild-type and transplastomic tobacco lines (Fig. 4). No significant difference was found in transcript pattern and mRNA accumulation levels between the transplastomic lines only having the aadA marker and those additionally carrying the psbJ deletion indicating that the deletion in the psbJ coding region does not negatively affect synthesis or stability of psbE operon transcripts. Faithful expression of the engineered psbE operon in ΔpsbJ plants was subsequently also confirmed at the protein level by assaying accumulation of the cytochromeb559 α-subunit, the psbE gene product (see below). As established earlier foraadA transgenes inserted into the same genomic location (31.Bock R. Kössel H. Maliga P. EMBO J. 1994; 13: 4623-4628Crossref PubMed Scopus (169) Google Scholar,32.Bock R. Hermann M. Kössel H. EMBO J. 1996; 15: 5052-5059Crossref PubMed Scopus (133) Google Scholar), transplastomic plants carrying the aadA selectable marker gene but not the psbJ deletion were phenotypically normal and indistinguishable from wild-type tobacco plants in all subsequent biochemical and physiological tests (see below). By contrast, homoplasmic psbJ knockout plants grown on synthetic medium under standard light conditions (100 μmol quanta m−2 s−1) displayed a clear mutant phenotype. While young leaves were almost normally green (Fig. 5), older leaves were completely white and showed strong symptoms of photobleaching. However, this phenotype was much less severe than that observed previously for tobacco photosynthesis null mutants (33.Ruf S. Kössel H. Bock R. J. Cell Biol. 1997; 139: 95-102Crossref PubMed Scopus (112) Google Scholar, 34.Hager M. Biehler K. Illerhaus J. Ruf S. Bock R. EMBO J. 1999; 18: 5834-5842Crossref PubMed Scopus (111) Google Scholar), suggesting that the lack of the PsbJ protein may reduce but does not completely abolish photosynthetic activity. The mutant phenotype was identical in all independently generated transplastomic lines and has remained stable during vegetative propagation, providing additional proof for the homoplasmy of the lines. When grown under extreme low light conditions (2 μmol quanta m−2 s−1), no bleaching of mature leaves occurred in ΔpsbJ plants (Fig. 5), confirming that the severe phenotype under standard light conditions is caused by the mutant's high sensitivity to light, which appears to result in the accumulation of photooxidative damage over time. The highly light-sensitive phenotype of the ΔpsbJ lines was indicative of inefficient electron transfer reactions, potentially leading to the production of free radicals that in turn cause photooxidative damage to chloroplast membranes and proteins. In order to determine the efficiency of photosynthetic electron transport, we comparatively analyzed PSII and PSI functions in wild-type andΔpsbJ plants. In view of the light-sensitive phenotype of the psbJ knockout plants and the strong dependence of photobleaching on leaf age, we included young and mature leaves from plants grown at 2 μmol quanta m−2 s−1 as well as similar leaf material from plants raised at 100 μmol quanta m−2 s−1 (Figure 5, Figure 6, Figure 7). We define here “young” as the first (or at most the second, depending on the size of the youngest visible leaf) leaf from the tip of the plant and “mature” as a relatively expanded leaf (second, third, or fourth from the tip), which, in the case of the ΔpsbJ plants grown at 100 μmol quanta m−2 s−1 must not yet be photobleached.Figure 7Test for PSI function by far red spectroscopy. Selective excitation of the PSI reaction center chlorophyll P700 results in a transition from its reduced to its oxidized state and hence reveals intact PSI photochemistry in both the wild type and the mutant. However, rereduction of P700 by white light pulses and, hence, by electrons released in PSII reveals remarkable differences. The 200-ms light pulse on mature mutant leaves grown at 2 μmol quanta m−2 s−1 as well as all pulses on mature mutant leaves grown at 100 μmol quanta m−2s−1 cannot rereduce the PSI reaction center chlorophylls and instead result in an apparent overoxidation of the P700 pool. Note that plants grown at 100 μmol quanta m−2s−1 generally possess more PSI than plants grown at 2 μmol quanta m−2 s−1, which is likely to reflect light-induced up-regulation of photosynthesis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We first measured PSII activity by chlorophyll fluorescence at room temperature. The minimum fluorescence F0 was determined by exposure of dark-adapted leaves to measuring light of low intensity (Fig. 6). Subsequently, maximum fluorescence Fm was obtained by illumination with two saturating light pulses each resulting in complete reduction of the primary quinone-type acceptor of PSII, QA. High variable fluorescence (Fvar =Fm − F0) was detected for both young wild-type and young ΔpsbJ leaves (Fig. 6), strongly suggesting that ΔpsbJ plants synthesize functional PSII units capable of reducing the primary PSII acceptor QA. In contrast, mature leaves from ΔpsbJplants grown at 100 μmol quanta m−2 s−1showed almost no variable fluorescence. Fvar was also drastically reduced in mature leaves from ΔpsbJplants grown under extreme low light conditions (Fig. 6) mainly caused by a strong increase in minimum fluorescence,F0. This may indicate a disproportion between the photon-capturing capacity of the PSII antenna and the transfer of absorbed light energy to the PSII reaction center resulting in dramatically enhanced chlorophyll fluorescence emission. The ratio (Fm −F0)/Fm can serve as a measure of the maximum quantum yield of PSII-driven photochemistry. In young leaves from ΔpsbJ plants, this ratio (and hence the maximum photochemical capacity of PSII) was found to be only slightly reduced as compared with wild-type leaves, suggesting that the absence of the PsbJ protein does not dramatically impair the electron transfer reactions in PSII. By contrast, mature leaves from ΔpsbJplants were severely affected (Fig. 6), indicating that the PsbJ protein is directly or indirectly involved in the protection of PSII from light-induced damage or destabilization. When wild-type leaves were exposed to continuous actinic light of low intensity, flashes of saturating white light superimposed on the actinic light resulted in a fluorescence rise, which approximately reached the initial value of Fm, and strong photochemical fluorescence quenching occurred (Fig. 6). By contrast, in young leaves from ΔpsbJ plants, the fluorescence rise induced by the saturating flashes clearly did not reach the initialFm value, and mature leaves from plants grown at 100 μmol quanta m−2 s−1 even lacked almost any PSII photochemistry. These results lend further support to the idea that, in the absence of the PsbJ protein, the photosynthetic apparatus is highly light-sensitive. In order to measure the efficiency of electron transfer to downstream components of the photosynthetic electron transport chain, we next set out to determine the activity of PSI. PSI function in wild-type and mutant plants was deduced from absorption measurements at 830 nm. Absorption changes at 830 nm correlate with the redox state of the PSI reaction center chlorophyll, P700. In the dark, P700 is present in its reduced form (19.Harbinson J. Hedley C.L. Plant Physiol. 1993; 103: 649-660Crossref PubMed Scopus (65) Google Scholar). Illumination of dark-adapted leaves with far red light selectively excites PSI, thereby converting P700 in its oxidized form (Fig. 7). This PSI-induced absorption shift is observed for the wild type as well as for all leaves from ΔpsbJ plants, including the mature leaves grown under normal light (100 μmol quanta m−2s−1), which had almost no measurable PSII activity, confirming that the knockout of psbJ primarily affects PSII. However, in all leaf samples from the mutant, the intensity of the absorption change was significantly lower than in the equivalent wild-type sample, suggesting that ΔpsbJ plants may have fewer active PSI units than the wild type (Fig. 7). When short pulses of white light are superimposed onto the continuous far red light, electrons are released from PSII and transferred to PSI, where they lead to a rereduction of P700, which again can be monitored as an absorption change at 830 nm (Fig. 7). This PSII-dependent rereduction of P700 was observed for young mutant leaves but was significantly impaired in mature leaves fromΔpsbJ plants (Fig. 7). Altogether, these physiological data suggest that, whileΔpsbJ plants are capable of synthesizing principally functional photosystems, (i) PSII function appears to be highly light-sensitive in a leaf age-dependent manner and (ii) PSI levels or activity may be reduced in the absence of the PsbJ polypeptide. Having established that the light reactions of photosynthesis still function to some extent in ΔpsbJ plants, we were interested to know whether or not the electron transfer capacity in the mutant was sufficient to sustain photoautotrophic growth. We therefore transferred cuttings from ΔpsbJ plants to sucrose-free synthetic medium. In this way, continued growth was made dependent on net photosynthetic carbon fixation. From our observations with the h" @default.
• W2022337592 created "2016-06-24" @default.
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• W2022337592 date "2002-04-01" @default.
• W2022337592 modified "2023-10-17" @default.
• W2022337592 title "Lack of the Small Plastid-encoded PsbJ Polypeptide Results in a Defective Water-splitting Apparatus of Photosystem II, Reduced Photosystem I Levels, and Hypersensitivity to Light" @default.
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