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(American Journal of Botany. 2004;91:1304-1311.)
© 2004 Botanical Society of America, Inc.

Physiology and Development

Inhibition of peptide deformylase in Nicotiana tabacum leads to decreased D1 protein accumulation, ultimately resulting in a reduction of photosystem II complexes1

Cai-Xia Hou2, Lynnette M. A. Dirk3 and Mark A. Williams2

2Department of Horticulture, Agricultural Science Center North, University of Kentucky, Lexington, Kentucky 40546 USA; 3Plant Science Building, University of Kentucky, Lexington, Kentucky 40546 USA

Received for publication October 8, 2003. Accepted for publication May 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Eukaryotic homologs of bacterial peptide deformylases were recently found in several vascular plants and may be essential in chloroplast protein processing. Treating tobacco seedlings with the peptide deformylase inhibitor actinonin resulted in leaf chlorosis and reduced growth and development, indicative of a systemic movement of the inhibitor. Photosystem II (PSII) activity was reduced, manifested as a significant decrease in the maximum quantum efficiency of photosystem II. Accumulation and assembly of nascent D1 protein into PSII monomers was also reduced, eventually leading to PSII disassembly and leaf necrosis. Processing and assembly of D1 protein in tobacco was a major and potentially critical target of peptide deformylase inhibition. These results confirm that N-terminal deformylation is an essential step in the accumulation and assembly of PSII subunit polypeptides in the chloroplasts of vascular plants.

Key Words: actinonin • D1 protein • peptide deformylase • photosynthesis • PSII complex


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Post-translational protein modifications are critical processes that can regulate and control protein stability and turnover as well as recognition and assembly of multi-subunit complexes and are essential in regulating protein responses to environmental changes (Nalivaeva and Turner, 2001 ; Wang et al., 2002 ). Among the various types of post-translational modifications, nascent N-terminal modifications are one of the more diverse (Nalivaeva and Turner, 2001 ) and are considered to be the prerequisite for other processing events (Kendall et al., 1990 ; Krishna and Wold, 1993 ; Solbiati et al., 1999 ). N-formyl-methionine is the initiating residue of protein translation in the organelles of all eukaryotic organisms examined and in the cytoplasm of prokaryotes, except Archaea (Mazel et al., 1997 ; Yuan et al., 2001 ). Deformylation is required for subsequent excision of the initiating methionine in the vast majority of proteins that do not retain methionine at their N termini; therefore, deformylation is an essential step in protein maturation. Although peptide deformylase was recently demonstrated to target human mitochondria (Lee et al., 2003 ; Nguyen et al., 2003 ; Serero et al., 2003 ), inhibitors of the enzyme have had little toxicity to normal human cell lines (Nguyen et al., 2003 ). Because genetic removal of peptide deformylase is lethal in eubacteria (Mazel et al., 1994 ), peptide deformylase has become an exciting new target for designing novel broad-spectrum antibacterial agents (Chen et al., 2000 ; Nguyen et al., 2003 ). The identification of two chloroplast-targeted peptide deformylases from the Arabidopsis genome (Williams et al., 2000 ) and their subsequent characterization (Giglione et al., 2000 ; Dirk et al., 2001 , 2002 ; Serero et al., 2001 ) have identified plant peptide deformylases as ideal molecular targets for the potential development of a new class of broad-spectrum herbicides (Dirk et al., 2001 ).

Actinonin, a pseudo-peptide hydroxamate derivative, is a highly potent and selective inhibitor of bacterial deformylases (Chen et al., 2000 ). In studies with purified, bacterially expressed, Arabidopsis deformylase, plant deformylases were also extremely sensitive to actinonin inhibition to an extent similar to that observed in bacteria (Dirk et al., 2001 ; Serero et al., 2001 ). Further investigations in vivo demonstrated that actinonin inhibited seed germination (Dirk et al., 2001 ), induced an albino phenotype in germinated seedlings (Dirk et al., 2001 ; Serero et al., 2001 ), and resulted in a stunting and slow bleaching of developing leaves after topical application (Dirk et al., 2001 ). Because N-terminal deformylated proteins in vascular plants are mainly located in the chloroplast (Giglione and Meinnel, 2001 ), these observations support the hypothesis that chloroplast-localized peptide deformylase is indispensable for plant growth and development (Hanson et al., 2000 ; Dirk et al., 2001 ; Serero et al., 2001 ).

Inhibition of peptide deformylase would theoretically result in an accumulation of proteins with N-formylated methionine residues. Although actinonin treatment was recently shown to induce an accumulation of proteins with N-formylated termini in prokaryotes (Solbiati et al., 2002 ; Bandow et al., 2003 ), there has been a lack of direct proof of the consequences of peptide deformylase inhibition in eukaryotic organisms. We previously suggested that inhibition of chloroplast-localized peptide deformylases would compromise co-translational protein processing and thus, potentially, protein function in all plant plastids (Dirk et al., 2001 ). However, because of the low abundance and extreme lability of peptide deformylase, studying the direct functions of peptide deformylases in plants has been difficult. During the preparation of this manuscript, actinonin treatment of Chlamydomonas reinhardtii was reported to result in a rapid degradation of all newly synthesized PSII complex-related proteins (Giglione et al., 2003 ), with the conclusion that chloroplast peptide deformylase is indeed the specific target of actinonin. Here we report on the responses of tobacco seedlings to actinonin treatment. Our data indicate that actinonin treatment results in a rapid decrease of nascent D1 protein accumulation, which ultimately results in PSII complex disassembly, a loss of PSII activity, and leaf death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Tobacco (KY160) and pea (cv. Laxton's Progress) plants were grown in a greenhouse in Metro Mix 280 (Scotts, Marysville, Ohio, USA) for 4–5 wk at 25°/20°C (day/night). Both natural and supplemental lights were used with a photo flux density (PFD) of at least 300 µmol · m–2 · s–1, given in a 17/7 h day/night cycle.

Actinonin treatment of tobacco plants
Young seedlings
Tobacco seeds were sterilized and germinated in Murashige and Skoog (MS) basal salts (Sigma, St. Louis, Missouri, USA) with 1% agar at 22°C with continuous light (50 µmol · m–2 · s–1 PFD) for 7 d. Seedlings were then transferred to the same medium supplemented with 0.4, 0.8, 1.6 or 3.2 mmol/L of actinonin (Sigma) and grown under the same conditions.

Older leaves
The first pair of true leaves of 19–28-d-old tobacco plants was painted daily with 50 µL of 5 mmol/L actinonin in 0.05% (v/v) Tween 20 (Sigma) for 6 d.

In vivo labeling of protein synthesis
Leaf discs (1.9 cm in diameter) from tobacco plants were incubated in petri dishes containing either 1 mmol/L actinonin and 0.05% (v/v) Tween 20 or 0.05% (v/v) Tween 20 only, for various times at a PFD of 100 µmol · m–2 · s–1. Approximately 1 mL of actinonin solution was used per leaf disc. Following the initial incubations, 35S-methionine (1175 Ci/mmol, NEN Life Science Products, Boston, Massachusetts, USA) was added to a final concentration of 7 µCi/mL. Leaf discs were washed with 0.05% (v/v) Tween 20 after a 2-h labeling, and thylakoid membranes were rapidly prepared by homogenizing the tissue in ice cold isolation buffer (330 mmol/L sucrose, 50 mmol/L HEPES buffer, pH 8.0, 10 mmol/L MgCl2, 5 mmol/L EDTA-Na2). After filtration through Miracloth (Calbiochem, La Jolla, California, USA), thylakoids were collected by centrifugation at 6000 x g for 2 min at 4°C, washed in 25 mmol/L Tris-HCl, pH 8.5, 10 mmol/L MgCl2, and finally suspended in storage buffer (10 mM HEPES buffer, pH 7.6, 0.1 mol/L sucrose, 5 mmol/L NaCl, 10 mmol/L MgCl2) (Pursiheimo et al., 2001 ).

Protein fractionation
Both thylakoid and soluble proteins were separated by SDS-PAGE (Laemmli, 1970 ) using 15% acrylamide gels containing 6 mol/L urea. Amounts loaded, as indicated in the figure legends, were based on the amount of chlorophyll (Porra et al., 1989 ) for thylakoid samples and on protein concentration (Bradford, 1976 ) for soluble samples. The separated polypeptides were either stained with Coomassie brilliant blue R250 or transferred to an Immobilon-P membrane (Millipore, Bedford, Massachusetts, USA) and exposed to a phosphor screen, which was scanned with a PhosphorImager SI gel and blot imaging system (Amersham Biosciences, Piscataway, New Jersey, USA). For immunological detection of D1 protein, the dry membrane was incubated with a D1-specific antibody (a kind gift from Dr. Eva-Mari Aro, University of Turku, Finland) and visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

Sample preparation and complex fractionation by blue-native PAGE was according to Thidholm et al. (2002) , with slight modifications. Briefly, thylakoid membranes corresponding to 15 µg of chlorophyll were washed with wash buffer (50 mmol/L Bis-Tris, pH 7.0, 0.33 mmol/L sorbitol) and resuspended in solubilization buffer (50 mmol/L Bis-Tris, pH 7.0, 750 mmol/L {epsilon}-amino-n-caproic acid and 20% (m/v) glycerol). Dodecyl-ß-d-maltoside was then added to a final concentration of 0.8% (v/v). Following a 50-min incubation on ice in the dark, solubilized supernatants were loaded onto a blue-native PAGE gel (4–12% gradient acrylamide) and electrophoresed at 4°C overnight.

Fluorescence measurement
Changes in the maximum quantum efficiency of photosystem II, given by Fv/Fm, (Fv = Fm – Fo; where Fv is variable fluorescence, Fm is maximum fluorescence, and Fo is minimal fluorescence), were monitored in actinonin-treated leaves with the Plant Efficiency Analyser (Hansatech Instruments, Norfolk, England) using a 5-s pulse of 3000 µmol · m–2 · s–1 PFD. Leaves or leaf discs were dark adapted for 30 min prior to measurements.

Statistical analyses
Chlorophyll fluorescence values (Fv/Fm) of leaves and leaf discs were subjected to analysis of variance (ANOVA; SAS 1999–2001 ) for treatment effects at each time point. If the analysis determined significant differences among means, the analysis of variance was rerun and included both Tukey's and Scheffe's mean separation tests ({alpha} = 0.05) for distinguishing among significantly deviating means. Additionally, Dunnett's test (Dunnett, 1964 ; {alpha} = 0.05) was used, within a chemical application treatment, to compare Fv/Fm values of leaf discs prior to high light treatment with those of discs 2 h after recovery from exposure to 0.5, 1, or 1.5 h of high light.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Effects of actinonin on young seedlings
To examine the effects of actinonin on seedling development, tobacco seeds were germinated and the seedlings grown for 7 d (two-cotyledon stage) on Murashige and Skoog medium. Seedlings were then transferred to actinonin-containing medium for an additional 7 d. At actinonin concentrations higher than 0.8 mmol/ L, the newly emerged true leaves had substantial bleaching (Fig. 1), whereas no significant bleaching occurred at or below 0.4 mmol/L. Similar to previous reports with Arabidopsis (Dirk et al., 2001 ; Serero et al., 2001 ), actinonin affected seedling development in a dose-dependent manner.



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  		Fig. 1. Effects of actinonin on tobacco seedling development. Seven-day-old tobacco seedlings germinated on Murashige and Skoog (MS) medium were transferred and grown for 7 d on MS medium with either 0, 0.4, 0.8, 1.6 or 3.2 mmol/L actinonin. Bleaching and stunting of the true leaves is apparent with increasing actinonin concentrations. The bar represents 1 cm

 
Similar actinonin treatments were also applied to older tobacco plants. The first pair of expanding true leaves of 19– 28-d-old greenhouse-grown tobacco plants was treated daily with 50 µL of a 5 mmol/L actinonin solution. Although phenotypes in the leaves painted for 3 d with actinonin did not change significantly (Fig. 2A), the maximal quantum yield of photosystem II (PSII), as monitored by chlorophyll fluorescence (Fv/Fm), had already decreased significantly after a single day of actinonin treatment compared with the control treatments (Fig. 2B). Although the control treatment also had a significant effect on chlorophyll fluorescence for most of the time monitored, the effect was stable over time and less severe than actinonin treatment even after just a single day (Fig. 2B). The entire experiment was replicated, and the data had identical trends (data not shown).



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  		Fig. 2. Effects of actinonin on phenotype (A) and photosystem II (B) of expanding tobacco leaves. The first pair of true leaves in 19–28-d tobacco plants were treated daily for 6 d with 50 µL 5 mmol/L actinonin in 0.05% Tween 20 (Act) or 50 µL Tween 20 (Ctrl); Untr denotes leaves receiving no treatment. (A) Phenotypic changes after 3 d or 6 d of actinonin treatment (top panels). Phenotypic changes in plants grown for three additional days after stopping the 6-d actinonin treatment (bottom panels). Bars represent 1 cm in each photograph; A/Act, painted with actinonin; C/Ctrl, Tween 20. (B) Maximum quantum efficiency of photosystem II measured by chlorophyll fluorescence (Fv/Fm) of the actinonin-treated tobacco leaves. Each point represents the mean (±SE; N = 3) of one representative experiment of two independently conducted. Error bars are smaller than the symbol

 
With further actinonin treatment and time, painted leaves gradually became bleached in a basipetal direction in accordance with tobacco leaf maturation (Fig. 2A). Although the actinonin treatment was confined to the first pair of true leaves, the plants had a pronounced overall stunting, indicating systemic movement of the compound. By day 5, the actinonin-treated leaves almost completely lost PSII activity as indicated by the very low Fv/Fm (Fig. 2B) and eventually died, even though actinonin treatment was stopped (data not shown).

Effects of actinonin on stability of protein complexes in thylakoid membranes
As just described and previously reported (Serero et al., 2001 ), actinonin treatment produced a significant phenotypic alteration in leaves; mainly bleaching, stunting, and eventual leaf necrosis. These observations, coupled with the drastic actinonin-induced reduction in photosystem II activity (Fig. 2B), focused our investigations on the effects of actinonin on the stability and compositional changes in chloroplast protein complexes.

SDS-PAGE profiles of thylakoid membrane proteins from actinonin-treated tobacco leaves had no apparent changes during the 6-d of treatment (Fig. 3A). Blue-native PAGE was then used to investigate the effects of actinonin treatment on the stability of thylakoid membrane protein complexes. This method has been an extremely useful tool to study intact membrane protein complexes by separation according to molecular mass (Schägger et al., 1994 ; Neff and Dencher, 1999 ). The primary protein complexes in tobacco thylakoid membranes were identified by comparing the tobacco protein profiles to those in pea thylakoid membranes, which have been documented (Fig. 3B, left panel; Thidholm et al., 2002 ). The only differences were that tobacco thylakoids have higher and lower amounts of PSII monomer and PSII supercomplexes, respectively, and the PSI complexes of tobacco thylakoids appear to be larger than that of pea thylakoids. After 6 d of actinonin treatment, the PSI complex and LHCII trimer in the thylakoid membrane profile were similar to the control samples, suggesting that actinonin treatment did not influence the stability of these complexes (Fig. 3B, right panel and densitometry). However, PSII monomers and other minor thylakoid membrane complexes gradually disassembled (Fig. 3B, right panel and densitometry), resulting in a severe overall disassembly of the membrane protein complexes in actinonin-treated leaves 3 d after the painting treatment was terminated (data not shown). Western analysis indicated that actinonin treatment resulted in a gradual decrease of total D1 protein in thylakoid membranes (Fig. 3C). Taken together, these data suggest that actinonin treatment induced the disassembly of thylakoid membrane complexes, particularly the PSII monomer complexes.



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  		Fig. 3. The SDS-PAGE profile of thylakoid membrane proteins from tobacco leaves during actinonin treatment. Tobacco leaves were treated daily with actinonin for 6 d, as described in Fig. 2 . Thylakoid membranes from days 3, 4, 5, and 6 actinonin-treated leaves were fractionated. (A) Thylakoid membrane proteins (5 µg chlorophyll/well) stained with Coomassie brilliant blue after separation by SDS-PAGE. (B) Blue-native PAGE profile of the thylakoid membrane protein complexes (15 µg chlorophyll/well). The volume of a band was measured using ImageQuant (Amersham Biosciences) on PhosphorImager gel and blot imaging system and corrected for a representative background. The decrease in protein relative to untreated control after 6 d of actinonin treatment was calculated for each reported band by the following formula: (volumebefore – volumeafter)/volumebefore. Pea: Profile of pea thylakoid protein complexes. Primary complexes are numbered and named on the left side of each panel. (C) Immunoblot of the thylakoid membrane proteins (1 µg chlorophyll/well) using D1 protein-specific antibody and colorimetric development. A plus (+) indicates actinonin treatment and a minus (–) indicates no actinonin treatment

 
Effects of actinonin on photoinhibition recovery
D1 protein synthesis and its co-translational assembly into the PSII complex is the prerequisite for recovery from photoinhibition in plants (Aro et al., 1993 ). We thus treated tobacco leaf discs with high light in the presence or absence of actinonin to investigate the effects of actinonin on the recovery from photoinhibition. Chloramphenicol, a known inhibitor of prokaryotic protein synthesis, was used as a control. The Fv/Fm of tobacco leaf discs pretreated with actinonin for 2 h under low light conditions remained unchanged compared with that of 0.05% (v/v) Tween 20-treated discs, whereas pretreatment with chloramphenicol resulted in a significant reduction in Fv/Fm (Fig. 4; statistically tested with Tukey's and Scheffe's mean separation tests, data not shown). The leaves were then illuminated with high light to induce a 50% photoinhibition of PSII, and the subsequent recovery capability under low light conditions was used to examine the effect of actinonin on this process (Mulo et al., 2003 ). With high light, the Fv/Fm in all treatments was reduced, though the decrease with actinonin was more similar to that of Tween 20 than the chloramphenicol treatment (Fig. 4). The illumination time (t1/2), which represents when Fv/Fm was reduced to 50% of that before high light treatment, was approximately 30 min for the chloramphenicol, 60 min for actinonin, and 90 min for Tween 20 treatments (Fig. 4). In the subsequent recovery process, leaf discs treated with Tween 20 were able to completely recover from 50% photoinhibition after 90 min of high light (statistically tested with Tukey's and Scheffe's mean separation tests, data not shown). The presence of chloramphenicol and actinonin, on the contrary, blocked the leaf discs' complete recovery from 50% photoinhibition after 30 min (for chloramphenicol) and 60 min (for actinonin) of high light (Fig. 4, statistics not shown). The failure in recovery from photoinhibition in the presence of actinonin clearly indicated that the D1 protein synthesis and assembly process was inhibited by actinonin in a manner apparently similar, albeit slower, to that observed with chloramphenicol.



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  		Fig. 4. Comparison of effects of actinonin and chloramphenicol on photoinhibition and PSII recovery after treatment of tobacco with high light. Leaf discs from 35-d-old tobacco plants were pretreated in low light (100 µmol · m–2 · s–1 photo flux density [PFD]) for 2 h with 0.05% (v/v) Tween 20 (squares), 1 mmol/L actinonin in 0.05% (v/v) Tween 20 (triangles), or 1 mmol/L chloramphenicol in 0.05% (v/v) Tween 20 (circles). As indicated by the arrow, samples were then shifted to high light (800 µmol · m–2 · s–1 PFD). After 30, 60, or 90 min, leaf discs were transferred back to low light (100 µmol · m–2 · s–1 PFD) for a 2-h recovery prior to measuring maximum quantum efficiency of photosystem II (Fv/Fm). Each point represents the mean (±SE; N = 3). Error bars are smaller than the size of the symbol

 
Effects of actinonin treatment on D1 protein synthesis and accumulation in vitro and in vivo
We next examined how the effects of actinonin on chloroplast protein synthesis resulted in disassembly of thylakoid membrane complexes and might inhibit recovery from photoinhibition. Although treating isolated intact chloroplasts from pea leaves with 1 mmol/L actinonin did not affect protein synthesis as 1 mmol/L chloramphenicol did (data not shown), the length of treatment (10 min) may have been insufficient to detect an effect of actinonin on protein synthesis. However, pretreatment with actinonin in an in vivo labeling experiment with tobacco leaf discs caused an intriguing change. Figure 5A shows the pattern of labeled thylakoid proteins from leaf discs treated with or without 1 mmol/L actinonin. The D1 protein is the most highly labeled thylakoid membrane protein under such conditions and is therefore a good indicator of perturbations in protein synthesis and accumulation. Incubation of the leaf discs with 1 mmol/L actinonin prior to labeling resulted in a drastic decrease in the incorporation of 35S-methionine into the D1 protein, and this decrease of label incorporation was evident after just 2 h of actinonin pretreatment (Fig. 5A and densitometry). Although other select thylakoid membrane proteins decreased as well (Fig. 5A, densitometry Random 1–3), the effects of actinonin were most pronounced on nascent D1 accumulation. The labeling of soluble proteins—such as the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, which undergoes extensive N-terminal processing (Houtz et al., 1989 )—were largely unaffected by actinonin treatment (Fig. 5B, densitometry RLSU and Random 1–2).



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  		Fig. 5. The effects of actinonin on D1 protein synthesis in leaf discs from tobacco seedlings. Leaf discs from 35-d-old tobacco plants were pretreated with 1 mmol/L actinonin in 0.05% (v/v) Tween 20 (+) or 0.05% (v/v) Tween 20 (–) for 2 or 4 h. 35S-methionine (7 µCi/mL) was then added to initiate a 2-h in vivo labeling of protein synthesis. (A) Phosphorimage of thylakoid membrane proteins (3 µg chlorophyll/well) separated by SDS-PAGE. (B) Phosphorimage of soluble proteins (10 µg protein/well) separated by SDS-PAGE. Label incorporation into D1 (A) and Rubisco large subunit (RLSU; B) and three randomly chosen bands per blot (R1–3) was quantified by the following formula: (volumebefore – volumeafter)/volumebefore. (C) Blue-native PAGE profile of thylakoid membrane protein complexes (left panel, 15 µg chlorophyll/well) and its corresponding phosphorimage (right panel). Complexes are named on the left side and quantified

 
To examine whether the actinonin-induced reduction of nascent D1 accumulation affected PSII complex integrity, thylakoid membranes from actinonin-treated plants were separated by blue-native PAGE. Figure 5C (left panel and densitometry) shows that a 2- or 4-h actinonin pretreatment did not result in a disassembly of the PSII monomer and other membrane complexes during the experiment. Notably, integration of the nascent D1 protein into the PSII monomer was clearly decreased at 4 h pretreatment (Fig. 5C, right panel). The dramatic loss was evident with as little as 2 h pretreatment (Fig. 5C, right panel).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Actinonin-induced bleaching in Arabidopsis (Dirk et al., 2001 ; Serero et al., 2001 ) and pea (Williams et al., 2000 ) leaves provides evidence suggesting that N-terminal deformylation is essential for plant development. Although actinonin produces similar effects in a wide variety of plants, the sensitivity appears to vary among plant species. Tobacco appears to be more resistant to the effects of actinonin compared to Arabidopsis and pea. Directly applying actinonin to young pea leaves eventually resulted in whole plant death (Williams et al., 2000 ), whereas such treatment of tobacco plants only resulted in stunted growth, with necrosis localized to actinonin-treated leaves (Fig. 2A). The developmental stage of the tobacco plant at the time of treatment affected susceptibility to actinonin; inhibition was very pronounced in early developmental stages, such as seed germination and young seedling growth (Fig. 1; Williams et al., 2000 ; Serero et al., 2001 ), yet insignificant in mature plants (data not shown).

The protein targets of peptide deformylase in plant cells are thought to be primarily restricted to about 74 plastid-encoded proteins and only seven putative mitochondrial proteins (Giglione and Meinnel, 2001 ). Most of the plastid-encoded targets are localized in thylakoid membranes and undergo N-deformylation (Giglione and Meinnel, 2001 ; Dirk et al., 2002 ). Blocking chloroplast deformylation by actinonin treatment would thus theoretically have a significant negative impact on chloroplast function. Painting tobacco leaves with actinonin for just 1 d significantly reduced PSII activity (Fv/Fm) in the treated leaves (Fig. 2B), although these leaves were still green. Accumulation of nascent D1 protein drastically decreased after just 2 h of actinonin pretreatment (Fig. 5A and C, right panel), while PSII activity remained unchanged (Fig. 4, triangles). Long-term (6 d) painting treatment caused a decrease in the total amount of D1 protein and a disassembly of PSII complexes (Fig. 3C and B). Interestingly, the accumulation and stability of the Rubisco large subunit was unaffected (Fig. 5B, right panel and densitometry) as has also been reported in Chlamydomonas (Giglione et al., 2003 ). That the effects of actinonin treatment were most pronounced in the D1 protein in tobacco but not in the D2 protein as discovered in Chlamydomonas (Giglione et al., 2003 ), is mainly due to the fact that the translation rate of D1 mRNA in vascular plants is 50– 100 times greater than those of other PSII proteins (Ohad et al., 1984 ). Thus, the effects of actinonin on D2 protein synthesis and accumulation were hard to investigate in our system. Our results are consistent with those from experiments using synthetic peptide substrates mimicking chloroplast proteins in the in vitro peptide deformylase reactions: namely, that D1 protein is one of the preferred substrates of peptide deformylase in vivo (Dirk et al., 2002 ).

In bacteria, peptide N-deformylation occurs as soon as the NH2 terminus of a nascent peptide chain emerges from the ribosome (Housman et al., 1972 ) and is thought to be a prerequisite for subsequent N-terminal modifications such as N-acetylation, N-methylation, and O-phosphorylation. The lethality of E. coli def null mutants has been attributed to the accumulation of essential N-formylated proteins in which the formyl group on the N-terminal residue is thought to inhibit protein folding or function (Mazel et al., 1994 ). Preventing the deformylation of nascent peptides has therefore been assumed to produce inactive chloroplast proteins (Serero et al., 2001 ). The co-translational assembly of nascent D1 peptides requires D2, CP47, and other cofactors as its assembly partners (Kim et al., 1991 ; Zhang et al., 1999 ), and the N-terminal portion of the D1 protein plays a critical role in the recognition and association of D1 with these assembly partners (Mullet et al., 1990 ; Zhang et al., 1999 ; Zhang and Aro, 2002 ). Thus, formyl-group retention could have prohibited D1 assembly as well as blocking its translation. Figure 3 (panel B and densitometry) demonstrated that after 6 d of actinonin treatment the amount of PSII complexes were reduced. Considering that nascent D1 peptides are known to co-translationally assemble with D2, CP47, and other cofactors (Kim et al., 1991 ; Zhang et al., 1999 ), blocking D1 translation and assembly would prevent PSII complex formation. Therefore, the eventual reduction in PS II complexes observed in Fig. 3 panel B could be caused by a disassembly of existing PSII complexes due to an inability to replace continually turned over D1 and a failure to replace these disassembled complexes with newly assembled ones; both of these events are originally initiated by the actinonin-induced retention of N-formyl methionine.

Like many other chloroplast-encoded proteins (Choquet et al., 2001 , 2003 ), the rate of D1 protein translation can be controlled by its assembly into PSII complexes (Zhang et al., 1999 ; Choquet and Vallon, 2000 ). This is likely the reason why both D1 accumulation (Figs. 3C and 5A, densitometry) and assembly (Fig. 5C, right panel, and Fig. 3B) decreased in the presence of actinonin. In Chlamydomonas, the actinonin-induced degradation of newly synthesized PSII proteins, such as D1, is a direct consequence of D2 instability and degradation (Giglione et al., 2003 ). However, in vascular plants nascent D1 proteins have been shown to directly assemble with complexes containing mature D2 and cytochrome b559 proteins (Müller and Eichacker, 1999 ), but not with nascent D2 peptides (Zhang et al., 1999 ). Therefore, the decrease of D1 protein accumulation and assembly in tobacco chloroplast is unlikely to be directly attributable to instability of the N-formylated nascent D2 protein.

Taken together, our data indicates that actinonin induced an inhibition of chloroplast peptide deformylation and likely resulted in retention of formylated termini in nascent PSII subunit proteins that prevented proper assembly into complexes. In vascular plants, D1 protein has a high turnover rate in PSII complexes to fulfill the maximum photosynthesis, and its synthesis, co-translational processing, and assembly are more tightly controlled than D2 and other PSII proteins (van Wijk and Eichacker, 1996 ). It is thus feasible that the inhibition of deformylation by actinonin induced a decrease of D1 protein accumulation via its assembly-mediated regulation, and such inhibition resulted in PSII complex disassembly and a loss of PSII activity, ultimately leading to a drastic reduction in photosynthesis and eventual leaf death.


   FOOTNOTES
 
1 We are grateful to Prof. Robert L. Houtz (R. L. H.) for essential suggestions to our work and critical reading of the manuscript. We are indebted to Dr. Bruce Downie for critical experimental assessment and for assistance with statistical analysis and densitometry. Additionally, anonymous reviewers are thanked for their comments, which have improved this manuscript. Funding for this project was provided by a Kentucky Tobacco Development Research Center Grant (no. 5-41176) to M. A. W., L. M. A. D., and R. L. H. and a National Science Foundation Award (no. MCB-0240165) to L. M. A. D., R. L. H., Anne-Frances Miller and M. A. W. Experiment Station manuscript number: 04-11-014. Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Aro E. M. I. Virgin B. Andersson 1993 Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochimica et Biophysica Acta 1143: 113-134[Medline]

Bandow J. E. D. Becher K. Büttner F. Hochgräfe C. Freiberg H. Brötz M. Hecker 2003 The role of peptide deformylase in protein biosynthesis: a proteomic study. Proteomics 3: 299-306[CrossRef][ISI][Medline]

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