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

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Article

The Genetic Manipulation of Isoprene Emissions inPoplar Plants Remodels the Chloroplast Proteome

Violeta Velikova, Andrea Ghirardo, Elisa Vanzo, JulianeMerl, Stefanie M Hauck, and Joerg-Peter Schnitzler

J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr401124z • Publication Date (Web): 22 Jan 2014

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The Genetic Manipulation of Isoprene Emissions in Poplar Plants

Remodels the Chloroplast Proteome

Violeta Velikova1,2, Andrea Ghirardo2, Elisa Vanzo2, Juliane Merl3, Stefanie M. Hauck3, Jörg-

Peter Schnitzler2*

1Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G.

Bonchev Str. Bl. 21, 1113 Sofia, Bulgaria

2Helmholtz Zentrum München, Institute of Biochemical Plant Pathology, Research Unit,

Environmental Simulation, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany

3Helmholtz Zentrum München, Research Unit Protein Science, Ingolstädter Landstr. 1, D-

85764 Neuherberg, Germany

ACS Paragon Plus Environment

Journal of Proteome Research

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

compounds.

ABSTRACT: Biogenic isoprene (2-methyl-1,3-butadiene) improves the integrity and

functionality of thylakoid membranes and scavenges reactive oxygen species (ROS) in plant

tissue under stress conditions. Based on available physiological studies, we hypothesized

that the suppression of isoprene production in the poplar plant by genetic engineering would

cause changes in the chloroplast protein pattern, which in turn would compensate for

changes in chloroplast functionality and overall plant performance under abiotic stress. To

test this hypothesis, we used a stable isotope-coded protein labeling technique in conjunction

with polyacrylamide gel electrophoresis and liquid chromatography tandem mass

spectrometry. We analyzed quantitative and qualitative changes in the chloroplast proteome

of isoprene-emitting (IE) and non-isoprene-emitting (NE) poplars. Here, we demonstrate that

suppression of isoprene synthase by RNA 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 a re-arrangement 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 with the

production of ROS.

1. INTRODUCTION

Plant volatile isoprenoids (isoprene, monoterpenes and sesquiterpenes) have developed

various functions in plants via evolution. These compounds play critical roles in plant

communication with herbivores1 and in plant defense mechanisms against biotic and abiotic

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stresses2 as well as modulate stress-induced signaling molecules3,4. Isoprene protects

photosynthetic processes against oxidative stresses elicited by high temperatures5-8, ozone9-

11 and drought12,13. Although the impact of volatile isoprenoids on abiotic stress protection

has been clearly demonstrated, the biophysical and biochemical mechanisms underlying the

induced protection are still unclear.

In the last decade, attempts have been made to analyze the functionality of isoprene using

molecular approaches. The role of isoprene in plant protection against thermal and oxidative

stresses has been studied using transgenic approaches either by the knock-down of the

natural isoprene emission in grey poplar (Populus x canescens)8 or by the introduction of this

trait in Arabidopsis thaliana14,15 and Nicotiana tabacum16, two naturally non-isoprene-emitting

species. Using both approaches, a positive effect of isoprene on plant stress resistance was

documented2. Detailed metabolomic analysis of transgenic poplars under various stress

conditions, e.g., ozone11, high temperature17, and atmospheric CO2 levels18 revealed

metabolome-wide re-arrangements of cellular metabolism between isoprene-emitting and

isoprene-suppressed poplars. However, these changes were also partially present under

control conditions. Moreover, recent evidence has demonstrated that isoprene improves the

integrity and functionality of thylakoid membranes under optimal conditions in transgenic

isoprene-emitting Arabidopsis19 and poplar20. Overall, the compensatory multiple stress

tolerance mechanisms in isoprene-suppressed poplar leaves most likely includes a

remodeling 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 the chloroplastic 2-C-methyl-D-erythritol-4-phosphate (MEP)

pathway, supplying the substrate dimethylallyl diphosphate (DMADP) to isoprene synthase

(ISPS, EC 4.2.3.27)21. Chloroplasts are specialized organelles harboring the photosynthetic

apparatus essential for the production of bioenergy and CO2 fixation and serve as metabolic

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hubs for various primary and secondary biosynthetic pathways essential for plant growth,

development and defense.

Chloroplasts have their own genome, containing approximately 120 genes. These genes

encode RNAs and proteins involved in gene expression as well as a variety of proteins that

function in photosynthesis22. Although the chloroplasts translate their own proteins, nuclear

genes encode approximately 90% of chloroplast proteins. These proteins are synthesized on

cytosolic ribosomes and are then imported into the chloroplast as complete polypeptide

chains22 via translocon complexes, followed by processing, folding and assembly by various

chaperone systems24-26. The chloroplast is surrounded by a double-membrane called the

inner and outer envelope membrane. In addition, a third membrane system is present inside

the chloroplast, known as the thylakoid membrane, which forms flattened discs or stacks

called thylakoid and grana, respectively. On thylakoid membranes, multi-subunit protein

complexes (photosystem I and II, “PSI” and “PSII”, the ATP synthase complex, and the

cytochrome b6f complex) are located27. The thylakoid membrane 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 best known for their role in photosynthesis, they

also host other essential 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 physiology and ultrastructural biology;

however, the knowledge regarding the chloroplast proteome is still scarce. To understand

metabolic processes in the chloroplasts, more information regarding selected proteins and

their functions is required. Advances in proteomic techniques in combination with increasing

genomic and transcriptomic information have enabled studies on chloroplast proteins,

providing additional information regarding their functional compartmentalization and

characterization29,30 .

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The present work is the first study specifically addressing changes in the chloroplast protein

profile of plants altered in their isoprene emission capability. The chloroplast proteome in

isoprene-emitting (IE: wild type and empty vector control, WT/EV) and non-isoprene-emitting

(NE: RA1/RA2) poplar lines was characterized. We applied an approach for quantitative and

qualitative proteomics based on stable isotope-coded protein labeling (ICPL) in combination

with polyacrylamide gel electrophoresis (PAGE) and liquid chromatography tandem mass

spectrometry (LC-MS/MS).31 We aimed to understand whether the absence of isoprene

production (i) triggers overall changes in the chloroplast proteome, (ii) affects the

composition of protein complexes of the photosynthetic electron transport chain, and alters

the enzyme abundance of chloroplast biosynthetic pathways, and (iii) how non-isoprene

emitting poplars adjust their structural proteome.

2. MATERIAL AND METHODS

Plant Materials and Growth Conditions

In the present study, two non-isoprene-emitting (NE) PcISPS-RNA interference (RNAi)

transgenic lines (RA1 and RA2) were compared to isoprene-emitting (IE) wild type (WT) and

the control of transgenic manipulation empty vector (EV) of grey poplars (Populus x

canescens; syn. Populus tremula x P. alba)8,11,17,18,20. The RA1 and RA2 lines correspond to

independently transformed lines emitting only 0.5-2% of isoprene compared to the emission

capacity of WT/EV8,20. The EV line was included in the experiments to ensure that

differences in the chloroplast proteome between NE and IE were due to specific alteration of

the PcISPS gene and not to a more general genetic manipulation effect. The plants were

grown in a greenhouse with ambient day/night temperature of 25°C/20°C, relative humidity of

60/50%, and 16 h day length. When the light intensity was less than 700 µmol m-2 s-1 of

photosynthetically active radiation (PAR), supplemental light was provided by high-pressure

sodium lamps (Philips SON T-AGRO 400W). The plants were planted into 2.2-L pots with

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soil substrate [25% v/v Fruhstorfer Einheitserde (Bayerische Gärtnereigenossenschaft,

Aschheim, Gemany), 25% v/v silica sand (particle size 1–3 mm) and 50% v/v perlite (Agriperl

Dämmstoff GmbH, Dortmund, Germany] and fertilizer [Triabon (Compo, Münster, Germany)

and Osmocote (Scotts Miracle-Gro, Marysville, USA) (1:1, v/v; 10 g per liter of soil)]. Four-

month old plants were used for the experiments.

Chloroplast Isolation

Fully expanded leaves (9th and 10th nodes from the apical meristem) from 4 to 5 plants were

collected. Approximately 10 g of finely chopped leaf material was homogenized in 40 mL of

semi-frozen isolation medium (350 mM sorbitol, 50 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 2

mM ascorbate, 0.1% BSA, 1 mM EDTA) for 3 x 10 sec pulses in a mechanical blender. The

homogenate was filtered through four layers of Miracloth (Calbiochem, Darmstadt,

Germany). The filtrate was centrifuged for 3 min at 4°C and 4,000 x g, and the pellet (“crude

chloroplasts”) was re-suspended in grinding medium (20 ml) and re-sedimented for 3 min at

4°C and 4,000 x g (including acceleration time), and again re-suspended. The suspension

was loaded on top of sucrose gradients (4 to 10 mL per gradient 40 and 80% sucrose) and

centrifuged at 10,000 x g at 4°C for 10 min. After centrifugation, the chloroplast layer at the

interface of the two gradients was collected, washed with 40 mL of re-suspension medium

(50 mM HEPES-KOH, pH 7.5, 1 mM MgCl2, 2 mM EDTA), and centrifuged for 10 min at

5,000 x g at 4ºC. The chloroplast pellet was re-suspended in a final volume of 1 to 2 mL. This

chloroplast fraction was used to perform the ICPL reaction and PAGE for subsequent label-

free comparisons.

The protein content of the chloroplast suspension was measured according to Bradford32 and

the chlorophyll content according to Lichtenthaler and Wellburn33. Before measuring the

protein level in the samples, the chlorophyll was removed by 3-times washing procedure with

methanol. In average, 5.0 ± 0.08 !g protein (n = 9) corresponded to 10 µg chlorophyll. For

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normalization of protein loading on the gel, a protein equivalent corresponding to 10 µg

chlorophyll was applied.

SDS-PAGE and Sample Preparation for Label-Free Comparative Analysis

The chloroplast proteins were separated on a SDS-PAGE gradient (4-16%) gel. Prior SDS-

PAGE the chloroplast proteins (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 temperature in the dark. The electrophoresis was performed in the dark at 4°C

and constant voltage (100 V) for about 4:30 – 5:00 h. After electrophoresis, 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 kDa 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 GmbH, Planegg, Germany) and stored at -20°C

prior to LC-MS/MS analysis.

LC-MS/MS Analysis

Dried peptides were resuspended in 2% acetonitrile/0.1% trifluoric acid. The samples were

centrifuged 1000 x g at 4°C for 5 min. LC-MS/MS analysis was performed as previously

described by using an Ultimate 3000 nano-HPLC (Dionex, Thermo Scientific, Bremen,

Germany)34,35. Briefly, every sample was automatically injected and loaded onto the trap

column. After 5 min, the peptides were eluted and separated on the analytical column (75 µm

i.d. x 15 cm, Acclaim PepMap100 C18, 3 µm, 100 Å Dionex, Idstein, Germany) by an

acetonitrile gradient at 300 nl min-1 flow rate (60 min gradients for the label-free analysis, 170

min gradients for the ICPL analysis). From the MS pre-scan, the 10 most abundant peptide

ions were fragmented by collision-induced dissociation in the linear ion trap if they showed

an intensity of at least 200 counts and if they were at least +2 charged. During fragmentation,

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a high-resolution (60,000 full-width half maximum) MS spectrum was acquired in the LTQ

OrbitrapXL (Thermo Scientific, Bremen, Germany) with a mass range from 300 to 1,500 Da

and a dynamic exclusion of 30 sec or 60 sec for the label-free and the ICPL analysis,

respectively.

Label-Free Analysis

The acquired spectra for the two label-free datasets (11-13 kDa and 50 kDa gel slices) were

loaded into the Progenesis LC-MS software (version 2.5, Nonlinear) for label-free

quantification and were analyzed as previously described34,35. Features with only one charge

or more than eight charges were excluded from data analysis. Raw abundances of the

remaining features were normalized to allow correction for factors resulting from

experimental variation. All MS/MS spectra were exported as a Mascot generic file (mgf) and

used for peptide identification with MASCOT (version 2.3.02) in the Populus trichocarpa

protein database (version 4, 17236452 residues, 45036 sequences). The search parameters

included 10 ppm peptide mass and 0.6 Da MS/MS tolerance, one missed cleavage was

allowed, cysteine carbamidomethylation was set as the fixed modification, methionine

oxidation and asparagine or glutamine deamidation were allowed as variable modifications.

Using a MASCOT ion score cut-off of 30 and an appropriate significance threshold p, a

MASCOT-integrated decoy database search calculated a false discovery rate (FDR) of <1%.

For each dataset, the peptide assignments were re-imported into the Progenesis LC-MS

software. After summing the abundances of all the peptides allocated to each protein, the

identification and quantification results were exported and are shown in Tables 1A and 1B.

Log2 ratios of [!(NE)/!(IE)] from protein abundances were calculated and are reported in

Table 1A and 1B.

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 ICPLTM Duplex- and Quadruplex-Kits (SERVA Electrophoresis

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GmbH, Heidelberg, Germany) following the manufacturer’s instructions. For the quatruplex

analysis with two biological replicates, 50 µg of the isotope-labeled proteins from the four

different samples (ICPL-0 = WT, ICPL-4 = EV, ICPL-6 = RA1, ICPL-10 = RA2) were

combined, and the proteins were precipitated using ice-cold 80% acetone. The same was

repeated for two biological replicates with duplex analysis paring WT/RA1 and EV/RA2. The

resulting 200 !g of protein per biological replicate was separated by 1D SDS-PAGE. After

protein staining with Coomassie Brilliant Blue each lane was cut into 5 slices and subjected

to in-gel digestion with trypsin (Sigma Aldrich) as previously described.34 LC-MS/MS analysis

was performed as described above.

The acquired MS/MS spectra were searched against the Populus trichocarpa database

(Version: 2.3, 45036 sequences) using the Mascot search engine (version 2.3.02; Matrix

Science) with the following parameters: a precursor mass error tolerance of 10 ppm and a

fragment tolerance of 0.6 D. One missed cleavage was allowed. The complete list of protein

descriptions, protein group accessions, sequence of peptides, quan usage, ion score,

number of missed cleavages, and the molecular mass of the MH+ ion is given in the

Supplemental Table S3. Carbamidomethylation was set as the fixed modification. ICPL-0,

ICPL-4, ICPL-6 and ICPL-10 for the lysine residues of the peptides were set as variable

modifications. Data processing for the identification and quantitation of the ICPL-duplex- and

quadruplex-labeled proteins was performed using Proteome Discoverer version 1.3.0.339

(Thermo Scientific). The Mascot Percolator algorithm was used for the discrimination

between correct and incorrect spectrum identifications36 with a maximum q value of 0.01.

Proteins were further filtered using the following parameters: high peptide confidence and at

least 2 peptides per protein (count only rank 1 peptides and count peptide only in top scored

proteins). Peptide lists were exported, and the abundances of all unique peptides allocated to

one respective protein were summed to determine the intensities of the individual and

differentially labeled proteins for the biological replicates. Protein lists were also exported,

containing the ICPL ratios for the individual proteins in the different samples.

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Blue Native PAGE (BN-PAGE)

BN-PAGE is an excellent tool to analyze proteins and protein complexes in their native form.

Thylakoid isolation was performed under dim light at 4°C following the protocol of Järvi et

al.37 modified for poplar leaf material. Thylakoids were isolated from fresh leaves ground in

ice-cold grinding buffer (350 mM sorbitol; 50 mM HEPES/KOH, pH 7.5; 5 mM MgCl2; 2 mM

ascorbate; 1 mM EDTA and 0.1% BSA). The suspension was filtered through two layers of

Miracloth followed by centrifugation at 5,000 x g at 4°C for 5 min. The pellet was re-

suspended in buffer (50 mM HEPES/KOH, pH 7.5; 1 mM MgCl2 and 2 mM EDTA), followed

by centrifugation at 5,000 x g at 4°C for 5 min. The pellet was re-suspended in a small aliquot

of storage buffer (50 mM HEPES/KOH, pH 7.5; 100 mM Sorbitol and 10 mM MgCl2).

The thylakoid membranes (10 µg chlorophyll) were re-suspended in ice-cold sample buffer (1

M 6-aminocapronic acid; 100 mM BisTris/HCl, pH 7.0; 100 mM NaCl, 20% glycerol, 0.1%

SERVA Blue G) (Serva Electrophoresis) with 0.25 mg mL-1 Pefabloc (Sigma-Aldrich,

Deisenhofen, Germany). Prior to native-PAGE, the thylakoid membranes were solubilized in

1.5% dodecyl maltoside (Invitrogen, Darmstadt, Germany) in the dark for 5 min on ice. The

BN-PAGE samples were supplemented with a one-tenth volume of Serva Blue G buffer

(Serva Electrophoresis, Heidelberg, Germany). Thylakoid membrane proteins were

separated on 4 – 16% Bis-Tris gel (Novex by LifeTechnologies, Darmstadt, Germany).

Electrophoresis was performed at 0°C with a gradual increase in the voltage as follow: 75 V

for 30 min, 100 V for 30 min, 125 V for 30 min, 150 V for 60 min (total running time 2:30 h).

Anode and cathode buffer were commercially obtained from Serva Electrophoresis. After

electrophoresis, the gel was fixed in 40% methanol and 10% acetic acid solution, followed by

a destaining procedure in 8% acetic acid solution. ImageJ v.1.47 was utilized to quantify the

bands.

Acid-Urea-PAGE of Histones

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The chloroplast suspension was used to analyze histones by acetic-acid-urea-

polyacrylamide gel electrophoresis following the protocol38. Histone proteins were separated

using 15% TBE-Urea gels (Novex by LifeTechnologies, Darmstadt, Germany). Prior to

separation, the samples were solubilized in a TBE-Urea sample buffer (Novex by

LifeTechnologies, Darmstadt, Germany) for 5 min at room temperature. Acetic acid (5%) was

used as a running buffer. Electrophoresis was performed at room temperature at a constant

voltage (300 V) for 4:30 h. Proteins were visualized with silver stain.39 For quantification of

the bands, ImageJ v.1.47 was utilized.

Statistics

Proteomic differences of ICPL chloroplast samples were analyzed using a multivariate data

analysis approach with Principal Component Analysis (PCA) and Orthogonal Partial Least

Square regression (OPLS) statistical methods from the software packages ‘SIMCA-P’ (v.

13.0.0.0, Umetrics, Umeå, Sweden). The results were validated by ‘full cross validation’40,41

using a 95% confidence level. PCA was performed on a 119-by-12 matrix of ICPL data, i.e.

using as X-variables (centered and scaled with 1 s.d.-1) the summed peptide intensities

(normalized per protein content) of the 119 proteins from the 12 samples (four lines: WT, EV,

RA1, RA2; n=3 biological replicates for each line). OPLS was used to select discriminant

proteins that significantly distinguish NE from IE samples. OPLS was calculated by defining

as Y-variable the ability of the samples to emit isoprene, with NE = 0 and IE = 1. The

regression model was tested for significance by CV-ANOVA41. Proteins showing Variable of

Importance for the Projection (VIP) greater than 1 and uncertainty bars of jack-knifing

method42 smaller than the respective VIP value were defined as discriminant proteins.

Proteins in the NE lines were classified down- or up-regulated proteins when log2 of

[!(NE)/!(IE)] MS peak sum was < -1 or > +1, respectively, otherwise unchanged.

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3. RESULTS and DISCUSSION

1D-PAGE Shows Differences in the Chloroplast Protein Profiles 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 obvious

differences in protein abundance in chloroplast extracts from two NE (RA1/RA2) and two IE

(WT/EV) lines (Figure 1). The analysis was conducted in duplicate using independent

chloroplast isolations and including a technical replicate, resulting in 3 1-DE gels. The

presence of the two bands at 21 and 14 kDa (Figure 1) are indicative for chloroplast integrity

during isolation. It was demonstrated that these bands disappear from the fraction when

chloroplasts are broken.43

Prominent bands present in extracts of NE plants at "50 kDa and "11-13 kDa were visually

absent in the IE chloroplast extracts (Figure 1, red arrows). We excised and subjected these

bands to LC-MS/MS and label-free quantitative analysis. The annotation of the peptides

isolated from the 50 kDa protein band of the NE and IE extracts led to the identification of 15

proteins in total (Table 1A). The most abundant protein identified in the 50 kDa band of NE

extracts compared to the IE was serine hydroxymethyl transferase (SHMT, EC 2.1.2.1, log2

= 7.2). Significant (p = 0.004, t-test) up-regulation of SHMT was confirmed later using ICPL

analysis (Figure 3, Table 2, Supplemental Figure S1). SHMT was also observed earlier in

chloroplasts preparations from poplar44. However, plants possess SHMT isoforms in the

cytoplasm and mitochondria45. Cytosolic and mitochondrial SHMTs play a primary role in

folate-dependent pathways of C1 metabolism46. They catalyze the reversible, simultaneous

conversions of L-serine to glycine and tetrahydrofolate to 5,10-methylenetetrahydrofolate47,48.

There is an experimental evidence that the chloroplastic isoform of SHMT is an important

enzyme involved in the photorespiratory pathway49. Photorespiration is a well-known process

mitigating photooxidative damage by functioning as an electron sink to prevent the over-

reduction of the photosynthetic electron transport chain in the thylakoid membranes as well

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as photoinhibition of PSII50. This process is initiated in the 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 effect51. Together with SHMT, aldehyde dehydrogenase (ALDH, EC

1.2.1.3) was another highly abundant protein in NE lines (log2 = 6.8). Earlier transcriptomic

analyses in the same poplar lines showed that ALDH transcript levels were lower in

unstressed NE (log2 = -1.34), indicating that the transcript levels and protein abundance

were not strictly correlated.17 ALDHs belong to a family of NAD(P)+-dependent enzymes that

play major roles in the detoxification of aldehydes generated in plant cells exposed to abiotic

stress. Arabidopsis plants over-expressing ALDH3 show improved tolerance to oxidative

stress (caused by excess of NaCl, heavy metals, methyl viologen, and H2O2) compared to

WT plants. Stress tolerance in these transgenic plants was accompanied by a reduction of

H2O2 and malondialdehyde derived from cellular lipid peroxidation52,53. We also found

catalase (EC 1.11.1.6), one of the main H2O2-metabolizing enzymes54, at a higher

abundance in chloroplast extracts of NE compared to IE (log2 = 5.2, Table 1A). Our findings

that NE extracts have an enhanced abundance of proteins related to antioxidative stress

mechanisms are consistent with observed metabolic differences between NE and IE poplars

under ambient CO2 concentrations18.

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 1A) may affect the cyclic and non-cyclic electron flow in NE

chloroplasts. This finding suggests that these chloroplasts may have higher demands for

ATP and reducing power compared to IE55.

The suppression of isoprene biosynthesis in the poplar also affected proteins related to the

light reactions in photosynthesis (PsbO, LHC, ATP synthase (EC 3.6.3.15)) and primary

metabolic processes such as hydroxyproline-rich glycoprotein, phosphoglycerate kinase (EC

2.7.2.3) and fructose-bisphosphate aldolase (EC 4.1.2.13). These proteins were more

abundant in NE chloroplasts compared to IE chloroplasts (Table 1A).

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Interestingly, in the excised 50 kDa band, two proteins had lower abundance in the NE plants

compared to the IE plants (Table 1A). These two proteins are the hydroperoxide lyase (EC

4.1.2.-, log2 = -2.1) and a protein belonging to the NmrA-like family (EC 1.3.1.-., log2 = -2.7).

Hydroperoxide lyases (HPLs) are members of the cytochrome P450 family and catalyze the

cleavage of fatty acid hydroperoxides to aldehydes and oxoacids56. HPLs mediate the

formation of green leaf volatiles (GLVs syn. Lipoxygenase (LOX) products) through oxylipin

metabolism56. Data from HPL-depleted transgenic potato lines strongly suggest that the

constitutive activity of this branch of the oxylipin biosynthetic pathway influences plant

defense processes57. NmrA acts as a negative transcriptional regulator involved in N

metabolism and is a member of the short-chain dehydrogenase reductase superfamily (EC

1.1.1.300). In addition, NmrA has the ability to discriminate between the oxidized and

reduced forms of dinucleotides, a feature that is linked to a possible role in redox sensing58, a

function 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 is characterized by an enrichment of ribosomal proteins in NE (Table

Overall, using the comparative 1-DE proteomic analysis combined with LC-MS/MS has

shown that the proteomes of NE and IE chloroplasts significantly differ.

ICPL Analysis Reveals Overall Protein Changes in NE and IE Poplars

The initial 1-DE analysis provided the first hints regarding the differences in the chloroplast

protein pattern of NE and IE plants. However, the analysis did not show sufficient resolution

to obtain a general overview of the chloroplast proteome, neither to study post-translational

modifications nor to quantify changes in protein abundance. Therefore, we applied ICPL

analysis, which is based on isotopic labeling of all free amino groups in proteins. This method

enables quantitative proteome profiling of highly complex protein mixture31 and has never

been previously used for plant chloroplasts.

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ICPL labeling allowed the quantification of 119 chloroplastic proteins, which were annotated

by searching against in the Populus trichocarpa genome sequences (Phytozome v9.1,

http://www.phytozome.net). A complete listing of all identified accessions is provided in

Supplemental Table S1. The full data of protein identification is listed in Supplemental Table

To determine the functions of the labeled chloroplastic proteins, a MapManBin search

(http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx) was performed using the accession

numbers of Arabidopsis. We clustered the 119 proteins in 8 functional categories. The main

group (29.4% of the total number of proteins) comprised proteins associated with

photosynthetic light reactions, proton transport, oxidation-reduction, the Calvin cycle and the

oxidative phosphopentose pathway. ‘Ribosomal proteins’ represented the next prominent

group (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 functionally annotated or are still

not yet identified (Figure 2). ISPS and other enzymes of the MEP-pathway were not

identified, possibly due to their relative low abundance in comparison to the annotated

proteins.

The sub-plastidial localization of the ICPL-identified proteins was annotated using the

ChloroP database (www.grenoble.prabi.fr/at_chloro/)30,59. The results are summarized in

Table 1A and 1B, Table 2 and Supplemental Table S2. Histone proteins were excluded by

the ChloroP database because the corresponding genes of these histone-like proteins 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). To understand the details in the

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relationship between individual chloroplast proteins and the suppression of ISPS, OPLS was

additionally 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 protein

variation 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 and are negatively correlated to

proteins involved in photosynthesis and several proteins with structural functions. The

importance of these proteins to discriminate between NE and IE samples is shown in Table

Up-regulated chloroplast proteins in NE plants

The finding that histone proteins are much more abundant in chloroplasts of NE plants was

confirmed by the quantification results using acid urea-PAGE (Supplemental Figure S2A, B).

Our data show that proteins identified as linker histone H1 and H5 family members, and

histone 2A and histone H2B were significantly up-regulated in the NE lines (Table 2, Figure

3, 4). Linker histones are usually structural chromatin components that generally repress the

accessibility of the genomic DNA60. The functions of histones related to isoprene are so far

unknown; therefore, it is difficult to interpret these results. The up-regulation of histones in

NE chloroplasts may be involved in specific developmental pathways and may even play a

role in the adaptation to water or thermal stress60,61. By dint of epigenetic memory, plants can

more efficiently respond to future stressful conditions. The up-regulation of histone proteins

in chloroplasts of NE plants compared to the IE controls has to be addressed in future

studies.

Ribosomal proteins were also significantly up-regulated in the NE plants compared to the IE

plants (Table 2, Figure 3, 4) both in the ICPL and in the label-free analysis of the 11-13 kDa

band. In addition to functioning in protein biosynthesis within the ribosome of chloroplasts,

several ribosomal proteins are involved in other cellular processes62. The up-regulation of

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ribosomal proteins in our study may be indicative for a substantial increase in protein

turnover in NE plants, which supports the higher abundance of metabolites associated with

amino acids and nitrogen metabolism in NE plants18.

Down-regulated chloroplast proteins in NE plants

Proteins with a structural role

We classified 16% of the identified and quantified proteins into a group comprising proteins

with a structural role (Figure 2). This group includes (1) cyclophilins, (2) FKBP-type peptidyl-

prolylcis-trans isomerases (EC 5.2.1.8), (3) PAP fibrillin, (4) thylakoid formation 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). The first four proteins

discriminate NE from IE chloroplast proteins (Table 2, Figure 3, 4). FKBP-type and THF were

found to be significantly down-regulated in the NE lines (log2 = -1.5, p= 0.027 and log2 = -

1.5, p= 0.017, respectively) (Table 2). The lower abundance of the THF in the NE plants may

influence the assembly of PSII63. In Arabidopsis, THF 1 is a re-modeling factor of PSII–LHCII

complexes and is involved in the repair cycle of PSII upon photo damage64. Cyclophilins and

PAP fibrillin contents negatively 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 are collectively referred to as immunophilins65. Cyclophilins stabilize the cis-trans

transition state of proteins and accelerate isomerization, which is an important process

involved in protein folding66, the assembly and the stabilization of multi-domain proteins,

including PSII63. Recent studies showed that cyclophilins and FKBPs play roles as

chaperonins and in cell signaling67,68. Based on the possible function of chloroplast

immunophilins as chaperonin as demonstrated by Gupta et al.67, we speculate that these

proteins may be involved in protein import and/or refolding processes in chloroplasts. The

lower abundance of cyclophilins in NE chloroplasts may result in the reduced assembly and

stability of photosynthetic complexes.

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The PAP fibrillin family comprises plastidic lipid-associated proteins (PAPs) and putative

fibrillins. PAPs are involved in the coat formation process of lipoprotein particles69. This coat

may contain receptors important for the attachment of lipoprotein particles to the thylakoid

membrane as well as regulatory proteins that may function in the transfer of lipids to and

from the thylakoid membranes. The down-regulation of PAP fibrillin in NE may influence the

lipid environment and affect thylakoid membrane fluidity. In Arabidopsis, fibrillin-related

proteins accumulate in response to water stress, and depending on plant species and/or

stress conditions, these proteins are associated with different plastid structure-like-fibrils70,

plastoglobuli71 and thylakoid membranes72. It is currently under discussion that fibrillins may

stabilize carotenoid-accumulating structures70. Due to fibrillin association with plastoglobuli,

their lower level in NE chloroplasts may impair the storage of lipophilic compounds as

suggested by Pozueta-Romero et al71. Fibrillins in the poplar chloroplast may be associated

with stromal lamellae thylakoids that participate in the structural stabilization of thylakoids,

which has already been demonstrated in Arabidopsis where these fibrillins help prevent

damage resulting from osmotic or oxidative stress73.

Proteins related to photosynthesis

Blue native-PAGE was applied to understand the dynamics in the composition of thylakoid

membrane protein complexes. When the thylakoid membranes were resolved by BN-PAGE,

the protein patterns of the two groups of poplar lines were similar in content and intensity of

the individual bands (Supplemental Figure S3A). However, after semi-quantitative analysis of

the individual protein bands, it became clear that the levels of PSI, the PSII dimer, ATP

synthase, the PSII monomer, and the cytochrome b6f complex were slightly reduced in NE

compared to IE chloroplasts (Supplemental Figure S3B).

As expected, the largest functional group of proteins identified in the ICPL dataset (Figure 2,

3) was related to photosynthesis. Most of these proteins were subunits of photosynthetic

complexes (PSI, and PsbP and PsbQ of PSII), and proteins of the ATP synthase family as

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well of the LHC (light-harvesting complex) were also present. Ferredoxin-NADP+ reductase

(EC 1.18.1.2), plastid NADH:ubiquinone oxidoreductase I (NDH-1, EC 1.6.5.3) with

oxidoreductase activity and the cytochrome b6f complex involved in photosynthetic electron

transfer reactions were additionally identified by LC-MS/MS analysis (Supplementary Table

S1). Most of the labeled proteins related to photosynthesis were strongly down-regulated in

NE chloroplasts (Figure 3, 4 and Table 2). Two intrinsic protein subunits of PSII, PsbP and

PsbQ were even strongly down-regulated in NE chloroplasts (Table 2). PsbP together with

PsbO stabilize the intrinsic D1 and D2 proteins in the PS II reaction center. In plant mutants

lacking PsbO, PsbP or both, a strong decrease in D1 or D2 content was demonstrated74.

PsbQ contributes to the general assembly of the PSII complex75. Based on these findings,

the reduced electron transport rate (ETR), accompanied by an increased non-photochemical

quenching (NPQ) of NE leaves experienced to abiotic stress can be explained8,17,20. Indeed,

photosynthetic gas exchange analyses clearly demonstrated a lower net CO2 assimilation

rate in transgenic NE poplars17,18. Moreover, transcripts found in NE plants related to

photosynthesis/light harvesting processes were found to also be down-regulated

constitutively under unstressed conditions17. In contrast, the extrinsic manganese-containing

protein (PsbO-2) was found to be up-regulated in the chloroplasts of both NE lines (Table 1A,

Supplemental Table S1). PsbO-2 is involved in the reassembly of the manganese cluster of

PSII after disassembly of the complex and binds to the D1 protein and allows the correct

assembly of the manganese cluster76. Two chlorophyll a/b binding proteins with function in

light harvesting were also more abundant in NE chloroplasts (Table 1A). The previous gene

expression analysis of poplar leaves showed that chlorophyllase 2 transcripts, a key enzyme

in the chlorophyll metabolism, were up-regulated in NE17. The up-regulation of chlorophyllase

2 may indicate higher turnover of chlorophyll and quick degradation of potentially phototoxic

breakdown intermediates in NE plants77.

We further observed a significant down-regulation of the cytochrome b6f complex in the NE

lines (Table 2). The cytochrome b6f complex occupies a central position in the sequence of

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photosynthetic electron transport carriers. This membrane complex mediates the transfer of

electrons between the PSII and PSI photosystems while transferring protons from the

chloroplast stroma across the thylakoid membrane into the lumen78. Electron transport via

cytochrome b6f is responsible for creating the proton gradient that drives the synthesis of

ATP in chloroplasts. The down-regulation of the cytochrome b6f complex in NE chloroplasts

suggests a disturbed production of ATP in these isoprene-suppressed lines. In fact, we

observed that extrinsic subunits of ATP synthase were also down-regulated in NE lines

supporting our assumption (Table 2). Significant down-regulation of ATP and cytochrome b6f

complex 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

observations showing that the electron transport rate in the leaves of NE plants was severely

impaired after exposure to sun flecks and recovered much slower compared to the IE

leaves17.

Proteins related to redox regulation and oxidative stress defense

Chloroplasts undergo tremendous changes in redox potential during the day/night cycle and

during variation in metabolic demand for NADPH and ATP. The redox regulation plays a

central role in many chloroplastic functions79. A multi-layered antioxidative defense system,

which keeps the production of ROS under strict control, is present in the chloroplasts. This

system includes enzymatic and non-enzymatic antioxidative elements80-82. Many systematic

proteome analyses of plants, combined with functional studies, have shown that numerous

proteins associated with lumenal, peripheral, and integral thylakoid proteins are involved in

antioxidant defense and/or repair of the thylakoid system83. These proteins include

peroxiredoxins, thioredoxins84,85, Fe- and Cu, Zn-superoxide dismutases82, enzymes and

structural proteins involved in the biosynthesis and the binding of carotenoids and/or

quenching of excess light energy86.

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Indeed, in the chloroplasts of NE lines, the PCA and OPLS analyses highlighted the

significant down-regulation of two proteins involved in the oxidative stress response,

peroxiredoxins (EC 1.11.1.15) and ascorbate peroxidase (EC 1.11.1.11) (log2 = -0.8,

p=0.003, and log2 = -2.4, p=0.037, respectively) (Table 2, Figure 3). At high H2O2

concentrations, the function of peroxiredoxin may become inactivated through over-

oxidation87. This inactivation has been proposed to explain the signaling function of H2O2 in

eukaryotes87 and ascorbate availability has been shown to limit violaxanthin de-epoxidase

(EC 1.10.99.3) activity in the thylakoid lumen88. Our results show a repression of violaxanthin

de-epoxidase in chloroplasts of NE plants compared to the IE controls (Supplemental Table

S1). In addition, the down-regulation of ferredoxin-NADP reductase (EC 1.18.1.2) in NE lines

also limited the regeneration of oxidized ascorbate (Supplemental Table S1).

Fibrillins, which belong to a family of thylakoid-bound proteins, play a role in the stress

response, including oxidative stress89,90. Fibrillins are not enzymatically active but form a

protein coat of lipid-rich particles named plastoglobuli (PGs) that are associated with

thylakoids91,92. PGs also contain various quinones and "-tocopherol as well as a significant

set of proteins likely involved in the metabolism of isoprenoid-derived molecules (i.e.,

quinones), lipids, and carotenoid cleavage. PGs are likely to play a role in various metabolic

pathways and oxidative stress defense91,92. Overall, our results demonstrate that isoprene

suppression initiates various stress response reactions. Indeed, previous biochemical and

metabolomic analyses showed higher levels of total ascorbate and #-tocopherol as well as

enhanced lipid peroxidation in unstressed leaves of NE poplars17.

Not annotated proteins

In our study involving poplar chloroplasts, a set of 13 proteins without assigned function but

with identified full length genes were detected (Figure 3 and in the Supplementary Table S1).

Twelve out of 13 share very high similarity (more than 70%) with Arabidopsis proteins. These

proteins were related to thylakoid lumen protein (TLP18.3), pentapeptide repeats, BNR/Asp-

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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 NE lines (log2 = -1.5 and -2.4, respectively, p = 0.036

and 0.035, t-test) (Table 2). Recently, it was shown that the TLP18.3 protein is involved in

the regulation of both the degradation/synthesis steps of the PSII D1 protein and in the

assembly of the PSII monomers in the grana stack93. Under standard growth conditions, the

absence of TLP18.3 protein does not lead to a severe collapse of the PSII complexes,

suggesting a redundancy of proteins assisting the repair steps of PSII to secure its

functionality. The Arabidopsis mutants lacking the TLP18.3 protein possess a higher

susceptibility of PSII to photoinhibition.

4. CONCLUSION

We aimed to elucidate whether the suppression of isoprene biosynthesis and emission

modifies the abundance of chloroplast proteins and how this modification may influence the

plant functionality. The lack of PcISPS and consequently of isoprene implied that the down-

regulation of proteins related to photosynthesis light reactions, redox regulation and oxidative

stress defense, and several proteins with structural activity that are responsible for lipid

metabolism alteration occurred (Figure 5). These changes were the consequences of

alternative defense mechanisms such as photorespiration and non-photochemical quenching

that needed to compensate for the absence of isoprene. Indeed, the lower amounts of

peroxiredoxin and ascorbate peroxidase indicated their over-oxidation in the presence of

increased levels of ROS. Overall, the present proteomic analysis revealed that the absence

of isoprene in poplar leaves remodels the chloroplast protein profile to cope against oxidative

stress. The present data strongly support the idea that isoprene improves thylakoid

membrane structure and regulates the production of ROS.

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ASSOCIATED CONTENT

Supporting Information

Supplemental Table S1 provides all proteins identified using the ICPL technique; Log 2

ratios, description and functional annotation of the proteins are listed. Supplemental Table S2

provides sub-plastidial localization of the ICPL-identified proteins. Supplemental Table S3

provides the full data of protein identification: peptide sequence, ion score, number of missed

cleavage, and exact molecular mass (MH+) of each peptide. Supplemental Figure S1

provides score and loading plots of PCA of chloroplasts proteins identified using the ICPL

technique. Supplemental Figure S2 provides representative Acid-urea-PAGE of histones in

chloroplasts isolated from IE (WT/EV) and NE (RA1/RA2) poplar leaves (A) as well as the

quantification of band intensities performed with ImageJ (B). Supplemental Figure S3

provides representative blue native-PAGE of poplar thylakoid membrane protein complexes

isolated from IE (WT/EV) and NE (RA1/RA2) poplar leaves (A) as well as the quantification

of band intensities performed using ImageJ (B). This material is available free of charge via

the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION

Corresponding Author

*E-mail: jp.schnitzler@helmholtz-muenchen.de. Phone: +49 89 3187 2413. Fax: +49 89 3187

The authors declare no competing financial interest.

Funding Sources

The present study was supported by Alexander-von-Humboldt-Foundation (individual

fellowship to VV).

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ACKNOWLEDGEMENTS

We would like to thank to Werner Heller and Katja Block (both Helmholtz Zentrum München)

for critical reading, discussions and comments on the manuscript.

ABBREVIATIONS

ALDH, aldehyde dehydrogenase; ATP, adenosine triphosphate; BN-PAGE, blue native

polyacrylamide gel electrophoresis; DMADP, dimethylallyl diphosphate; ETR, electron

transport rate; HPL, hydroperoxide lyases; ICPL, isotope-coded protein labeling; 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 adenine dinucleotide phosphate; NE, non-isoprene

emitting; OPLS, Orthogonal Partial Least Square regression;PAGE, polyacrylamide gel

electrophoresis; PAPs, plastidic lipid-associated proteins; PCA – Principal Component

Analysis; PGs, plastoglobuli; PSI – photosystem I; PSII - photosystem II; SDS-PAGE –

sodium dodecyl sulfate polyacrylamide gel electrophoresis; SHMT - serine hydroxymethyl

transferase; VIP - Variable of Importance for the Projection; THF, thylakoid formation protein;

TLP, thylakoid lumen protein; WT, wild type

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FIGURE LEGENDS

Figure 1. Representative Coomassie blue-stained polyacrylamide gel displaying 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 NE grey poplar

plants. Red arrows indicate the excised bands that were further subjected to LC-MS/MS and

Label-free analyses.

Figure 2. Functional summary of the 119 chloroplast proteins identified using the ICPL

technique in NE (RA1/RA2) and IE (WT/EV) grey poplar lines. Identified proteins are

clustered in 8 categories, according to their functions.

Figure 3. Score (A) and loading (B) plots of Orthogonal Partial Least Squares (OPLS) of

chloroplast proteins identified using the ICPL technique and analyzed by LC-MS/MS (x-axis =

PC1 = 22%; y-axis = PC2 = 21%). A – NE (RA1/RA2) – blue triangles, IE (WT/EV) – red

circles; B - each functional group of proteins is indicated with different symbols, zoomed

symbols with a dot represent the significantly different proteins between the IE and NE grey

poplar plants; red circle – histones; yellow square – ribosomal; blue diamond – structural;

green triangle-up – photosynthesis; purple star – metabolism; dark red circle – stress; dark

grey square – others; grey triangle-down – unknown (not annotated or not identified).

Figure 4. The number of proteins with low (grey bars) or high (dark red bars) levels in NE

grey poplar lines. Suppression of the PcISPS protein by RNAi caused changes in protein

regulation of both NE lines (RA1/RA2) compared to the IE lines (WT/EV). Low or high

abundance of chloroplast proteins was counted when log2 [!(NE)/!(IE)] was < -1 or >1,

respectively. In total, the abundance of 31 proteins out of 119 was unchanged. The

chloroplast proteins were grouped based on their function.

Figure 5. Sub-organelle structure of NE poplar chloroplasts. The suppression of isoprene

biosynthesis modified the protein profiles. Blue symbols with the arrow pointing down

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indicate the spatial loci where proteins were down-regulated and red symbols with the arrow

pointing up indicate the spatial loci where proteins were up-regulated. Isoprene suppression

caused significant down-regulation of specific proteins related to photosynthetic electron

transport, redox regulation and oxidative stress defense, and several proteins with structural

role, which responsible for the alteration of lipid metabolism. 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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LEGENDS OF SUPPLEMENTARY FIGURES AND TABLES

Supplemental Figure S1. Score (A) and loading (B) plots of Principal Component Analyses

(PCA) of chloroplasts proteins identified using the ICPL technique and analyzed by LC-

MS/MS (x-axis = PC1 = 27%; y-axis = PC2 = 21% of explained variance). A - blue triangles -

NE (RA1/RA2), red circles - IE (WT/EV) poplar plants; B - each functional group of proteins

is indicated with different symbols; red circle – histones; yellow square – ribosomal; blue

diamond – structural; green triangle-up – photosynthesis; purple star – metabolism; dark red

circle – stress; dark grey square – others; grey triangle-down – unknown (not annotated or

not identified).

Supplemental Figure S2. Isoprene suppression caused the up-regulation of histone

proteins. (A) Representative acid-urea-PAGE of histones in chloroplasts isolated from IE

(WT/EV) and NE (RA1/RA2) grey poplar leaves. Chloroplast histones were stained with

Silver. (B) The quantification of histone band intensities was performed with using ImageJ.

Asterisk indicate statistically significant difference from WT (Student’s t-test, *P<0.05)

Supplemental Figure S3. (A) Representative blue native-PAGE of poplar thylakoid

membrane protein complexes isolated from IE (WT/EV) and NE transgenic grey poplar

leaves (RA1/RA2). (B) The quantification of band intensities was performed using ImageJ.

Asterisk indicate statistically significant difference from WT (Student’s t-test, *P<0.05)

Supplemental Table S1. Chloroplast proteins identified by isotope-coded protein labeling in

IE (ET/EV) and in NE (RA1/RA2) grey poplar leaves. Proteins were analyzed with LC-

MS/MS. Log2 ratios are presented. Down- (negative numbers) or up-regulated (positive

numbers) proteins were counted when log2 was < -1 or >1, respectively. Empty spaces

mean that in one of the samples the protein was not found. MW - molecular mass, kDa; A -

Coverage, %; B - Unique peptides used for identification.

Supplemental Table S2. Chloroplast proteins identified by isotope-coded protein labeling in

IE and NE grey poplar leaves. Associated information is extracted from PPDB

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(http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx; http://www.grenoble.prabi.fr/at_chloro).

Empty spaces mean that an information was not found. TargetP – prediction of subcellular

localization using TargetP, Curated localization (PPDB) – curated subcellular or sub-

plastidial localization as stated in PPDB.

Supplemental Table S3. Full data set for protein identification (Protein Description, Protein

Group Accessions, Sequence of Peptides, QuanUsage, IonScore, # of Missed Cleavages,

molecular mass MH+ [Da]) by isotope-coded protein labeling in IE and NE grey poplar

leaves. The acquired MS/MS spectra were searched against the Populus trichocarpa

genome database (Phytozome v9.1, http://www.phytozome.net) using the Mascot search

engine (version 2.3.02; Matrix Science) with the following parameters: a precursor mass

error tolerance of 10 ppm and a fragment tolerance of 0.6 D. One missed cleavage was

allowed.

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Table 1A. Chloroplast proteins identified in bands (!50kDa) excised from 1-DE gel. Log 2 ratios are given to show different expression level of

extracted proteins. Down- (negative numbers) or up-regulated (positive numbers) proteins were counted when log2 = "(NE)/"(IE) was < -1 or >1,

respectively, in NE 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.

Accession A B C Log2 Description Function TargetP Localization

POPTR_0005s13860.1 2 67 0.02 2.5 PsbO - manganese-stabilizing protein OEC C THY

POPTR_0001s21740.1 3 161 0.02 3.1 Chl a/b binging protein Light harvesting C THY

POPTR_0019s09140.1 7 325 0.01 2.8 Chl a/b binging protein Light harvesting C THY

POPTR_0010s12680.1 POPTR_0008s12550.1

11 533 0.01 3.7 ATP synthase Proton-transporting ATP C THY

Metabolism

POPTR_0008s00350.4 POPTR_0002s10990.1 POPTR_0008s00350.2 POPTR_0008s00350.3

4 630 0.03 7.2

Serine hydroxymethyl transferase L-serine metabolism C

POPTR_0005s11500.1 2 99 0.01 2.7 Oxidoreductase NAD-binding domain Oxidoreductase activity C STR-THY

POPTR_0014s09220.1 2 99 0.02 3.7 Hydroxyproline-rich glycoprotein Hydrolyzation C THY

POPTR_0008s08410.1 POPTR_0008s08400.1

2 340 0.01 3.7 Phosphoglycerate kinase Glycolysis C STR-THY

POPTR_0004s16920.1 POPTR_0007s13800.1

2 119 0.02 2.0 Fructose-bisphosphate aldolase Glycolysis C STR

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Table 1!. (Continued from previous page)

POPTR_0009s04180.1 6 425 0.02 -2.7 NmrA-like family Nitrogen metabolism C THY-ENV

POPTR_0014s15390.1 14 882 0.01 -2.1 Hydroperoxide lyase, Cit P450 Electron carrier activity

Stress related proteins

POPTR_0018s11820.1 4 167 0.01 6.8 Aldehyde dehydrogenase family Oxidoreductase activity

POPTR_0002s01080.1 POPTR_0005s10340.1 POPTR_0005s27300.1 POPTR_0007s08910.1

6 275 0.01 5.2

Catalase Response to oxidative stress

Peroxisome

Others

POPTR_0001s08770.1 2 83 0.03 2.4 Elongation factor Tu GTP binding domain GTPase activity C STR-THY

Not identified

POPTR_2555s00200.1 5 356 0.01 4.8 Not identified - - -

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Table 1B. Chloroplast proteins identified in bands (!15kDa) excised from 1-DE gel. Log 2 ratios are given to show different expression level of

extracted proteins. Down- (negative numbers) or up-regulated (positive numbers) proteins were counted when log2 = "(NE)/"(IE) was < -1 or >1,

respectively, in NE grey poplar plants (RA1/RA2). A - Peptides used for quantitition; B - Confidence score; C - Anova (p). The information about

subcellular 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.

Ribosomal proteins

POPTR_0004s16450.1 2 71 0.02 7.4 S25 ribosomal protein Structural constituent of ribosome C

POPTR_0007s04160.1 2 425 0.01 2.1 PSI reaction center PSI C THY

POPTR_0004s09910.1 2 141 0.05 1.9 Rubisco small subunit Rubisco activity C ENV-STR

POPTR_0002s18110.1 3 104 0.02 2.7 TM phosphoprotein No functional annotations C

POPTR_0001s25670.1 POPTR_0009s04820.1

2 99 0.01 2.5 Thylakoid phosphoprotein No functional annotations C THY

Proteins with transporting and binding functions

POPTR_0008s02430.1 2 84 0.002 4.6 Copper chaperone Metal ion transport and binding - -

POPTR_0002s01740.1 3 171 0.005 -4.1 Copper binding proteins Electron carrier activity C THY

Not identified

POPTR_0001s42970.1 7 446 0.01 2.9 Not identified - - -

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Table 2. Variable Importance for the Projection (VIP) for each discriminant protein that significantly separate non-isoprene emitting (NE) from

isoprene emitting (IE) Populus canescens samples in the OPLS model (p = 0.021; CV-ANOVA). Analysis was performed on ICPL data obtained

by isolating chloroplast proteins from two NE (RA1/RA2) and two IE (WT/EV) lines. Discriminant proteins from OPLS model were classified in

order of importance by means of VIP scores. High VIP score indicates high importance. SE = Standard Error of jack-knifing method; Log2 =

[!!(NE)/!!(IE)] shown in negative or positive numbers for down- or up-regulated proteins, respectively, related to isoprene suppressed plants (RA1 and RA2);

Significant differences between RA1/RA2 and WT/EV were tested by t-test (* = p < 0.05 and ** = p < 0.01). Associated information is extracted from

PPDB (http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx, http://www.grenoble.prabi.fr/at_chloro). TargetP – prediction of subcellular localization

using TargetP; Curated localization (PPDB) – curated subcellular or sub-plastidial localization as stated in PPDB).

Accession VIP SE Description Function Log2 TargetP Curated localization (PPDB)

Histone proteins

POPTR_0008s13170.1 1.926 0.640 Linker histone H1 and H5 family DNA synthesis 3.6 ** -

POPTR_0010s23720.1 1.924 0.618 Histone H2B DNA synthesis 3.5 ** -

POPTR_0006s08230.1 1.829 0.732 Histone 2A DNA synthesis 2.8 ** -

POPTR_0015s04390.1 1.472 0.901 Linker histone H1 and H5 family DNA synthesis 5.5 * C

POPTR_0011s13490.1 1.349 0.895 Histone 2A DNA synthesis 1.7 * C

POPTR_0012s04580.1 1.303 0.917 Linker histone H1 and H5 family DNA synthesis 2.2 * C

Ribosomal proteins

POPTR_0018s03390.1 1.629 0.842 Zinc knuckle Nucleic acid binding 4.6 ** C Plastid ribosome

POPTR_0018s11170.1 1.552 0.886 Ribosomal protein L17 Protein biosynthesis 2.3 * C Plastid ribosome

POPTR_0006s23770.1 1.484 0.840 50S Ribosomal protein L4/L1 family Protein biosynthesis 2.7 * C Plastid ribosome

POPTR_0002s04420.1 1.461 1.010 Ribosomal protein S19 Protein biosynthesis 12.8 * C Plastid ribosome

POPTR_0001s33710.1 1.435 0.998 Ribosomal L27 protein Protein biosynthesis 3.6 * C Plastid ribosome

POPTR_0006s11420.1 1.368 0.926 Ribosomal protein L17 Protein biosynthesis 3.5 ns C Plastid ribosome

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Table 2. (Continued from the previous page)

Accession VIP SE Description Function Log2 TargetP Curated location (PPDB)

POPTR_0004s22660.1 1.275 1.066 50S Ribosomal protein L9 Protein biosynthesis 1.6 ns C Plastid ribosome

POPTR_0007s14040.1 1.201 1.072 Ribosomal Proteins L2 Protein biosynthesis 11.2 ns C Plastid ribosome

POPTR_0016s08250.1 1.120 1.118 Zinc knuckle Nucleic acid binding 12.7 ns C Plastid ribosome

POPTR_0014s15120.1 1.114 0.971 60S Ribosomal protein L6 Protein biosynthesis 3.0 ns C Plastid ribosome

Proteins with structural activity

POPTR_0001s12230.1 1.217 1.013 FKBP-type Protein folding - 1.5 * C Thylakoid-periferal-lumenal-side

POPTR_0019s14050.1 1.183 0.846 Thylakoid formation protein TM organization and biogenesis

- 1.5 * C Envelop; plastid stroma; thylakoid-periferal-stromal-side

POPTR_0001s02040.1 1.156 0.867 PAP fibrillin Structural molecule activity

- 0.8 ns C Plastoglobules; thylakoid-periferal-stromal-side

POPTR_0001s41530.1 1.154 1.118 Cyclophilin type Protein folding - 1.7 ns C Thylakoid-periferal-lumenal-side

POPTR_0002s22560.1 1.679 0.841 ATPase family ATPase activity - 2.9 * C Thylakoid-integral

POPTR_0001s44210.1 1.660 1.106 PsbQ - oxygen evolving enhancer protein 3

OEC -0.9 ** C Thylakoid-periferal-lumenal-side

POPTR_0004s01470.1 1.604 0.402 ATP synthase Proton-transporting ATP synthase complex

- 1.8 * C Thylakoid-periferal-stromal-side

POPTR_0002s05660.1 1.331 1.015 PsbP OEC - 2.4 * C Thylakoid-periferal-lumenal-side

POPTR_0003s14870.1 1.300 1.042 PSI reaction center subunit III PSI - 1.7 ns C Thylakoid-integral

POPTR_0013s14520.1 1.270 1.002 Cyt b6-F complex Fe-S subunit Oxidoreductase activity

- 2.4 * C Thylakoid-periferal-lumenal-side

POPTR_0014s13560.1 1.249 1.246 ATPase family - with various cellular activities

ATPase activity -4.9 ns C Thylakoid-integral

POPTR_0019s11720.1 1.197 0.848 ATP synthase delta (OSCP) subunit

Proton-transporting ATP synthase complex

- 0.7 ** C Thylakoid-periferal-stromal-side

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Table 2. (Continued from the previous page)

Accession VIP SE Description Function Log2 Target P Curated location (PPDB)

POPTR_0004s03160.1 1.187 1.066 PsbQ - oxygen evolving enhancer protein 3

OEC -1.7 * C Thylakoid-periferal-lumenal-side

Metabolism

POPTR_0008s00350.4 1.888 0.957 Serine hydroxymethyltransferas L-serine metabolism 7.7 ** M Mitochondria

POPTR_0002s10990.1 1.615 1.082 Serine hydroxymethyltransferas L-serine metabolism 7.4 * - -

POPTR_0001s25160.1 1.260 1.009 NmrA-like family Nitrogen metabolism - 4.4 ns C Thylakoid

POPTR_0001s34960.1 1.210 1.033 Trypsin, Serine protease Proteolysis and peptidolysis

- 1.7 * C Thylakoid-peripheral-lumenal-side

Stress related proteins

POPTR_0006s13980.1 1.238 0.897 Peroxiredoxin, AhpC/TSA family Antioxidant activity - 0.8 ** C Plastid

POPTR_0005s17350.1 1.477 0.652 L-ascorbate peroxidase Response to oxidative stress

- 2.4 * C Thylakoid-peripheral-lumenal-side

Other proteins

POPTR_0018s08850.1 1.198 1.124 Peptidase S26 Signal peptidase - 2.9 * C Thylakoid-peripheral-lumenal-side

No functional annotation or not identified

POPTR_0013s02740.1 1.426 0.630 Thylakoid lumen 18.3 kDa protein

Unknown function - 1.6 * C Thylakoid-peripheral-lumenal-side

POPTR_0005s04090.1 1.399 0.672 Thylakoid lumen 18.3 kDa protein

Unknown function - 1.5 * C Thylakoid-peripheral-lumenal-side

POPTR_0008s05900.1 1.170 1.162 Thylakoid lumenal 15 kDa protein

Family not named - 2.4 * C Thylakoid-peripheral-lumenal-side

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Figure 1

96x112mm (300 x 300 DPI)

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Figure 2

127x88mm (300 x 300 DPI)

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Figure 3

249x375mm (300 x 300 DPI)

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Figure 4

108x70mm (300 x 300 DPI)

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Figure 5

164x131mm (300 x 300 DPI)

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Graphical Abstract

254x190mm (72 x 72 DPI)

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

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

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