Genetic Manipulation of Isoprene Emissions in Poplar Plants Remodels the Chloroplast Proteome

Genetic Manipulation of Isoprene Emissions in Poplar PlantsRemodels the Chloroplast ProteomeVioleta Velikova,†,‡ Andrea Ghirardo,‡ Elisa Vanzo,‡ Juliane Merl,§ Stefanie M. Hauck,§

and Jorg-Peter Schnitzler*,‡

†Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. Bl. 21, 1113 Sofia, Bulgaria‡Helmholtz Zentrum Munchen, Institute of Biochemical Plant Pathology, Research Unit, Environmental Simulation, IngolstadterLandstr. 1, D-85764 Neuherberg, Germany§Helmholtz Zentrum Munchen, Research Unit Protein Science, Ingolstadter Landstr. 1, D-85764 Neuherberg, Germany

*S Supporting Information

ABSTRACT: Biogenic isoprene (2-methyl-1,3-butadiene) improves theintegrity and functionality of thylakoid membranes and scavenges reactiveoxygen species (ROS) in plant tissue under stress conditions. On the basisof available physiological studies, we hypothesized that the suppression ofisoprene production in the poplar plant by genetic engineering wouldcause changes in the chloroplast protein pattern, which in turn wouldcompensate for changes in chloroplast functionality and overall plantperformance under abiotic stress. To test this hypothesis, we used a stableisotope-coded protein-labeling technique in conjunction with polyacryla-mide gel electrophoresis and liquid chromatography tandem massspectrometry. We analyzed quantitative and qualitative changes in thechloroplast proteome of isoprene-emitting and non isoprene-emittingpoplars. Here we demonstrate that suppression of isoprene synthase byRNA interference resulted in decreased levels of chloroplast proteins involved in photosynthesis and increased levels of histones,ribosomal proteins, and proteins related to metabolism. Overall, our results show that the absence of isoprene triggers arearrangement of the chloroplast protein profile to minimize the negative stress effects resulting from the absence of isoprene.The present data strongly support the idea that isoprene improves/stabilizes thylakoid membrane structure and interferes withthe production of ROS.

KEYWORDS: proteomics, photosynthesis, PSI, PSII, abiotic stress, defense, volatile organic compounds

1. INTRODUCTION

Plant volatile isoprenoids (isoprene, monoterpenes, andsesquiterpenes) have developed various functions in plants viaevolution. These compounds play critical roles in plantcommunication with herbivores1 and in plant defensemechanisms against biotic and abiotic stresses2 as well asmodulate stress-induced signaling molecules.3,4 Isopreneprotects photosynthetic processes against oxidative stresseselicited by high temperatures,5−8 ozone,9−11 and drought.12,13

Although the impact of volatile isoprenoids on abiotic stressprotection has been clearly demonstrated, the biophysical andbiochemical mechanisms underlying the induced protection arestill unclear.In the past decade, attempts have been made to analyze the

functionality of isoprene using molecular approaches. The roleof isoprene in plant protection against thermal and oxidativestresses has been studied using transgenic approaches either bythe knock-down of the natural isoprene emission in grey poplar(Populus × canescens)8 or by the introduction of this trait inArabidopsis thaliana14,15 and Nicotiana tabacum,16 two naturallynon isoprene-emitting species. Using both approaches, a

positive effect of isoprene on plant stress resistance wasdocumented.2 Detailed metabolomic analysis of transgenicpoplars under various stress conditions, for example, ozone,11

high temperature,17 and atmospheric CO2 levels,18 revealedmetabolome-wide rearrangements of cellular metabolismbetween isoprene-emitting and isoprene-suppressed poplars.However, these changes were also partially present undercontrol conditions. Moreover, recent evidence has demon-strated that isoprene improves the integrity and functionality ofthylakoid membranes under optimal conditions in transgenicisoprene-emitting Arabidopsis19 and poplar.20 Overall, thecompensatory multiple stress tolerance mechanisms inisoprene-suppressed poplar leaves most likely include aremodeling of the proteome of photosynthetically active cells,which in turn may influence plant functionality.The present study focuses on the chloroplast proteome of

poplar because isoprene biosynthesis proceeds through thechloroplastic 2-C-methyl-D-erythritol-4-phosphate (MEP) path-

Received: November 17, 2013Published: January 22, 2014

Article

pubs.acs.org/jpr

© 2014 American Chemical Society 2005 dx.doi.org/10.1021/pr401124z | J. Proteome Res. 2014, 13, 2005−2018

pubs.acs.org/jpr

way, supplying the substrate dimethylallyl diphosphate(DMADP) to isoprene synthase (ISPS, EC 4.2.3.27).21

Chloroplasts are specialized organelles harboring the photo-synthetic apparatus essential for the production of bioenergyand CO2 fixation and serve as metabolic hubs for variousprimary and secondary biosynthetic pathways essential for plantgrowth, development, and defense.Chloroplasts have their own genome, containing ∼120 genes.

These genes encode RNAs and proteins involved in geneexpression as well as a variety of proteins that function inphotosynthesis.22 Although the chloroplasts translate their ownproteins, nuclear genes encode ∼90% of chloroplast proteins.These proteins are synthesized on cytosolic ribosomes and arethen imported into the chloroplast as complete polypeptidechains via translocon complexes,23 followed by processing,folding, and assembly by various chaperone systems.24−26 Thechloroplast is surrounded by a double-membrane called theinner and outer envelope membrane. In addition, a thirdmembrane system is present inside the chloroplast, known asthe thylakoid membrane, which forms flattened discs or stackscalled thylakoid and grana, respectively. On thylakoidmembranes, multisubunit protein complexes (photosystems Iand II, “PSI” and “PSII”, the ATP synthase complex, and thecytochrome b6 f complex) are located.27 The thylakoidmembrane is the location where solar energy is collected,converted, and stored in the form of chemical compounds(ATP and NADPH).27 Although the chloroplasts are bestknown for their role in photosynthesis, they also host otheressential metabolic processes, such as the synthesis of lipids,pigments, phenylquinones, aromatic amino acids, vitamins,secondary metabolites such as isoprenoids and alkaloids, starch,a wide set of signaling molecules, plant hormone precursors,and others.28

Chloroplast studies are a classical field in plant physiologyand ultrastructural biology; however, the knowledge regardingthe chloroplast proteome is still scarce. To understandmetabolic processes in the chloroplasts, more informationregarding selected proteins and their functions is required.Advances in proteomic techniques in combination withincreasing genomic and transcriptomic information haveenabled studies on chloroplast proteins, providing additionalinformation regarding their functional compartmentalizationand characterization.29,30

The present work is the first study specifically addressingchanges in the chloroplast protein profile of plants altered intheir isoprene emission capability. The chloroplast proteome inisoprene-emitting (IE: wild type and empty vector control,WT/EV) and non isoprene-emitting (NE: RA1/RA2) poplarlines was characterized. We applied an approach forquantitative and qualitative proteomics based on stableisotope-coded protein labeling (ICPL) in combination withpolyacrylamide gel electrophoresis (PAGE) and liquidchromatography tandem mass spectrometry (LC−MS/MS).31

We aimed to understand whether the absence of isopreneproduction (i) triggers overall changes in the chloroplastproteome and (ii) affects the composition of protein complexesof the photosynthetic electron transport chain and alters theenzyme abundance of chloroplast biosynthetic pathways and(iii) how non isoprene-emitting poplars adjust their structuralproteome.

2. MATERIAL AND METHODS

Plant Materials and Growth Conditions

In the present study, two NE PcISPS-RNA interference (RNAi)transgenic lines (RA1 and RA2) were compared with IE WTand the control of transgenic manipulation EV of grey poplars(Populus × canescens; syn. Populus tremula × P. alba).8,11,17,18,20

The RA1 and RA2 lines correspond to independentlytransformed lines emitting only 0.5 to 2% of isoprene comparedwith the emission capacity of WT/EV.8,20 The EV line wasincluded in the experiments to ensure that differences in thechloroplast proteome between NE and IE were due to specificalteration of the PcISPS gene and not to a more general geneticmanipulation effect. The plants were grown in a greenhousewith ambient day/night temperature of 25/20 °C, relativehumidity of 60/50%, and 16 h day length. When the lightintensity was <700 μmol m−2 s−1 of photosynthetically activeradiation (PAR), supplemental light was provided by high-pressure sodium lamps (Philips SON T-AGRO 400W). Theplants were planted into 2.2 L pots with soil substrate [25% v/vFruhstorfer Einheitserde (Bayerische Gartnereigenossenschaft,Aschheim, Gemany), 25% v/v silica sand (particle size 1 to 3mm), and 50% v/v perlite (Agriperl Dammstoff, Dortmund,Germany] and fertilizer [Triabon (Compo, Munster, Germany)and Osmocote (Scotts Miracle-Gro, Marysville, OH) (1:1 v/v;10 g per liter of soil)]. Four month old plants were used for theexperiments.

Chloroplast Isolation

Fully expanded leaves (9th and 10th nodes from the apicalmeristem) from four to five plants were collected. Approx-imately 10 g of finely chopped leaf material was homogenizedin 40 mL of semifrozen isolation medium (350 mM sorbitol, 50mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 2 mM ascorbate,0.1% BSA, 1 mM EDTA) for 3 × 10 s pulses in a mechanicalblender. The homogenate was filtered through four layers ofMiracloth (Calbiochem, Darmstadt, Germany). The filtrate wascentrifuged for 3 min at 4 °C and 4000g, and the pellet (“crudechloroplasts”) was resuspended in grinding medium (20 mL)and resedimented for 3 min at 4 °C and 4000g (includingacceleration time) and again resuspended. The suspension wasloaded on top of sucrose gradients (4 to 10 mL per gradient 40and 80% sucrose) and centrifuged at 10 000g at 4 °C for 10min. After centrifugation, the chloroplast layer at the interfaceof the two gradients was collected, washed with 40 mL ofresuspension medium (50 mM HEPES-KOH, pH 7.5, 1 mMMgCl2, 2 mM EDTA), and centrifuged for 10 min at 5000g at 4°C. The chloroplast pellet was resuspended in a final volume of1 to 2 mL. This chloroplast fraction was used to perform theICPL reaction and PAGE for subsequent label-free compar-isons.The protein content of the chloroplast suspension was

measured according to Bradford,32 and the chlorophyll contentaccording to Lichtenthaler and Wellburn.33 Before measuringthe protein level in the samples, the chlorophyll was removedby three times washing procedure with methanol. On average,5.0 ± 0.08 μg protein (n = 9) corresponded to 10 μg ofchlorophyll. For normalization of protein loading on the gel, aprotein equivalent corresponding to 10 μg chlorophyll wasapplied.

Journal of Proteome Research Article

dx.doi.org/10.1021/pr401124z | J. Proteome Res. 2014, 13, 2005−20182006

SDS-PAGE and Sample Preparation for Label-FreeComparative Analysis

The chloroplast proteins were separated on a SDS-PAGEgradient (4−16%) gel. Prior to SDS-PAGE, the chloroplastproteins (10 μg chlorophyll) were solubilized in a sample buffer(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerin and 5%mercaptoethanol) by incubation for 30 min at room temper-ature in the dark. The electrophoresis was performed in thedark at 4 °C and constant voltage (100 V) for 4.5 to 5 h. Afterelectrophoresis, the gel was fixed in 15% trichloracetic acid for

10 min and stained with Coomassie Brilliant Blue R-250. The

respective gel-bands at ∼50 and ∼11−13 kDa were excised in

all lanes (three replicates of IE and NE, respectively) and

subjected to in-gel digestion using trypsin as previously

described.34 The supernatants containing the eluted tryptic

peptides were dried in a speedvac (UniEquip Laboratory

Instruments, Planegg, Germany) and stored at −20 °C prior to

LC−MS/MS analysis.

Table 1. (A) Chloroplast Proteins Identified in Bands ((A) ∼50 kDa and (B) ∼11−13 kDa) Excised from 1-DE Gel. Log2Ratios Are Given to Show Different Expression Level of Extracted Proteinsa

accession A B C Log 2 description function TargetP localization

(A)

Proteins Related to PhotosynthesisPOPTR_0005s13860.1 2 67 0.02 2.5 PsbO - manganese-stabilizing protein OEC C THYPOPTR_0001s21740.1 3 161 0.02 3.1 Chl a/b binging protein light harvesting C THYPOPTR_0019s09140.1 7 325 0.01 2.8 Chl a/b binging protein light harvesting C THYPOPTR_0010s12680.1 11 533 0.01 3.7 ATP synthase proton-transporting ATP C THYPOPTR_0008s12550.1MetabolismPOPTR_0008s00350.4 4 630 0.03 7.2 serine hydroxymethyl transferase L-serine metabolism CPOPTR_0002s10990.1POPTR_0008s00350.2POPTR_0008s00350.3POPTR_0005s11500.1 2 99 0.01 2.7 oxidoreductase NAD-binding domain oxidoreductase activity C STR-THYPOPTR_0014s09220.1 2 99 0.02 3.7 hydroxyproline-rich glycoprotein hydrolyzation C THYPOPTR_0008s08410.1 2 340 0.01 3.7 phosphoglycerate kinase glycolysis C STR-THYPOPTR_0008s08400.1POPTR_0004s16920.1 2 119 0.02 2.0 fructose-bisphosphate aldolase glycolysis C STRPOPTR_0007s13800.1POPTR_0009s04180.1 6 425 0.02 −2.7 NmrA-like family nitrogen metabolism C THY-ENVPOPTR_0014s15390.1 14 882 0.01 −2.1 hydroperoxide lyase, Cit P450 electron carrier activityStress-Related ProteinsPOPTR_0002s01080.1 6 275 0.01 5.2 catalase response to oxidative stress peroxisomePOPTR_0005s10340.1POPTR_0005s27300.1POPTR_0007s08910.1POPTR_0018s11820.1 4 167 0.01 6.8 aldehyde dehydrogenase family oxidoreductase activityOthersPOPTR_0001s08770.1 2 83 0.03 2.4 elongation factor Tu GTP binding domain GTPase activity C STR-THYNot IdentifiedPOPTR_2555s00200.1 5 356 0.01 4.8 not identified

(B)

Ribosomal ProteinsPOPTR_0004s16450.1 2 71 0.02 7.4 S25 ribosomal protein structural constituent of ribosome CProteins Related to PhotosynthesisPOPTR_0007s04160.1 2 425 0.01 2.1 PSI reaction center PSI C THYPOPTR_0004s09910.1 2 141 0.05 1.9 RuBisCO small subunit RuBisCO activity C ENV-STRPOPTR_0002s18110.1 3 104 0.02 2.7 TM phosphoprotein no functional annotations CPOPTR_0001s25670.1 2 99 0.01 2.5 thylakoid phosphoprotein no functional annotations C THYPOPTR_0009s04820.1Proteins with Transporting and Binding FunctionsPOPTR_0008s02430.1 2 84 0.002 4.6 copper chaperone metal ion transport and bindingPOPTR_0002s01740.1 3 171 0.005 −4.1 copper binding proteins electron carrier activity C THYNot IdentifiedPOPTR_0001s42970.1 7 446 0.01 2.9 not identified

aDown- (negative numbers) or up-regulated (positive numbers) proteins were counted when log 2 = Σ(NE)/Σ(IE) was <−1 or >1, respectively, inNE grey poplar plants (RA1/RA2). A, Peptides used for quantitition; B, confidence score; C, Anova (p). The information about sub-cellular or sub-plastidial localization is extracted from http://www.grenoble.prabi.fr/at_chloro; THY, localization in the thylakoids; STR, localization in the stroma;ENV, localization in the envelope.



http://www.grenoble.prabi.fr/at_chloro

LC−MS/MS Analysis

Dried peptides were resuspended in 2% acetonitrile/0.1%trifluoric acid. The samples were centrifuged 1000g at 4 °C for5 min. LC−MS/MS analysis was performed as previouslydescribed by using an Ultimate 3000 nano-HPLC (Dionex,Thermo Scientific, Bremen, Germany).34,35 In brief, everysample was automatically injected and loaded onto the trapcolumn. After 5 min, the peptides were eluted and separated onthe analytical column (75 μm i.d. × 15 cm, AcclaimPepMap100 C18, 3 μm, 100 Å Dionex, Idstein, Germany) byan acetonitrile gradient at 300 nL min−1 flow rate (60 mingradients for the label-free analysis, 170 min gradients for theICPL analysis). From the MS prescan, the 10 most abundantpeptide ions were fragmented by collision-induced dissociationin the linear ion trap if they showed an intensity of at least 200counts and if they were at least +2 charged. Duringfragmentation, a high-resolution (60 000 full width at half-maximum) MS spectrum was acquired in the LTQ OrbitrapXL(Thermo Scientific, Bremen, Germany) with a mass range from300 to 1500 Da and a dynamic exclusion of 30 or 60 s for thelabel-free and the ICPL analysis, respectively.Label-Free Analysis

The acquired spectra for the two label-free data sets (11−13and 50 kDa gel slices) were loaded into the Progenesis LC−MSsoftware (version 2.5, nonlinear) for label-free quantificationand were analyzed as previously described.34,35 Features withonly one charge or more than eight charges were excluded fromdata analysis. Raw abundances of the remaining features werenormalized to allow correction for factors resulting fromexperimental variation. All MS/MS spectra were exported as aMascot generic file (mgf) and used for peptide identificationwith MASCOT (version 2.3.02) in the Populus trichocarpaprotein database (version 4, 1 7236 452 residues, 45 036sequences). The search parameters included 10 ppm peptidemass and 0.6 Da MS/MS tolerance, one missed cleavage wasallowed, cysteine carbamidomethylation was set as the fixedmodification, and methionine oxidation and asparagine orglutamine deamidation were allowed as variable modifications.Using a MASCOT ion score cutoff of 30 and an appropriatesignificance threshold p, a MASCOT-integrated decoy databasesearch calculated a false discovery rate (FDR) of <1%. For eachdata set, the peptide assignments were reimported into theProgenesis LC−MS software. After summing the abundances ofall peptides allocated to each protein, the identification andquantification results were exported and are shown in Table1A,B. Log 2 ratios of [Σ(NE)/Σ(IE)] from protein abundanceswere calculated and are reported in Table 1A,B.Isotope-Coded Protein Labeling and Analyses

Stable isotope labeling of chloroplast proteins isolated from IE(WT, EV) and NE (RA1, RA2) was performed using the ICPLDuplex- and Quadruplex-Kits (SERVA Electrophoresis, Heidel-berg, Germany) following the manufacturer’s instructions. Forthe quatruplex analysis with two biological replicates, 50 μg ofthe isotope-labeled proteins from the four different samples(ICPL-0 = WT, ICPL-4 = EV, ICPL-6 = RA1, ICPL-10 = RA2)was combined, and the proteins were precipitated using ice-cold 80% acetone. The same was repeated for two biologicalreplicates with duplex analysis paring WT/RA1 and EV/RA2.The resulting 200 μg of protein per biological replicate wasseparated by 1D SDS-PAGE. After protein staining withCoomassie Brilliant Blue, each lane was cut into five slices andsubjected to in-gel digestion with trypsin (Sigma Aldrich), as

previously described.34 LC−MS/MS analysis was performed aspreviously described.The acquired MS/MS spectra were searched against the

Populus trichocarpa database (Version: 2.3, 45 036 sequences)using the Mascot search engine (version 2.3.02; MatrixScience) with the following parameters: a precursor masserror tolerance of 10 ppm and a fragment tolerance of 0.6 D.One missed cleavage was allowed. The complete list of proteindescriptions, protein group accessions, sequence of peptides,quan usage, ion score, number of missed cleavages, and themolecular mass of the MH+ ion is given in the SupplementalTable S3 in the Supporting Information. Carbamidomethyla-tion was set as the fixed modification. ICPL-0, ICPL-4, ICPL-6,and ICPL-10 for the lysine residues of the peptides were set asvariable modifications. Data processing for the identificationand quantitation of the ICPL-duplex- and quadruplex-labeledproteins was performed using Proteome Discoverer version1.3.0.339 (Thermo Scientific). The Mascot Percolator algo-rithm was used for the discrimination between correct andincorrect spectrum identifications36 with a maximum q value of0.01. Proteins were further filtered using the followingparameters: high peptide confidence and at least two peptidesper protein (count only rank one peptides and count peptideonly in top scored proteins). Peptide lists were exported, andthe abundances of all unique peptides allocated to onerespective protein were summed to determine the intensitiesof the individual and differentially labeled proteins for thebiological replicates. Protein lists were also exported, containingthe ICPL ratios for the individual proteins in the differentsamples.

Blue Native PAGE (BN-PAGE)

BN-PAGE is an excellent tool to analyze proteins and proteincomplexes in their native form. Thylakoid isolation wasperformed under dim light at 4 °C following the protocol ofJarvi et al.37 modified for poplar leaf material. Thylakoids wereisolated from fresh leaves ground in ice-cold grinding buffer(350 mM sorbitol; 50 mM HEPES/KOH, pH 7.5; 5 mMMgCl2; 2 mM ascorbate; 1 mM EDTA and 0.1% BSA). Thesuspension was filtered through two layers of Miracloth,followed by centrifugation at 5000g at 4 °C for 5 min. Thepellet was resuspended in buffer (50 mM HEPES/KOH, pH7.5; 1 mM MgCl2 and 2 mM EDTA), followed bycentrifugation at 5000g at 4 °C for 5 min. The pellet wasresuspended in a small aliquot of storage buffer (50 mMHEPES/KOH, pH 7.5; 100 mM Sorbitol; and 10 mM MgCl2).The thylakoid membranes (10 μg chlorophyll) were

resuspended in ice-cold sample buffer (1 M 6-aminocapronicacid; 100 mM BisTris/HCl, pH 7.0; 100 mM NaCl, 20%glycerol, 0.1% SERVA Blue G) (Serva Electrophoresis) with0.25 mg mL−1 Pefabloc (Sigma-Aldrich, Deisenhofen, Ger-many). Prior to native-PAGE, the thylakoid membranes weresolubilized in 1.5% dodecyl maltoside (Invitrogen, Darmstadt,Germany) in the dark for 5 min on ice. The BN-PAGE sampleswere supplemented with a one-tenth volume of Serva Blue Gbuffer (Serva Electrophoresis, Heidelberg, Germany). Thyla-koid membrane proteins were separated on 4−16% Bis-Tris gel(Novex by LifeTechnologies, Darmstadt, Germany). Electro-phoresis was performed at 0 °C with a gradual increase in thevoltage as follow: 75 V for 30 min, 100 V for 30 min, 125 V for30 min, and 150 V for 60 min (total running time 2:30 h).Anode and cathode buffer were commercially obtained fromServa Electrophoresis. After electrophoresis, the gel was fixed in



40% methanol and 10% acetic acid solution, followed by adestaining procedure in 8% acetic acid solution. ImageJ v.1.47was utilized to quantify the bands.

Acid-Urea-PAGE of Histones

The chloroplast suspension was used to analyze histones byacetic-acid−urea−polyacrylamide gel electrophoresis followingthe protocol.38 Histone proteins were separated using 15%TBE-Urea gels (Novex by LifeTechnologies, Darmstadt,Germany). Prior to separation, the samples were solubilizedin a TBE-Urea sample buffer (Novex by LifeTechnologies) for5 min at room temperature. Acetic acid (5%) was used as arunning buffer. Electrophoresis was performed at roomtemperature at a constant voltage (300 V) for 4:30 h. Proteinswere visualized with silver stain.39 For quantification of thebands, ImageJ v.1.47 was utilized.

Statistics

Proteomic differences of ICPL chloroplast samples wereanalyzed using a multivariate data analysis approach withPrincipal Component Analysis (PCA) and Orthogonal PartialLeast Square regression (OPLS) statistical methods from thesoftware packages ‘SIMCA-P’ (v. 13.0.0.0, Umetrics, Umea,Sweden). The results were validated by “full cross valida-tion”40,41 using a 95% confidence level. PCA was performed ona 119-by-12 matrix of ICPL data, that is, using as X variables(centered and scaled with 1 s.d.−1) the summed peptideintensities (normalized per protein content) of the 119 proteinsfrom the 12 samples (four lines: WT, EV, RA1, RA2; n = 3biological replicates for each line). OPLS was used to selectdiscriminant proteins that significantly distinguish NE from IEsamples. OPLS was calculated by defining as Y variable theability of the samples to emit isoprene, with NE = 0 and IE = 1.The regression model was tested for significance by CV-ANOVA.41 Proteins showing Variable of Importance for theProjection (VIP) greater than 1 and uncertainty bars of jack-knifing method42 smaller than the respective VIP value weredefined as discriminant proteins.Proteins in the NE lines were classified down- or up-

regulated proteins when log 2 of [Σ(NE)/Σ(IE)] MS peak sumwas <−1 or >+1, respectively, otherwise unchanged.

3. RESULTS AND DISCUSSION

1D-PAGE Shows Differences in the Chloroplast ProteinProfiles of NE and IE Poplars

The comparative study of chloroplast proteins using one-dimensional gel electrophoresis (1-DE) and subsequent LC−MS/MS and label-free proteome analysis showed obviousdifferences in protein abundance in chloroplast extracts fromtwo NE (RA1/RA2) and two IE (WT/EV) lines (Figure 1).The analysis was conducted in duplicate using independentchloroplast isolations and including a technical replicate,resulting in 3 1-DE gels. The presence of the two bands at21 and 14 kDa (Figure 1) are indicative for chloroplast integrityduring isolation. It was demonstrated that these bandsdisappear from the fraction when chloroplasts are broken.43

Prominent bands present in extracts of NE plants at ∼50 and∼11−13 kDa were visually absent in the IE chloroplast extracts(Figure 1, red arrows). We excised and subjected these bandsto LC−MS/MS and label-free quantitative analysis. Theannotation of the peptides isolated from the 50 kDa proteinband of the NE and IE extracts led to the identification of 15proteins in total (Table 1). The most abundant protein

identified in the 50 kDa band of NE extracts compared with theIE was serine hydroxymethyl transferase (SHMT, EC 2.1.2.1,log 2 = 7.2). Significant (p = 0.004, t test) up-regulation ofSHMT was confirmed later using ICPL analysis (Figure 3,Table 2, Supplemental Figure S1 in the SupportingInformation). SHMT was also observed previously inchloroplasts preparations from poplar.44 However, plantspossess SHMT isoforms in the cytoplasm and mitochondria.45

Cytosolic and mitochondrial SHMTs play a primary role infolate-dependent pathways of C1 metabolism.46 They catalyzethe reversible, simultaneous conversions of L-serine to glycineand tetrahydrofolate to 5,10-methylenetetrahydrofolate.47,48

There is an experimental evidence that the chloroplasticisoform of SHMT is an important enzyme involved in thephotorespiratory pathway.49 Photorespiration is a well-knownprocess mitigating photooxidative damage by functioning as anelectron sink to prevent the over-reduction of the photo-synthetic electron transport chain in the thylakoid membranesas well as photoinhibition of PSII.50 This process is initiated inthe chloroplasts by the oxygenase activity of ribulose-1,5-bisphosphate-carboxylase/-oxygenase (RuBisCO, EC 4.1.1.39).Photorespiration generates various molecules such as H2O2,glycine, and serine that further increase the dissipatory effect.51

Together with SHMT, aldehyde dehydrogenase (ALDH, EC1.2.1.3) was another highly abundant protein in NE lines (log 2= 6.8). Previous transcriptomic analyses in the same poplarlines showed that ALDH transcript levels were lower inunstressed NE (log 2 = −1.34), indicating that the transcriptlevels and protein abundance were not strictly correlated.17

ALDHs belong to a family of NAD(P)+-dependent enzymesthat play major roles in the detoxification of aldehydesgenerated in plant cells exposed to abiotic stress. Arabidopsisplants overexpressing ALDH3 show improved tolerance tooxidative stress (caused by excess of NaCl, heavy metals, methylviologen, and H2O2) compared with WT plants. Stresstolerance in these transgenic plants was accompanied by areduction of H2O2 and malondialdehyde derived from cellularlipid peroxidation.52,53 We also found catalase (EC 1.11.1.6),

Figure 1. Representative Coomassie blue-stained polyacrylamide geldisplaying the protein pattern in chloroplasts isolated from IE (WT,EV) lines and two NE lines (RA1, RA2) of grey poplar plants.Chloroplast protein patterns were different between the IE and NEgrey poplar plants. Red arrows indicate the excised bands that werefurther subjected to LC−MS/MS and label-free analyses.



Table2.

VariableIm

portance

fortheProjection(V

IP)forEachDiscrim

inantProtein

ThatSignificantlySeparatesNon

isop

rene-Emitting

(NE)from

Isop

rene-Emitting

(IE)

Populus

canescensSamples

intheOPLS

Mod

el(p

=0.021;

CV-ANOVA)a

accession

VIP

SEdescrip

tion

functio

nLo

g2

TargetP

curatedlocalization(PPD

B)

Histone

Proteins

POPT

R_0

008s13170.1

1.926

0.640

linkerhistoneH1andH5family

DNAsynthesis

3.6**

POPT

R_0

010s23720.1

1.924

0.618

histoneH2B

DNAsynthesis

3.5**

POPT

R_0

006s08230.1

1.829

0.732

histone2A

DNAsynthesis

2.8**

POPT

R_0

015s04390.1

1.472

0.901


DNAsynthesis

5.5*

CPO

PTR_0

011s13490.1

1.349

0.895

histone2A

DNAsynthesis

1.7*

CPO

PTR_0

012s04580.1

1.303

0.917


DNAsynthesis

2.2*

CRibosom

alProteins

POPT

R_0

018s03390.1

1.629

0.842

zinc

knuckle

nucleicacid

binding

4.6**

Cplastid

ribosom

ePO

PTR_0

018s11170.1

1.552

0.886

ribosom

alproteinL1

7proteinbiosynthesis

2.3*

Cplastid

ribosom

ePO

PTR_0

006s23770.1

1.484

0.840

50Srib

osom

alproteinL4

/L1family

proteinbiosynthesis

2.7*

Cplastid

ribosom

ePO

PTR_0

002s04420.1

1.461

1.010

ribosom

alproteinS19

proteinbiosynthesis

12.8*

Cplastid

ribosom

ePO

PTR_0

001s33710.1

1.435

0.998

ribosom

alL2

7protein

proteinbiosynthesis

3.6*

Cplastid

ribosom

ePO

PTR_0

006s11420.1

1.368

0.926

ribosom

alproteinL1

7proteinbiosynthesis

3.5ns

Cplastid

ribosom

ePO

PTR_0

004s22660.1

1.275

1.066

50Srib

osom

alproteinL9

proteinbiosynthesis

1.6ns

Cplastid

ribosom

ePO

PTR_0

007s14040.1

1.201

1.072

ribosom

alproteins

L2proteinbiosynthesis

11.2ns

Cplastid

ribosom

ePO

PTR_0

016s08250.1

1.120

1.118

zinc

knuckle

nucleicacid

binding

12.7ns

Cplastid

ribosom

ePO

PTR_0

014s15120.1

1.114

0.971

60Srib

osom

alproteinL6

proteinbiosynthesis

3.0ns

Cplastid

ribosom

eProteins

with

StructuralActivity

POPT

R_0

001s12230.1

1.217

1.013

FKBP-type

proteinfolding

−1.5*

Cthylakoid-periferal-lumenal-side

POPT

R_0

019s14050.1

1.183

0.846

thylakoidform

ationprotein

TM

organizatio

nandbiogenesis

−1.5*

Cenvelop;

plastid

stroma;thylakoid-periferal-strom

al-side

POPT

R_0

001s02040.1

1.156

0.867

PAPfibrillin

structuralmoleculeactivity

−0.8ns

Cplastoglobules;thylakoid-periferal-strom

al-side

POPT

R_0

001s41530.1

1.154

1.118

cyclophilin

type

proteinfolding

−1.7ns


Proteins

Related

toPh

otosynthesis

POPT

R_0

002s22560.1

1.679

0.841

ATPase

family

ATPase

activity

−2.9*

Cthylakoid-integral

POPT

R_0

001s44210.1

1.660

1.106

PsbQ

-oxygenevolving

enhancer

protein3

OEC

−0.9**


POPT

R_0

004s01470.1

1.604

0.402

ATPsynthase

proton-transportingATPsynthase

complex

−1.8*

Cthylakoid-periferal-strom

al-side

POPT

R_0

002s05660.1

1.331

1.015

PsbP

OEC

−2.4*


POPT

R_0

003s14870.1

1.300

1.042

PSIreactio

ncenter

subunitIII

PSI

−1.7ns

Cthylakoid-integral

POPT

R_0

013s14520.1

1.270

1.002

Cyt

b 6-F

complex

Fe−Ssubunit

oxidoreductase

activity

−2.4*


POPT

R_0

014s13560.1

1.249

1.246

ATPase

family

-with

vario

uscellularactivities

ATPase

activity

-4.9

nsC

thylakoid-integral

POPT

R_0

019s11720.1

1.197

0.848

ATPsynthase

delta

(OSC

P)subunit

proton-transportingATPsynthase

complex

−0.7**

Cthylakoid-periferal-strom

al-side

POPT

R_0

004s03160.1

1.187

1.066

PsbQ

-oxygen

evolving

enhancer

protein3

OEC

−1.7*


Metabolism

POPT

R_0

008s00350.4

1.888

0.957

serin

ehydroxym

ethyltransferas

L-serin

emetabolism

7.7**

Mmito

chondria

POPT

R_0

002s10990.1

1.615

1.082

serin

ehydroxym

ethyltransferas

L-serin

emetabolism

7.4*

POPT

R_0

001s25160.1

1.260

1.009

NmrA-like

family

nitrogen

metabolism

−4.4ns

Cthylakoid

POPT

R_0

001s34960.1

1.210

1.033

trypsin,

serin

eprotease

proteolysisandpeptidolysis

−1.7*

Cthylakoid-perip

heral-lum

enal-side

Stress-Related

Proteins

POPT

R_0

006s13980.1

1.238

0.897

peroxiredoxin,

AhpC/T

SAfamily

antio

xidant

activity

−0.8**

Cplastid

POPT

R_0

005s17350.1

1.477

0.652

L-ascorbateperoxidase

response

tooxidativestress

−2.4*

Cthylakoid-perip

heral-lum

enal-side



one of the main H2O2-metabolizing enzymes,54 at a higherabundance in chloroplast extracts of NE compared with IE (log2 = 5.2, Table 1). Our findings that NE extracts have anenhanced abundance of proteins related to antioxidative stressmechanisms are consistent with observed metabolic differencesbetween NE and IE poplars under ambient CO2 concen-trations.18

The up-regulation of a protein of the NAD/FAD-oxidoreductase protein family (EC 2.4.1.2 and EC 2.4.1.3; log2 = 2.7, Table 1) may affect the cyclic and noncyclic electronflow in NE chloroplasts. This finding suggests that thesechloroplasts may have higher demands for ATP and reducingpower compared with IE.55

The suppression of isoprene biosynthesis in the poplar alsoaffected proteins related to the light reactions in photosynthesis(PsbO, LHC, ATP synthase (EC 3.6.3.15)) and primarymetabolic processes such as hydroxyproline-rich glycoprotein,phosphoglycerate kinase (EC 2.7.2.3), and fructose-bisphos-phate aldolase (EC 4.1.2.13). These proteins were moreabundant in NE chloroplasts compared with IE chloroplasts(Table 1).Interestingly, in the excised 50 kDa band, two proteins had

lower abundance in the NE plants compared with the IE plants(Table 1). These two proteins are the hydroperoxide lyase (EC4.1.2.-, log 2 = −2.1) and a protein belonging to the NmrA-likefamily (EC 1.3.1.-., log 2 = −2.7). Hydroperoxide lyases(HPLs) are members of the cytochrome P450 family andcatalyze the cleavage of fatty acid hydroperoxides to aldehydesand oxoacids.56 HPLs mediate the formation of green leafvolatiles (GLVs syn. Lipoxygenase (LOX) products) throughoxylipin metabolism.56 Data from HPL-depleted transgenicpotato lines strongly suggest that the constitutive activity of thisbranch of the oxylipin biosynthetic pathway influences plantdefense processes.57 NmrA acts as a negative transcriptionalregulator involved in N metabolism and is a member of theshort-chain dehydrogenase reductase superfamily (EC1.1.1.300). In addition, NmrA has the ability to discriminatebetween the oxidized and reduced forms of dinucleotides, afeature that is linked to a possible role in redox sensing,58 afunction that may also be of importance in poplars.The proteomic analysis of the second band (∼11−13 kDa),

visually differing in abundance between NE and IE, ischaracterized by an enrichment of ribosomal proteins in NE(Table 1B).Overall, using the comparative 1-DE proteomic analysis

combined with LC−MS/MS has shown that the proteomes ofNE and IE chloroplasts significantly differ.

ICPL Analysis Reveals Overall Protein Changes in NE and IEPoplars

The initial 1-DE analysis provided the first hints regarding thedifferences in the chloroplast protein pattern of NE and IEplants. However, the analysis did not show sufficient resolutionto obtain a general overview of the chloroplast proteome,neither to study post-translational modifications nor to quantifychanges in protein abundance. Therefore, we applied ICPLanalysis, which is based on isotopic labeling of all free aminogroups in proteins. This method enables quantitative proteomeprofiling of highly complex protein mixture31 and has neverbeen previously used for plant chloroplasts.ICPL labeling allowed the quantification of 119 chloroplastic

proteins, which were annotated by searching against in thePopulus trichocarpa genome sequences (Phytozome v9.1,T

able

2.continued

accession

VIP

SEdescrip

tion

functio

nLo

g2

TargetP

curatedlocalization(PPD

B)

Other

Proteins

POPT

R_0

018s08850.1

1.198

1.124

peptidaseS26

signalpeptidase

−2.9*

Cthylakoid-perip

heral-lum

enal-side

NoFu

nctio

nalAnnotationor

Not

Identified

POPT

R_0

013s02740.1

1.426

0.630

thylakoidlumen

18.3kD

aprotein

unknow

nfunctio

n−1.6*

Cthylakoid-perip

heral-lum

enal-side

POPT

R_0

005s04090.1

1.399

0.672

thylakoidlumen

18.3kD

aprotein

unknow

nfunctio

n−1.5*

Cthylakoid-perip

heral-lum

enal-side

POPT

R_0

008s05900.1

1.170

1.162

thylakoidlumenal15

kDaprotein

family

notnamed

−2.4*

Cthylakoid-perip

heral-lum

enal-side

aAnalysiswasperformed

onICPL

dataobtained

byisolatingchloroplastp

roteinsfrom

twoNE(RA1/RA2)

andtwoIE

(WT/EV)lines.D

iscriminantp

roteinsfrom

OPL

Smodelwereclassified

inorderof

importance

bymeans

ofVIP

scores.H

ighVIP

scoreindicateshigh

importance.SE=standard

errorof

jack-knifing

method;

Log2

=[Σ(N

E)/Σ(

IE)]

show

nin

negativeor

positivenumbersfordown-

orup-regulated

proteins,respectively,relatedto

isoprene

suppressed

plants(RA1andRA2);S

ignificant

differencesbetweenRA1/RA2andWT/EVweretested

byttest(*

=p<0.05

and**

=p<0.01).

Associatedinform

ationis

extractedfrom

PPDB

(http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx,

http://w

ww.grenoble.prabi.fr/at_chloro).

TargetP,predictio

nof

subcellularlocalizationusing

TargetP;Curated

localization(PPD

B),curatedsubcellularor

sub-plastid

iallocalizationas

stated

inPP

DB.



http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx

http://www.grenoble.prabi.fr/at_chloro

http://www.phytozome.net). A complete listing of all identifiedaccessions is provided in Supplemental Table S1 in theSupporting Information. The full data of protein identificationis listed in Supplemental Table S3 in the SupportingInformation.To determine the functions of the labeled chloroplastic

proteins, a MapManBin search (http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx) was performed using the accessionnumbers of Arabidopsis. We clustered the 119 proteins in 8functional categories. The main group (29.4% of the totalnumber of proteins) comprised proteins associated withphotosynthetic light reactions, proton transport, oxidation−reduction, the Calvin cycle, and the oxidative phosphopentosepathway. ‘Ribosomal proteins’ represented the next prominentgroup (19.3%), followed by the category of ‘Structural role’(16.0%) summarizing proteins involved in protein synthesis,binding, and folding. Proteins clustered in the ‘Metabolism’group (12.6%) are assigned to various metabolic processes.‘Histones’ represented 7.6% of the overall number of proteins.Only a few proteins were related to ‘Stress’ (1.7%) and ‘Others’(2.5%). A total of 10.9% labeled proteins were not functionallyannotated or are still not yet identified (Figure 2). ISPS andother enzymes of the MEP-pathway were not identified,possibly due to their relative low abundance in comparison withthe annotated proteins.

The subplastidial localization of the ICPL-identified proteinswas annotated using the ChloroP database (www.grenoble.prabi.fr/at_chloro/).30,59 The results are summarized in Tables1A,B and 2 and Supplemental Table S2 in the SupportingInformation. Histone proteins were excluded by the ChloroPdatabase because the corresponding genes of these histone-likeproteins are still unknown.All identified proteins were subjected to PCA, which clearly

revealed two distinct protein profiles in the NE and IE lines(Supplemental Figure S1 in the Supporting Information). Tounderstand the details in the relationship between individualchloroplast proteins and the suppression of ISPS, OPLS wasadditionally employed. NE samples were significantly (p =0.021; CV-ANOVA) separated from IE by means of the first-principal component (PC1). PC1 accounted for 22% of proteinvariation and explained 93% of the variation in the Y-variables,

which were defining the two NE and IE groups (Figure 3A).The loading plot shows that the NE samples are positively

correlated to histones, ribosomal, and metabolism proteins andare negatively correlated to proteins involved in photosynthesisand several proteins with structural functions. The importanceof these proteins to discriminate between NE and IE samples isshown in Table 2.

Up-Regulated Chloroplast Proteins in NE Plants. Thefinding that histone proteins are much more abundant inchloroplasts of NE plants was confirmed by the quantificationresults using acid urea-PAGE (Supplemental Figure S2A,B inthe Supporting Information). Our data show that proteinsidentified as linker histone H1 and H5 family members andhistone 2A and histone H2B were significantly up-regulated inthe NE lines (Table 2, Figures 3 and 4). Linker histones areusually structural chromatin components that generally repressthe accessibility of the genomic DNA.60 The functions of

Figure 2. Functional summary of the 119 chloroplast proteinsidentified using the ICPL technique in NE (RA1/RA2) and IE (WT/EV) grey poplar lines. Identified proteins are clustered in eightcategories, according to their functions.

Figure 3. Score (A) and loading (B) plots of Orthogonal Partial LeastSquares (OPLS) of chloroplast proteins identified using the ICPLtechnique and analyzed by LC−MS/MS (x axis = PC1 = 22%; y axis =PC2 = 21%). (A) NE (RA1/RA2), blue triangles; IE (WT/EV), redcircles. (B) Each functional group of proteins is indicated withdifferent symbols. Zoomed symbols with a dot represent thesignificantly different proteins between the IE and NE grey poplarplants. Red circle, histones; yellow square, ribosomal; blue diamond,structural; green triangle-up, photosynthesis; purple star, metabolism;dark red circle, stress; dark gray square, others; gray triangle-down,unknown (not annotated or not identified).



http://www.phytozome.net



www.grenoble.prabi.fr/at_chloro/

www.grenoble.prabi.fr/at_chloro/

histones related to isoprene are so far unknown; therefore, it isdifficult to interpret these results. The up-regulation of histonesin NE chloroplasts may be involved in specific developmentalpathways and may even play a role in the adaptation to water orthermal stress.60,61 By means of epigenetic memory, plants canmore efficiently respond to future stressful conditions. The up-regulation of histone proteins in chloroplasts of NE plantscompared with the IE controls has to be addressed in futurestudies.Ribosomal proteins were also significantly up-regulated in the

NE plants compared with the IE plants (Table 2, Figures 3 and4) both in the ICPL and in the label-free analysis of the 11−13kDa band. In addition to functioning in protein biosynthesiswithin the ribosome of chloroplasts, several ribosomal proteinsare involved in other cellular processes.62 The up-regulation ofribosomal proteins in our study may be indicative for asubstantial increase in protein turnover in NE plants, whichsupports the higher abundance of metabolites associated withamino acids and nitrogen metabolism in NE plants.18

Down-Regulated Chloroplast Proteins in NE Plants.Proteins with a Structural Role. We classified 16% of theidentified and quantified proteins into a group comprisingproteins with a structural role (Figure 2). This group includes(1) cyclophilins, (2) FKBP-type peptidyl-prolyl-cis-transisomerases (EC 5.2.1.8), (3) PAP fibrillin, (4) thylakoidformation protein (THF), (5) ubiquitin, (6) the TCT-1/cpn60 chaperonin family, (7) the peptidase family (EC 3.4.-.-),and (8) chromatin binding protein (Supplemental Table S1 inthe Supporting Information). The first four proteins discrim-inate NE from IE chloroplast proteins (Table 2, Figures 3 and4). FKBP-type and THF were found to be significantly down-regulated in the NE lines (log 2 = −1.5, p = 0.027 and log 2 =−1.5, p = 0.017, respectively) (Table 2). The lower abundanceof the THF in the NE plants may influence the assembly ofPSII.63 In Arabidopsis, THF 1 is a remodeling factor of PSII−LHCII complexes and is involved in the repair cycle of PSIIupon photo damage.64 Cyclophilins and PAP fibrillin contentsnegatively correlate with the NE plant (Figure 3 and Table 2),although statistical significance was not found (t-test, p = 0.112

and 0.086, respectively). Cyclophilins and FKBPs arecollectively referred to as immunophilins.65 Cyclophilinsstabilize the cis−trans transition state of proteins and accelerateisomerization, which is an important process involved inprotein folding,66 the assembly, and the stabilization ofmultidomain proteins, including PSII.63 Recent studies showedthat cyclophilins and FKBPs play roles as chaperonins and incell signaling.67,68 On the basis of the possible function ofchloroplast immunophilins as chaperonin as demonstrated byGupta et al.,67 we speculate that these proteins may be involvedin protein import or refolding processes in chloroplasts. Thelower abundance of cyclophilins in NE chloroplasts may resultin the reduced assembly and stability of photosyntheticcomplexes.The PAP fibrillin family comprises plastidic lipid-associated

proteins (PAPs) and putative fibrillins. PAPs are involved in thecoat-formation process of lipoprotein particles.69 This coat maycontain receptors important for the attachment of lipoproteinparticles to the thylakoid membrane as well as regulatoryproteins that may function in the transfer of lipids to and fromthe thylakoid membranes. The down-regulation of PAP fibrillinin NE may influence the lipid environment and affect thylakoidmembrane fluidity. In Arabidopsis, fibrillin-related proteinsaccumulate in response to water stress, and depending on plantspecies or stress conditions, these proteins are associated withdifferent plastid structure-like-fibrils,70 plastoglobuli,71 andthylakoid membranes.72 It is currently under discussion thatfibrillins may stabilize carotenoid-accumulating structures.70

Because of fibrillin association with plastoglobuli, their lowerlevel in NE chloroplasts may impair the storage of lipophiliccompounds, as suggested by Pozueta-Romero et al.71 Fibrillinsin the poplar chloroplast may be associated with stromallamellae thylakoids that participate in the structural stabilizationof thylakoids, which has already been demonstrated inArabidopsis, where these fibrillins help prevent damage resultingfrom osmotic or oxidative stress.73

Proteins Related to Photosynthesis. Blue native-PAGE wasapplied to understand the dynamics in the composition ofthylakoid membrane protein complexes. When the thylakoidmembranes were resolved by BN-PAGE, the protein patterns ofthe two groups of poplar lines were similar in content andintensity of the individual bands (Supplemental Figure S3A inthe Supporting Information). However, after semiquantitativeanalysis of the individual protein bands, it became clear that thelevels of PSI, the PSII dimer, ATP synthase, the PSII monomer,and the cytochrome b6 f complex were slightly reduced in NEcompared with IE chloroplasts (Supplemental Figure S3B inthe Supporting Information).As expected, the largest functional group of proteins

identified in the ICPL data set (Figures 2 and 3) was relatedto photosynthesis. Most of these proteins were subunits ofphotosynthetic complexes (PSI, and PsbP and PsbQ of PSII),and proteins of the ATP synthase family as well of the LHC(light-harvesting complex) were also present. Ferredoxin-NADP+ reductase (EC 1.18.1.2), plastid NADH/ubiquinoneoxidoreductase I (NDH-1, EC 1.6.5.3) with oxidoreductaseactivity, and the cytochrome b6 f complex involved inphotosynthetic electron-transfer reactions were additionallyidentified by LC−MS/MS analysis (Supplementary Table S1 inthe Supporting Information). Most of the labeled proteinsrelated to photosynthesis were strongly down-regulated in NEchloroplasts (Figures 3 and 4 and Table 2). Two intrinsicprotein subunits of PSII, PsbP, and PsbQ were even strongly

Figure 4. Number of proteins with low (gray bars) or high (dark redbars) levels in NE grey poplar lines. Suppression of the PcISPS proteinby RNAi caused changes in protein regulation of both NE lines (RA1/RA2) compared with the IE lines (WT/EV). Low or high abundanceof chloroplast proteins was counted when log 2 [Σ(NE)/Σ(IE)] was<−1 or >1, respectively. In total, the abundance of 31 proteins out of119 was unchanged. The chloroplast proteins were grouped based ontheir function.



down-regulated in NE chloroplasts (Table 2). PsbP togetherwith PsbO stabilize the intrinsic D1 and D2 proteins in the PSII reaction center. In plant mutants lacking PsbO, PsbP, orboth, a strong decrease in D1 or D2 content wasdemonstrated.74 PsbQ contributes to the general assembly ofthe PSII complex.75 On the basis of these findings, the reducedelectron transport rate (ETR), accompanied by an increasednonphotochemical quenching (NPQ) of NE leaves experiencedin abiotic stress can be explained.8,17,20 Indeed, photosyntheticgas exchange analyses clearly demonstrated a lower net CO2assimilation rate in transgenic NE poplars.17,18 Moreover,transcripts found in NE plants related to photosynthesis/light-harvesting processes were found to also be down-regulatedconstitutively under unstressed conditions.17 In contrast, theextrinsic manganese-containing protein (PsbO-2) was found tobe up-regulated in the chloroplasts of both NE lines (Table 1,Supplemental Table S1 in the Supporting Information). PsbO-2 is involved in the reassembly of the manganese cluster of PSIIafter disassembly of the complex and binds to the D1 proteinand allows the correct assembly of the manganese cluster.76

Two chlorophyll a/b binding proteins with function in lightharvesting were also more abundant in NE chloroplasts (Table1). The previous gene expression analysis of poplar leavesshowed that chlorophyllase 2 transcripts, a key enzyme in thechlorophyll metabolism, were up-regulated in NE.17 The up-regulation of chlorophyllase 2 may indicate higher turnover ofchlorophyll and quick degradation of potentially phototoxicbreakdown intermediates in NE plants.77

We further observed a significant down-regulation of thecytochrome b6 f complex in the NE lines (Table 2). Thecytochrome b6 f complex occupies a central position in thesequence of photosynthetic electron transport carriers. Thismembrane complex mediates the transfer of electrons betweenthe PSII and PSI photosystems while transferring protons fromthe chloroplast stroma across the thylakoid membrane into thelumen.78 Electron transport via cytochrome b6 f is responsiblefor creating the proton gradient that drives the synthesis ofATP in chloroplasts. The down-regulation of the cytochromeb6 f complex in NE chloroplasts suggests a disturbed productionof ATP in these isoprene-suppressed lines. In fact, we observedthat extrinsic subunits of ATP synthase were also down-regulated in NE lines, supporting our assumption (Table 2).Significant down-regulation of ATP and cytochrome b6 fcomplex proteins (p = 0.014 and 0.016, respectively, t-test)indicates substantial negative changes in electron transport.These data are again in accordance with previous observationsshowing that the electron-transport rate in the leaves of NEplants was severely impaired after exposure to sun flecks andrecovered much slower compared with the IE leaves.17

Proteins Related to Redox Regulation and OxidativeStress Defense. Chloroplasts undergo tremendous changes inredox potential during the day/night cycle and during variationin metabolic demand for NADPH and ATP. The redoxregulation plays a central role in many chloroplastic functions.79

A multilayered antioxidative defense system, which keeps theproduction of ROS under strict control, is present in thechloroplasts. This system includes enzymatic and nonenzymaticantioxidative elements.80−82 Many systematic proteome anal-yses of plants, combined with functional studies, have shownthat numerous proteins associated with lumenal, peripheral, andintegral thylakoid proteins are involved in antioxidant defenseor repair of the thylakoid system.83 These proteins includeperoxiredoxins, thioredoxins,84,85 Fe-, Cu-, Zn-superoxide

dismutases,82 enzymes, and structural proteins involved in thebiosynthesis and the binding of carotenoids or quenching ofexcess light energy.86

Indeed, in the chloroplasts of NE lines, the PCA and OPLSanalyses highlighted the significant down-regulation of twoproteins involved in the oxidative stress response, peroxiredox-ins (EC 1.11.1.15) and ascorbate peroxidase (EC 1.11.1.11)(log 2 = −0.8, p = 0.003, and log 2 = −2.4, p = 0.037,respectively) (Table 2, Figure 3). At high H2O2 concentrations,the function of peroxiredoxin may become inactivated throughoveroxidation.87 This inactivation has been proposed to explainthe signaling function of H2O2 in eukaryotes,87 and ascorbateavailability has been shown to limit violaxanthin de-epoxidase(EC 1.10.99.3) activity in the thylakoid lumen.88 Our resultsshow a repression of violaxanthin de-epoxidase in chloroplastsof NE plants compared with the IE controls (SupplementalTable S1 in the Supporting Information). In addition, thedown-regulation of ferredoxin-NADP reductase (EC 1.18.1.2)in NE lines also limited the regeneration of oxidized ascorbate(Supplemental Table S1 in the Supporting Information).Fibrillins, which belong to a family of thylakoid-bound

proteins, play a role in the stress response, including oxidativestress.89,90 Fibrillins are not enzymatically active but form aprotein coat of lipid-rich particles named plastoglobuli (PGs)that are associated with thylakoids.91,92 PGs also containvarious quinones and α-tocopherol as well as a significant set ofproteins likely involved in the metabolism of isoprenoid-derivedmolecules (i.e., quinones), lipids, and carotenoid cleavage. PGsare likely to play a role in various metabolic pathways andoxidative stress defense.91,92 Overall, our results demonstratethat isoprene suppression initiates various stress-responsereactions. Indeed, previous biochemical and metabolomicanalyses showed higher levels of total ascorbate and α-tocopherol as well as enhanced lipid peroxidation in unstressedleaves of NE poplars.17

Not Annotated Proteins. In our study involving poplarchloroplasts, a set of 13 proteins without assigned function butwith identified full-length genes was detected (Figure 3 and inthe Supplementary Table S1 in the Supporting Information).Twelve out of 13 share very high similarity (more than 70%)with Arabidopsis proteins. These proteins were related tothylakoid lumen protein (TLP18.3), pentapeptide repeats,BNR/Asp-box repeat related to PSII stability/assembly,Rhodonese-like domain, which relates to cell cycle control,and the NDH-dependent cyclic electron flow. Interestingly,TLP 18.3 and TLP 15 were significantly down-regulated in NElines (log 2 = −1.5 and −2.4, respectively, p = 0.036 and 0.035,t-test) (Table 2). Recently, it was shown that the TLP18.3protein is involved in the regulation of both the degradation/synthesis steps of the PSII D1 protein and in the assembly ofthe PSII monomers in the grana stack.93 Under standardgrowth conditions, the absence of TLP18.3 protein does notlead to a severe collapse of the PSII complexes, suggesting aredundancy of proteins assisting the repair steps of PSII tosecure its functionality. The Arabidopsis mutants lacking theTLP18.3 protein possess a higher susceptibility of PSII tophotoinhibition.

4. CONCLUSIONSWe aimed to elucidate whether the suppression of isoprenebiosynthesis and emission modifies the abundance ofchloroplast proteins and how this modification may influencethe plant functionality. The lack of PcISPS and consequently of



isoprene implied that the down-regulation of proteins related tophotosynthesis light reactions, redox regulation, and oxidativestress defense and several proteins with structural activity thatare responsible for lipid metabolism alteration occurred (Figure5). These changes were the consequences of alternative defensemechanisms such as photorespiration and nonphotochemicalquenching that needed to compensate for the absence ofisoprene. Indeed, the lower amounts of peroxiredoxin andascorbate peroxidase indicated their overoxidation in thepresence of increased levels of ROS. Overall, the presentproteomic analysis revealed that the absence of isoprene inpoplar leaves remodels the chloroplast protein profile to copeagainst oxidative stress. The present data strongly support theidea that isoprene improves thylakoid membrane structure andregulates the production of ROS.

■ ASSOCIATED CONTENT

*S Supporting Information

Supplemental Table S1 provides all proteins identified usingthe ICPL technique; Log 2 ratios, description, and functionalannotation of the proteins are listed. Supplemental Table S2provides subplastidial localization of the ICPL-identifiedproteins. Supplemental Table S3 provides the full data ofprotein identification: peptide sequence, ion score, number ofmissed cleavage, and exact molecular mass (MH+) of eachpeptide. Supplemental Figure S1 provides score and loadingplots of PCA of chloroplasts proteins identified using the ICPLtechnique. Supplemental Figure S2 provides representativeacid-urea-PAGE of histones in chloroplasts isolated from IE(WT/EV) and NE (RA1/RA2) poplar leaves (A) as well as thequantification of band intensities performed with ImageJ (B).Supplemental Figure S3 provides representative blue native-PAGE of poplar thylakoid membrane protein complexesisolated from IE (WT/EV) and NE (RA1/RA2) poplar leaves

(A) as well as the quantification of band intensities performedusing ImageJ (B). This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*E-mail: [email protected]. Phone: +49 893187 2413. Fax: +49 89 3187 4431.Funding

The present study was supported by Alexander-von-Humboldt-Foundation (individual fellowship to V.V.).Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Werner Heller and Katja Block (both HelmholtzZentrum Munchen) for critical reading, discussions, andcomments on the manuscript.

■ ABBREVIATIONSALDH, aldehyde dehydrogenase; ATP, adenosine triphosphate;BN-PAGE, blue native polyacrylamide gel electrophoresis;DMADP, dimethylallyl diphosphate; ETR, electron transportrate; HPL, hydroperoxide lyases; ICPL, isotope-coded proteinlabeling; IE, isoprene emitting; ISPS, isoprene synthase; LC−MS/MS, liquid chromatography tandem mass spectrometry;LHC, light-harvesting complex; MEP, 2-C-methyl-D-erythritol-4-phosphate pathway; NADP(H), nicotinamide adeninedinucleotide phosphate; NE, non isoprene emitting; OPLS,orthogonal partial least square regression; PAGE, polyacryla-mide gel electrophoresis; PAPs, plastidic lipid-associatedproteins; PCA, principal component analysis; PGs, plastoglo-buli; PSI, photosystem I; PSII, photosystem II; SDS-PAGE,

Figure 5. Suborganelle structure of NE poplar chloroplasts. The suppression of isoprene biosynthesis modified the protein profiles. Blue symbolswith the arrow pointing down indicate the spatial loci where proteins were down-regulated and red symbols with the arrow pointing up indicate thespatial loci where proteins were up-regulated. Isoprene suppression caused significant down-regulation of specific proteins related to photosyntheticelectron transport, redox regulation and oxidative stress defense, and several proteins with structural role, which responsible for the alteration of lipidmetabolism. The chloroplast proteome of transgenic poplar were further characterized by a higher abundance of histones and ribosomal proteins,which may be linked to a higher protein turnover in NE chloroplasts.



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sodium dodecyl sulfate polyacrylamide gel electrophoresis;SHMT, serine hydroxymethyl transferase; VIP, variable ofimportance for the projection; THF, thylakoid formationprotein; TLP, thylakoid lumen protein; WT, wild type

■ REFERENCES(1) Dicke, M.; Baldwin, I. T. The evolutionary context for herbivore-induced plant volatiles: beyond the “cry for help. Trends Plant Sci.2010, 15, 167−175.(2) Loreto, F.; Schnitzler, J. P. Abiotic stresses and induced BVOCs.Trends Plant Sci. 2010, 15, 154−166.(3) Velikova, V.; Pinelli, P.; Pasqualini, S.; Reale, L.; Ferranti, F.;Loreto, F. Isoprene decreases the concentration of nitric oxide inleaves exposed to elevated ozone. New Phytol. 2005, 166, 419−426.(4) Velikova, V.; Sharkey, T. D.; Loreto, F. Stabilization of thylakoidmembranes in isoprene-emitting plants reduces formation of reactiveoxygen species. Plant Signaling Behav. 2012, 7 (1), 139−141.(5) Sharkey, T. D.; Singsaas, E. L. Why plants emit isoprene. Nature1995, 374, 769.(6) Singsaas, E. L.; Lerdau, M.; Winter, K.; Sharkey, T. D. Isopreneincreases thermotolerance of isoprene-emitting species. Plant Physiol.1997, 115, 1413−1420.(7) Velikova, V.; Loreto, F.; Tsonev, T.; Brilli, F.; Edreva, A. Isopreneprevents the negative consequences of high temperature stress inPlatanus orientalis leaves. Funct. Plant Biol. 2006, 33, 931−940.(8) Behnke, K.; Ehlting, B.; Teuber, M.; Bauerfeind, M.; Louis, S.;Hansch, R.; Polle, A.; Bohlmann, J.; Schnitzler, J.-P. Transgenic, non-isoprene-emitting poplars don’t like it hot. Plant J. 2007, 51, 485−499.(9) Loreto, F.; Mannozzi, M.; Maris, C.; Nascetti, P.; Ferranti, F.;Pasqualini, S. Ozone quenching properties of isoprene and itsantioxidant role in plants. Plant Physiol. 2001, 126, 993−1000.(10) Loreto, F.; Velikova, V. Isoprene produced by leaves protectsthe photosynthetic apparatus against ozone damage, quenches ozoneproducts, and reduces lipid peroxidation of cellular membranes. PlantPhysiol. 2001, 127, 1781−1787.(11) Behnke, K.; Kleist, E.; Uerlings, R.; Wildt, J.; Rennenberg, H.;Schnitzler, J.-P. RNAi-mediated suppression of isoprene biosynthesisin hybrid poplar impacts ozone tolerance. Tree Physiol. 2009, 29, 725−736.(12) Brilli, F.; Barta, C.; Fortunati, A.; Lerdau, M.; Loreto, F.;Centritto, M. Response of isoprene emission and carbon metabolismto drought in white poplar (Populus alba) saplings. New Phytol. 2007,175, 244−254.(13) Ryan, A. C.; Hewitt, C. N.; Possell, M.; Vickers, C. E.; Purnell,A.; Mullineaux, P. M.; Davies, W. J.; Dodd, I. C. Isoprene emissionprotects photosynthesis but reduces plant productivity during droughtin transgenic tobacco (Nicotiana tabacum) plants. New Phytol. 2014,201, 205−216.(14) Sasaki, K.; Saito, T.; Lamsa, M.; Oksman-Caldentey, K.-M.;Suzuki, M.; Ohyama, K.; Muranaka, T.; Ohara, K.; Yazaki, K. Plantsutilize isoprene emission as a thermotolerance mechanism. Plant CellPhysiol. 2007, 48, 1254−1262.(15) Loivamaki, M.; Gilmer, F.; Fischbach, R. J.; Sorgel, C.; Bachl, A.;Walter, A.; Schnitzler, J.-P. Arabidopsis, a model to study biologicalfunctions of isoprene emission? Plant Physiol. 2007, 144, 1066−1078.(16) Vickers, C. E.; Possell, M.; Cojocariu, C. I.; Velikova, V. B.;Laothawornkitkul, J.; Ryan, A.; Mullineaux, P. M.; Hewitt, C. N.Isoprene synthesis protects transgenic plants from oxidative stress.Plant Cell Environ. 2009, 32, 520−531.(17) Behnke, K.; Loivamaki, M.; Zimmer, I.; Rennenberg, H.;Schnitzler, J. P.; Louis, S. Isoprene emission protects photosynthesis insunfleck exposed grey poplar. Photosynth. Res. 2010, 104, 5−17.(18) Way, D. A.; Ghirardo, A.; Kanawati, B.; Esperschutz, J.; Monson,R. K.; Jackson, R. B.; Schmitt-Kopplin, P.; Schnitzler, J. P. Increasingatmospheric CO2 reduces metabolic and physiological differencesbetween isoprene and non-isoprene-emitting poplars. New Phytol.2013, 200, 534−546.

(19) Velikova, V.; Varkonyi, Z.; Szabo, M.; Maslenkova, L.; Nogues,I.; Kovacs, L.; Peeva, V.; Busheva, M.; Garab, G.; Sharkey, T. D.;Loreto, F. Increased thermostability of thylakoid membranes inisoprene-emitting leaves probed with three biophysical techniques.Plant Physiol. 2011, 157, 905−916.(20) Behnke, K.; Ghirardo, A.; Janz, D.; Kanawati, B.; Esperschutz, J.;Zimmer, I.; Schmitt-Kopplin, P.; Niinemets, U.; Polle, A.; Schnitzler, J.P.; Rosenkranz, M. Isoprene function in two contrasting poplars undersalt and sunflecks. Tree Physiol. 2013, 33, 562−578.(21) Lichtenthaler, H. K. The 1-deoxy-d-xylulose-5-phosphatepathway of isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol.Plant Mol. Biol. 1999, 50, 47−65.(22) Cooper, G. M. Chloroplasts and Other Plastids. In The Cell: AMolecular Approach, 2nd ed.; ASM Press: Sunderland, MA, 2000(23) Jarvis, P. Targeting of nucleus-encoded proteins to chloroplastsin plants. New Phytol. 2008, 179, 257−285.(24) Jarvis, P.; Soll, J. Toc, tic, and chloroplast protein import.Biochim. Biophys. Acta 2002, 1590, 177−189.(25) Jarvis, P.; Robinson, C. Mechanisms of protein import androuting in chloroplasts. Curr. Biol. 2004, 14, R1064−R1077.(26) Soll, J.; Schleiff, E. Protein import into chloroplasts. Nat. Rev.Mol. Cell Biol. 2004, 5, 198−208.(27) Ort, D. R.; Yocum, C. F. Electron Transfer and EnergyTransduction in Photosynthesis: An Overview. In Oxygenic Photosyn-thesis: The Light Reactions. Advances in Photosynthesis; Ort, D. R.,Yocum, C. F., Eds.; Kluwer Academic Publishers: Dordrecht, TheNetherlands, 1996; Vol. 4, pp 1−9.(28) Finkemeier, I.; Leister, D. Plant Chloroplasts and Other Plastids.In Encyclopedia of Life Sciences (ELS); John Wiley & Sons, Ltd.:Chichester, U.K., 2010.(29) van Wijk, K. J.; Baginsky, S. Plastid proteomics in higher plants:current state and future goals. Plant Physiol. 2011, 155, 1578−1588.(30) Bruley, C.; Dupierris, V.; Salvi, D.; Rolland, N.; Ferro, M.AT_CHLORO: a chloroplast protein database dedicated to sub-plastidial localization. Front. Plant Sci. 2012, 3, Article 205(31) Prange, A.; Profrock, D. Chemical labels and natural elementtags for the quantitative analysis of bio-molecules. J. Anal. At. Spectrom.2008, 23, 432−459.(32) Bradford, M. M. A rapid and sensitive method for thequantitation of microgram quantities of protein utilising the principleof protein-dye binding. Anal. Biochem. 1976, 72, 248−254.(33) Lichtenthaler, H. K.; Wellburn, A. R. Determinations of totalcarotenoids and chlorophyll a and b of leaf extracts in differentsolvents. Biochem. Soc. Trans. 1983, 603, 591−592.(34) Merl, J.; Ueffing, M.; Hauck, S. M.; von Toerne, C. Directcomparison of MS-based label-free and SILAC quantitative proteomeprofiling strategies in primary retinal Muller cells. Proteomics 2012, 12,1902−1911.(35) Hauck, S. M.; Dietter, J.; Kramer, R. L.; Hofmaier, F.; Zipplies, J.K.; Amann, B.; Feuchtinger, A.; Deeg, C. A.; Ueffing, M. Decipheringmembrane-associated molecular processes in target tissue of auto-immune uveitis by label-free quantitative mass spectrometry. Mol. Cell.Proteomics 2010, 9, 2292−2305.(36) Brosch, M.; Hubbard, T.; Choudhary, J. Accurate and sensitivepeptide identification with Mascot Percolator. J. Proteome Res. 2009, 8(6), 3176−3181.(37) Jarvi, S.; Soursa, M.; Paakkapinen, V.; Aro, E.-M. Optimizednative gel systems for separation of thylakoid protein complexes: novelsuper- and mega-complexes. Biochem. J. 2011, 439, 207−214.(38) Waterborg, J. Acid-Urea-Triton Polyacrylamide Gel Electro-phoresis of Histones. In The Protein Protocols Handbook, 2nd ed.;Walker, J. M., Ed.; Springer Protocols Handbooks Series; HumanaPress, Inc.: Totowa, NJ, 2002; pp 103−111.(39) Chevallet, M.; Luche, S.; Rabilloud, T. Silver staining of proteinsin polyacrylamide gels. Nat. Protoc. 2006, 1 (4), 1852−1858.(40) Eriksson, L.; Johansson, E.; Kettaneh-Wold, N.; Trygg, J.;Wikstrom, C.; Wold, S. Multi- and Megavariate Data Analysis. Part I:Basic Principles and Applications; Umetrics Academy: Umea, Sweden,2006.



(41) Eriksson, L.; Trygg, J.; Wold, S. CV-ANOVA for significancetesting of PLS and OPLS models. J. Chemometrics 2008, 22, 594−600.(42) Efron, B.; Gong, G. A leisurely look at the bootstrap, the jack-knife, and cross-validation. Am. Stat. 1983, 37, 36−48.(43) Kley, J.; Heil, M.; Muck, A.; Svatos, A.; Boland, W. Isolatingintact chloroplasts from Arabidopsis samples for proteomic studies.Anal. Biochem. 2010, 398, 198−202.(44) Bohler, S.; Sergeant, K.; Hoffmann, L.; Dizengremel, P.;Hausman, J.-F.; Renaut, J.; Jolivet, Y. A difference gel electrophoresisstudy on thylakoids isolated from poplar leaves reveals a negativeimpact of ozone exposure on membrane proteins. J. Proteome Res.2011, 10, 3003−3011.(45) Besson, V.; Nauburger, M.; Rebeille, F.; Douce, R. Evidence forthree serine hydroxymethyltransferases in green leaf cells. Purificationand characterization of the mitochondrial and chloroplastic isoforms.Plant Physiol. Biochem. 1995, 33 (6), 665−673.(46) Cossins, E. A.; Chen, L. Folates and one-carbon metabolism inplants and fungi. Phytochemistry 1997, 45 (3), 437−452.(47) Appaji Rao, N.; Ambili, M.; Jala, V. R.; Subramanya, H. S.;Savithri, H. S. Structure-function relationship in serine hydroxymethyl-transferase. Biochim. Biophys. Acta 2003, 1647 (1−2), 24−29.(48) Stover, P.; Schirch, V. Serine hydroxymethyltransferase catalyzesthe hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahy-drofolate. J. Biol. Chem. 1990, 265 (24), 14227−14233.(49) Moreno, J. I.; Martín, R.; Castresana, C. Arabidopsis SHMT1, aserine xydroxymethyltransferase that functions in the photorespiratorypathway influences resistance to biotic and abiotic stress. Plant J. 2005,41, 451−463.(50) Karpinski, S.; Gabrys, H.; Mateo, A.; Karpinska, B.; Mullineaux,P. M. Light perception in plant disease defence signalling. Curr. Opin.Plant Biol. 2003, 6, 390−396.(51) Leegood, R. C.; Lea, P. J.; Adcock, M. D.; Hausler, R. E. Theregulation and control of photorespiration. J. Exp. Bot. 1995, 46,1397−1414.(52) Sunkar, R.; Bartels, D.; Kirch, H. H. Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana intransgenic plants improves stress tolerance. Plant J. 2003, 35 (4), 452−464.(53) Kotchoni, S. O.; Kuhns, C.; Ditzer, A.; Kirch, H. H.; Bartels, D.Over-expression of different aldehyde dehydrogenase genes inArabidopsis thaliana confers tolerance to abiotic stress and protectsplants against lipid peroxidation and oxidative stress. Plant CellEnviron. 2006, 29 (6), 1033−1048.(54) Mhamdi, A.; Queval, G.; Chaouch, S.; Vanderauwera, S.; VanBreusegem, F.; Noctor, G. Catalase function in plants: a focus onArabidopsis mutants as stress-mimic models. J. Exp. Bot. 2010, 61,4197−4220.(55) Karplus, P. A.; Bruns, C. M. Structure-function relations forferredoxin reductase. J. Bioenerg. Biomembr. 1994, 26 (1), 89−99.(56) Matsui, K. Green leaf volatiles: hydroperoxide lyase pathway ofoxylipin metabolism. Curr. Opin. Plant Biol. 2006, 9, 274−280.(57) Vancanneyt, G.; Sanz, C.; Farmaki, T.; Paneque, M.; Ortego, F.;Castanera, P.; Sanchez-Serrano, J. J. Hydroperoxide lyase depletion intransgenic potato plants leads to an increase in aphid performance.Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8139−8144.(58) Lamb, H. K.; Leslie, K.; Dodds, A. L.; Nutley, M.; Cooper, A.;Johnson, C.; Thompson, P.; Stammers, D. K.; Hawkins, A. R. Thenegative transcriptional regulator NmrA discriminates betweenoxidised and reduced dinucleotides. J. Biol. Chem. 2003, 278,32107−32114.(59) Ferro, M.; Brugiere, S.; Salvi, D.; Seigneurin-Berny, D.; Court,M.; Moyet, L.; Ramus, C.; Miras, S.; Mellal, M.; Le Gall, S.; Kieffer-Jaquinod, S.; Bruley, C.; Garin, J.; Joyard, J.; Masselon, C.; Rolland, N.AT_CHLORO, a comprehensive chloroplast proteome database withsubplastidial localization and curated information on envelopeproteins. Mol. Cell. Proteomics 2010, 9, 1063−1064.(60) Jerzmanowski, A.; Przewłoka, M.; Grasser, K. D. Linker histonesand HMG1 proteins of higher plants. Plant Biol. 2000, 2, 586−597.

(61) Boden, S. A.; Kavanova, M.; Finnegan, E. J.; Wigge, P. A.Thermal stress effects on grain yield in Brachypodium distachyonoccur via H2A.Z-nucleosomes. Genome Biol. 2013, 14, R65.(62) Stelzl, U.; Connell, S.; Nierhaus, K. H.; Wittmann-Liebold, B.Ribosomal proteins: role in ribosomal functions. Encycl. Life Sci. 2001,1−12.(63) Fu, A.; He, Z.; Cho, H. S.; Lima, A.; Buchanan, B. B.; Luan, S. Achloroplast cyclophilin functions in the assembly and maintenance ofphotosystem II in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A.2007, 104 (40), 15947−15952.(64) Huang, W.; Chen, Q.; Zhu, Y.; Hu, F.; Zhang, L.; Ma, Z.; He, Z.;Huang, J. Arabidopsis thylakoid formation 1 is a critical regulator fordynamics of PSII-LHCII complexes in leaf senescence and excess light.Mol. Plant. 2013, 6, 1673−1691.(65) Pratt, W. B.; Krishna, P.; Olsen, L. J. Hsp90-bindingimmunophilins in plants: the protein movers. Trends Plant Sci. 2001,6 (2), 54−58.(66) Gothel, S. F.; Marahiel, M. A. Peptidyl-prolyl cis-transisomerases, a superfamily of ubiquitous folding catalysts. Cell. Mol.Life Sci. 1999, 55, 423−436.(67) Gupta, R.; Mould, R. M.; He, Z.; Luan, S. A chloroplast FKBPinteracts with and affects the accumulation of Rieske subunit ofcytochrome bf complex. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (24),15806−15811.(68) Wang, P.; Heiman, J. The cyclophilis. Genome Biol. 2005, 6:Article 226.(69) Brehelin, C.; Kessler, F.; van Wijk, K. J. Plastoglobules: versatilelipoprotein particles in plastids. Trends Plant Sci. 2007, 12 (6), 260−266.(70) Deruere, J.; Romer, S.; d’Harlingue, A.; Backhaus, R. A.; Kuntz,M.; Camara, B. Fibril assembly and carotenoid overaccumulation inchromoplasts: A model for supramolecular lipoprotein structures.Plant Cell 1994, 6, 119−133.(71) Pozueta-Romero, J.; Rafia, F.; Houlne, G.; Cheniclet, C.; Carde,J. P.; Schantz, M.-L.; Schantz, R. A ubiquitous plant housekeepinggene, PAP, encodes a major protein component of bell pepperchromoplasts. Plant Physiol. 1997, 115, 1185−1194.(72) Pruvot, G.; Cuine, S.; Peltier, G.; Rey, P. Characterization of anovel drought-induced 34-kDa protein located in the thylakoids ofSolanum tuberosum L. plants. Planta 1996, 198 (3), 471−479.(73) Gillet, B.; Beyly, A.; Peltier, G.; Rey, P. Molecular character-ization of CDSP 34, a chloroplastic protein induced by water deficit inSolanum tuberosum L. plants, and regulation of CDSP 34 expression byABA and high illumination. Plant J. 1998, 16 (2), 257−262.(74) Yi, X.; Hargett, S.; Frankel, L.; Bricker, T. The effects ofsimultaneous RNAi suppression of PsbO and PsbP protein expressionin photosystem II of Arabidopsis. Photosynth. Res. 2008, 98 (1), 439−448.(75) Yi, X.; Hargett, S. R.; Frankel, L. K.; Bricker, T. M. The PsbQprotein is required in Arabidopsis for photosystem II assembly/stability and photoautotrophy under low light conditions. J. Biol. Chem.2006, 281 (36), 26260−26267.(76) Roose, J. L.; Pakrasi, H. B. The Psb27 protein facilitatesmanganese cluster assembly in photosystem II. J. Biol. Chem. 2008,283 (7), 4044−4050.(77) Hortensteiner, S.; Krautler, B. Chlorophyll breakdown in higherplants. Biochim. Biophys. Acta 2011, 1807, 977−88.(78) Kurisu, G.; Zhang, H.; Smith, J. L.; Cramer, W. A. Structure ofthe cytochrome b6f complex of oxygenic photosynthesis: tuning thecavity. Science 2003, 302 (5647), 1009−1014.(79) Scheibe, R.; Dietz, K. H. Reduction−oxidation network forflexible adjustment of cellular metabolism in photoautotrophic cells.Plant Cell Environ. 2012, 35, 202−216.(80) Mullineaux, P.; Karpinski, S. Signal transduction in response toexcess light: getting out of the chloroplast. Curr. Opin. Plant Biol. 2002,5, 43−48.(81) Apel, K.; Hirt, H. Reactive oxygen species: metabolism,oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004,55, 373−399.



(82) Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F.Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9 (10),490−498.(83) van Wijk, K. J. Plastid proteomics. Plant Physiol. Biochem. 2004,42, 963−977.(84) Dietz, K. J.; Horling, F.; Konig, J.; Baier, M. The function of thechloroplast 2-cysteine peroxiredoxin in peroxide detoxification and itsregulation. J. Exp. Bot. 2002, 53 (372), 1321−1329.(85) Buchanan, B. B.; Balmer, Y. Redox regulation: a broadeninghorizon. Annu. Rev. Plant Biol. 2005, 56, 187−220.(86) Bassi, R.; Caffarri, S. Lhc proteins and the regulation ofphotosynthetic light harvesting function by xanthophylls. Photosynth.Res. 2000, 64, 243−256.(87) Puerto-Galan, L.; Perez-Ruiz, J. M.; Ferrandez, J.; Cano, B.,Naranjo, B.; Najera, V. A.; Gonzalez, M.; Lindahl, A. M.; Cejudo, F. J.Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxicand signaling activities of hydrogen peroxide. Front Plant Sci. 2013, 4,Article 310.(88) Giacomelli, L.; Rudella, A.; Wijk, K. J. High light response of thethylakoid proteome in Arabidopsis wild type and the ascorbate-deficient mutant vtc2-2. A comparative proteomics study. Plant Physiol.2006, 141, 685−701.(89) Rey, P.; Gillet, B.; Romer, S.; Eymery, F.; Massimino, J.; Peltier,G.; Kuntz, M. Over-expression of a pepper plastid lipid-associatedprotein in tobacco leads to changes in plastid ultrastructure and plantdevelopment upon stress. Plant J. 2000, 21, 483−494.(90) Langenkamper, G.; Mana’h, N.; Broin, M.; Cuine, S.; Becuwe,N.; Kuntz, M.; Rey, P. Accumulation of plastid lipid-associatedproteins (fibrillin/CDSP34) upon oxidative stress, ageing and bioticstress in Solanaceae and in response to drought in other species. J. Exp.Bot. 2001, 52 (360), 1545−1554.(91) Vidi, P. A.; Kanwischer, M.; Baginsky, S.; Austin, J. R.; Csucs,G.; Dormann, P.; Kessler, F.; Brehelin, C. Tocopherol cyclase (VTE1)localization and vitamin E accumulation in chloroplast plastoglobulelipoprotein particles. J. Biol. Chem. 2006, 281, 11225−11234.(92) Ytterberg, A. J.; Peltier, J. B.; van Wijk, K. J. Protein profiling ofplastoglobules in chloroplasts and chromoplasts: a surprising site fordifferential accumulation of metabolic enzymes. Plant Physiol. 2006,140, 984−997.(93) Sirpio, S.; Allahverdiyeva, Y.; Suorsa, M.; Paakkarinen, V.;Vainonen, J.; Battchikova, N.; Aro, E.-M. TLP18.3, a novel thylakoidlumen protein regulating photosystem II repair cycle. Biochem. J. 2007,406, 415−425.



Genetic Manipulation of Isoprene Emissions in Poplar Plants Remodels the Chloroplast Proteome

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