Knocking Down of Isoprene Emission Modi es the Lipid Matrix ......1996; Brilli et al., 2007; Teuber...

Knocking Down of Isoprene Emission Modifies the LipidMatrix of Thylakoid Membranes and Influences theChloroplast Ultrastructure in Poplar1

Violeta Velikova, Constanze Müller, Andrea Ghirardo, Theresa Maria Rock, Michaela Aichler,Axel Walch, Philippe Schmitt-Kopplin, and Jörg-Peter Schnitzler*

Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria (V.V.); andResearch Unit Analytical BioGeoChemistry (C.M., T.M.R., P.S.-K.), Research Unit Environmental Simulation,Institute of Biochemical Plant Pathology (V.V., A.G., J.-P.S.), and Research Unit Analytical Pathology (M.A.,A.W.), Helmholtz Zentrum München, 85764 Neuherberg, Germany

ORCID ID: 0000-0002-9825-867X (J.-P.S.).

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses. Here, we studied the lipidcomposition of thylakoid membranes and chloroplast ultrastructure in isoprene-emitting (IE) and nonisoprene-emitting (NE) poplar(Populus 3 canescens). We demonstrated that the total amount of monogalactosyldiacylglycerols, digalactosyldiacylglycerols,phospholipids, and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amountof unsaturated fatty acids, particularly linolenic acid in NE chloroplasts, was associated with the reduced fluidity of thylakoidmembranes, which in turn negatively affects photosystem II photochemical efficiency. The low photosystem II photochemicalefficiency in NE plants was negatively correlated with nonphotochemical quenching and the energy-dependent component ofnonphotochemical quenching. Transmission electron microscopy revealed alterations in the chloroplast ultrastructure in NEcompared with IE plants. NE chloroplasts were more rounded and contained fewer grana stacks and longer stroma thylakoids, moreplastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in IEspecies, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.

Isoprene is globally the most abundant biogenichydrocarbon constitutively emitted from many plantspecies (Guenther et al., 2012). It has been proposed thatleaf isoprene emission is an important adaptation forplants, conferring tolerance to different environmentalconstraints (Vickers et al., 2009; Loreto and Schnitzler,2010; Loreto and Fineschi, 2014). However, biogenicisoprene emission represents a nontrivial carbon loss inplants, particularly under stress conditions (Fang et al.,1996; Brilli et al., 2007; Teuber et al., 2008; Ghirardo

et al., 2014), and the reason(s) why plants emit isopreneare still ambiguous, and the true role of isopreneemission remains elusive. Different approaches andtechniques have been used to determine whether andhow the cost of this expensive carbon emission ismatched by the accomplishment of the physiologicalfunction in planta. It has been shown that isoprenemight quench and/or regulate reactive oxygen andnitrogen species formation (Behnke et al., 2010a;Velikova et al., 2012), thereby indirectly providing ageneral antioxidant action (for review, see Vickers et al.,2009; Loreto and Schnitzler, 2010) and stabilizing thyla-koid membrane structures due to the lipophilic propertiesof this molecule (Sharkey et al., 2001; Velikova et al., 2011).

Protein and pigment-protein complexes are assem-bled and embedded in a lipid matrix, which has aunique lipid composition. The thylakoid lipid bilayer ofchloroplasts is characterized by a high proportion ofgalactolipids with one (monogalactosyldiacylglycerol[MGDG]) or two (digalactosyldiacylglycerol [DGDG])Gal molecules (Joyard et al., 2010). MGDGs are theprimary constituents (approximately 50%) of thylakoidmembrane glycerolipids, followed by DGDGs (approxi-mately 30%), sulfoquinovosyldiacylglycerol (approximately5%–12%), and phosphatidylglycerol (approximately 5%–12%; Kirchhoff et al., 2002). Galactolipids contain a largeproportion of polyunsaturated fatty acids, and conse-quently, the thylakoid membrane is a relatively fluid

1 This workwas supported by the Alexander vonHumboldt Foun-dation (to V.V.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Jörg-Peter Schnitzler ([email protected]).

V.V. conceived the original research plan, designed the experimentwith inputs from J.-P.S. and A.G., performed the experiments withsupport from A.G., C.M., T.M.R., and M.A., analyzed the data andindependently wrote the article with extensive inputs from A.G. andJ.-P.S.; C.M. carried out the lipid analyses and analyzed the data; A.G.performed the statistical analyses and wrote the article; T.M.R. pro-vided technical assistance to C.M.; M.A. performed the transmissionelectron microscopy study; A.W. and P.S.-K. supervised and comple-mented the writing; J.-P.S. conceived the original research plan, su-pervised the experiments, and wrote the article.

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system (Gounaris and Barber, 1983) compared with otherbiological membranes. The fluidity of the thylakoid mem-brane is essential for photosynthetic processes.

The thylakoid membranes are highly organized inter-nal membrane chloroplast systems that conduct the lightreactions of photosynthesis. These membranes com-prise pigments and proteins organized in complexes.Thylakoid membranes are arranged into stacked andunstacked regions called grana and stroma thylakoids,respectively, differentially enriched in PSI and PSII com-plexes (Mustárdy et al., 2008). The spatial separation ofthe PSI and PSII complexes in the stacked and unstackedmembrane regions and the macromolecular organizationof PSII in stacked grana thylakoids are self-organizingprocesses and important features to maintain the func-tional integrity of the photosynthetic apparatus (Kirchhoffet al., 2007).

It is not known how changes in the lipid matrix affectlipid-protein interactions and vice versa, and howmembrane macroorganization ensures the efficient dif-fusion of protein complexes associated with plant ad-aptation to the changing environment remains elusive.The isoprene impact on thylakoid intactness and func-tionality has been assessed using different biophysicaltechniques (Velikova et al., 2011). Thermoluminescencedata demonstrated that the position of the main peak(QB peak; Sane, 2004) was upshifted approximately 10°in isoprene-emitting (IE) plants, suggesting modificationsin the lipid environment due to the presence of isoprenein heterologous Arabidopsis (Arabidopsis thaliana) plantsexpressing the isoprene synthase gene from poplar(Populus spp.). It was also shown that isoprene improvesthe stability of PSII light-harvesting complex II (LHCII)through the modification of pigment-protein complexorganization in thylakoid membranes (Velikova et al.,2011). Moreover, we recently showed that knocking downisoprene emission in poplar remodels the chloroplastproteome (Velikova et al., 2014). The lack of isopreneresulted in the down-regulation of proteins associatedwith the light reactions of photosynthesis, redox regula-tion, and oxidative stress defenses and several proteinsresponsible for lipid metabolism (Velikova et al., 2014).

In this study, we focused on the lipid composition ofthylakoid membranes in IE and nonisoprene-emitting(NE) poplar (Populus 3 canescens) leaves. Specifically, wedetermined whether the translational suppression of iso-prene synthase in NE leaves influences the lipid matrix ofthylakoids and how this phenomenon affects membranestructure and function. Here, we provide evidence that thesuppression of isoprene biosynthesis in poplar (1) reducedtotal galactolipids, phospholipids (PLs), and linolenic fattyacid (18:3), (2) altered the chloroplast ultrastructure, and(3) stimulated photoprotective mechanisms.

RESULTS

Lipid, Fatty Acid, and Malondialdehyde Analyses

Chloroplast membranes isolated from NE poplarhad significantly lower (253%; P, 0.01) lipid contents

than the membranes of IE plants (Fig. 1). In both NE andIE plants, the major molecular species of MGDG were18:2-18:3 and 18:3 dimers. In DGDG, the major molec-ular species were n16:0-18:3 and 18:3 dimers. Linolenicacid (18:3) was the major fatty acid of both IE and NEchloroplast membranes, but the content of this fatty acidwas consistently much lower in NE than in IE plants(Table I; Supplemental Fig. S1). The fatty acid analysisalso revealed significantly lower palmitic (n16:0), linoleic(18:2), and stearic (n18:0) acid contents in NE plantscompared with IE plants. In the fraction of the PLs, thephosphatidic acid (18:1) levels were lower in NE plants.

The concentration of malondialdehyde (MDA), theprincipal product of polyunsaturated fatty acid perox-idation, was lower in chloroplasts isolated from NEplants than in those isolated from IE poplar (Fig. 2), as-sociated with a lower concentration of polyunsaturatedfatty acids in NE chloroplasts (Table I; SupplementalFigure S1).

Chloroplast Ultrastructure Observations and ProteinAbundance in Photosynthetic Membranes

To determine whether the different lipid concentra-tions and changes in lipid composition affect the chlo-roplast structure, thin leaf segments obtained from themiddle region of IE and NE leaves were subjected totransmission electron microscopy (TEM) analyses. Rep-resentative micrographs of chloroplasts from IE and NEspecimens are shown in Figures 3 and 4.

The typical elliptic shape of mesophyll chloroplastswas more oval in NE than in IE specimens (Fig. 3). Themesophyll cells of IE leaves are characterized by a well-developed inner membrane system, comprising granaof different sizes and relatively long stromal thylakoids.IE chloroplasts contained single, midsize starch granulesand less numerous peroxisomes, and these organelleswere associated with relatively small-sized mitochon-dria (Figs. 3, A and B, and 4, A and B).

Figure 1. Lipid contents in isolated chloroplasts of IE (wild type [WT]and empty vector [EV]) and NE (RNA interference transgenic lines,RA1 and RA2) poplar. Error bars display the SE (n = 4). Asterisks indi-cate significant differences from the wild type: **, P , 0.01.

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Conversely, the chloroplasts of NE plants were char-acterized by a less developed membrane system, withshorter and fewer grana stacks and longer stroma thy-lakoids (Fig. 4, C–E). NE chloroplasts contained moreplastoglobules and smaller starch grains than IE chlo-roplasts (Fig. 3, C and D). NE chloroplasts were also inclose structural contact with mitochondria through rel-atively large associative regions (Fig. 4C). A relativelylarge number of NE chloroplasts were undeveloped(data not shown).To further understand how the structural changes

were related to the protein enrichment in photosyntheticmembranes, we extracted mass spectrometry (MS) datafrom our recent proteome study (Velikova et al., 2014).The concentrations of PSI-PSI reaction centers (RCI) andPSII-PSII reaction centers (RCII) were strongly decreasedin NE chloroplasts compared with IE chloroplasts(Fig. 5, A and B). Lower protein abundance of PSII-RCIIcorrelated with fewer stacks (Fig. 4F). Chlorophyll con-centrations in the NE lines RA1 and RA2 were alsosignificantly reduced (Fig. 5C).

Chlorophyll Fluorescence

We measured light- and dark-adapted states of chlo-rophyll fluorescence in IE and NE poplar plants grownunder ambient greenhouse conditions (Fig. 6). Therewas no significant difference in maximal PSII activitybetween IE and NE plants (data not shown), suggestingthat the efficiency of PSII, when all reaction centers wereopen, was similarly high in both groups of plants.However, NE leaves exhibited significantly lower pho-tosystem II photochemical efficiency (FPSII) and PSIIredox state (qL) and higher nonphotochemical quench-ing (NPQ) and energy-dependent quenching (qE; Fig. 6).Importantly, the true efficiency of PSII (FPSII) waslower in NE compared with IE, indicating that asmaller fraction of the absorbed light energy was usedfor photochemistry. Indeed, the accurate indicator of the

PSII redox state, qL (Baker, 2008), was significantly lowerin NE, suggesting that the fraction of open PSII reactioncenters was much lower in these mutants (Figs. 5 and 7;Supplemental Fig. S2).

Multivariate Data Analyses

We examined the involvement of lipid content andfatty acid composition in NE and IE chloroplasts, com-pared with chlorophyll fluorescence measurements, MDAcontents, and previously described proteomic differ-ences (Velikova et al., 2014). Principal component analysis(PCA) showed that the isoprene emission traits reflectedthe largest variance of the measured data, indicatedby the separation between NE and IE samples in thefirst two principal components (Supplemental Fig.S2; explained variance, PC1 = 51%, PC2 = 16%), where

Table I. Fatty acid composition (mg mg21 chlorophyll) of the main lipid classes in C16 to C23 saturated (:0) and unsaturated (:1, :2, and :3) compoundsin chloroplasts of IE (wild type [WT] and empty vector [EV]) and NE (RA1 and RA2) poplar plant lines

Fatty acids are designated as the total number of carbon atoms followed by the number of double bonds and their location (omega) after the colon:n16:0, palmitic acid; 16:1, palmitoleic acid; n18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, linolenic acid; and n23:0, tricosanoicacid. Saturated straight-chain fatty acids are indicated with an n. Means6 SE are shown; n = 4. Asterisks and boldface indicate significant differencesfrom the wild type: *, P , 0.05; **, P , 0.01; and ***, P , 0.001.

Line Lipid Classes n16:0 16:1 n18:0 18:1 18:2 18:3 20:3 n23:0

WT MGDG 21.1 6 3.9 2.8 6 0.6 61.8 6 13.6 411.1 6 29.0DGDG 77.5 6 22.1 13.2 6 5.4 10.7 6 3.3 265.1 6 17.6 1.4 6 0.6PLs 17.7 6 3.6 3.5 6 1.2 4.1 6 0.5 12.9 6 4.7 7.6 6 1.3 151.9 6 16.5 4.0 6 1.0

EV MGDG 19.5 6 6.0 2.6 6 0.9 54.9 6 19.5 350.3 6 75.2DGDG 56.0 6 15.9 8.7 6 2.7 8.8 6 3.2 230.3 6 59.4 1.4 6 0.6PLs 17.8 6 2.2 2.4 6 0.4 4.3 6 0.2 13.8 6 6.4 9.1 6 0.5 120.9 6 3.0 4.1 6 1.9

RA1 MGDG 12.7 6 1.7* 1.7 6 0.2 38.0 6 8.9* 253.2 6 20.0*DGDG 37.8 6 9.0** 5.7 6 1.5* 5.7 6 1.6* 154.2 6 30.1*** 0.5 6 0.2*PLs 9.1 6 2.7 1.5 6 0.5 3.3 6 0.5 3.7 6 0.7 5.7 6 0.8 66.1 6 9.0* 0.7 6 0.1*

RA2 MGDG 11.9 6 1.1** 1.9 6 0.4 35.9 6 4.9* 236.0 6 8.8*DGDG 35.0 6 1.8** 5.4 6 0.4* 4.7 6 0.7* 139.5 6 1.5*** 0.5 6 0.1*PLs 11.5 6 1.2* 0.9 6 0.3 2.7 6 0.3** 3.6 6 1.3 3.2 6 0.4 72.6 6 6.6** 2.5 6 0.5

Figure 2. MDA levels in isolated chloroplasts of IE (wild type [WT] andempty vector [EV]) and NE (RA1 and RA2) poplar. Error bars displaythe SE (n = 4). Asterisks indicate significant differences from the wildtype: *, P , 0.05.

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Q2 is the fraction of Y variation predicted by the Xmodel and according to cross-validation, and R2(X) andR2(Y) are the fraction of X variation modeled, using theX and Y model, respectively. Additionally, there wereno appreciable differences between the two groups, asboth RA1 and RA2 lines and WT and EV lines clusteredtogether in the NE and IE groups, respectively.

We performed a discriminant analysis to determinewhich of the analyzed parameters were significantlyaffected by the suppression of isoprene biosynthesis andto assess the relative importance of these parameters indistinguishing NE from IE plants (Fig. 7). The OPLSanalysis indicated, overall, that differences between NEand IE chloroplasts reflect the lipid composition, fattyacid and MDA contents, chlorophyll fluorescence pa-rameters, and chloroplastic proteins associated withphotosynthesis or cell structure. Each singular factor hada different importance (Fig. 7C). Specifically, the mostimportant (high VIP values) variables negatively corre-lated with NE (positive and high correlation coefficientvalues) were MDA, qL, PL, saturated and unsaturated

fatty acids, MGDG and DGDG, and photosyntheticproteins (Fig. 7, B and C). Importantly, the unsaturatedfatty acid 18:3 (linolenic acid) was strongly negativelycorrelated with NE in all lipids (MGDG, DGDG, andPL). Additionally, linolenic acid was well correlatedwith the lipid degradation product MDA, detected inboth MGDG and DGDG. The PL content was highlycorrelated with photosynthetic proteins, namely PSIproteins, ATP synthase, cytochrome b6 f, and an intrinsicprotein of PSII oxygen-evolving complex, PsbP. Con-versely, NPQ and qE were strongly and positively cor-related with the NE genotype.

The computed OPLS model was reliable, resulting ina significant (P = 0.00061, cross-validated ANOVA)cross-validated predictive ability of Q2(Y) = 84% todistinguish NE from IE samples and a cross-validated

Figure 3. A to D, Transmission electron micrographs of representativechloroplast cross sections taken from the intact leaves of IE (wild type[WT] and empty vector [EV]; A and B) and NE (RA1 and RA2; C and D)poplar. E and F, Height-length ratio (E) and average number of starchgrains (F) in IE and NE chloroplasts. CW, Cell wall; GT, granal thyla-koids; M, mitochondrion; P, plastoglobuli; S, stroma; SI, starch gain.Bars = 1 mm at 6,3003 magnification.

Figure 4. A to D, Transmission electron micrographs of representativechloroplast cross sections taken from the intact leaves of IE (wild type[WT] and empty vector [EV]; A and B) and NE (RA1 and RA2; C and D)poplar. E and F, Average number of stacks per chloroplast (E) andcorrelation between PSII-RCII protein abundance (10 peptides; forprotein accession nos., see “Materials and Methods”) and number ofstacks (F). CW, Cell wall; GT, granal thylakoids; M, mitochondrion; P,plastoglobuli; S, stroma; SI, starch gain. Bars = 500 nm in A and Cat 10,0003 magnification; bars = 200 nm in B and D at 20,0003magnification.


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goodness of R2(X) = 44%, r2 = 93%, R2(Y) = 100% usingonly the first principal component.

DISCUSSION

Suppression of Isoprene Biosynthesis Decreases theChloroplastic Lipid Content and AltersChloroplast Ultrastructure

One of the proposed biological functions of isopreneis the stabilization of thylakoid membrane struc-tures through modification of the lipid environmentand organization of the pigment-protein complexes inthylakoid membranes (Velikova et al., 2011). Indeed,

several clear alterations were evident in thylakoid mem-brane lipids and fatty acid composition due to thetranslational suppression of isoprene synthase activityin poplar plants. The most important changes in NEchloroplasts were the absolute decrease in the contentsof galactolipids (MGDG and DGDG) and PLs throughthe down-regulation of the unsaturated fatty linolenicacid (18:3). The functional role of MGDGs in the bio-activity of various membrane proteins is well known(Lee, 2003, 2004): a mutant with a defective MGDGSYNTHASE1 is unable to produce photosyntheticallyactive membranes (Kobayashi et al., 2013). MGDGs areessential for the efficient activity of violaxanthin deep-oxidase (Yamamoto and Higashi, 1978). The ability ofthe lipid mixture to segregate into bilayer and non-bilayer phases might regulate the protein content inchloroplast membranes (Garab et al., 2000). Indeed, wecould demonstrate that the proteins related to photo-synthesis were strongly down-regulated in NE com-pared with IE plants (Figs. 7 and 8; Velikova et al., 2014).

DGDGs are the predominant bilayer lipid species inthylakoid membranes of higher plants (Joyard et al.,2010). They exert structural functions and improve thethermal stability of membranes, particularly at hightemperatures (Krumova et al., 2010). DGDGs bindto PSII (Loll et al., 2007) through the formation ofhydrogen bonds with Tyr in PSII (Gabashvili et al.,1998), and DGDGs are also important for binding ofextrinsic proteins required for the stabilization of theoxygen-evolving complex (Sakurai et al., 2007). Ourdata clearly indicate that the suppression of isoprenebiosynthesis significantly diminished the level ofDGDGs in poplar chloroplasts, which was accompa-nied by a reduction of the RCI and RCII concentrations(Fig. 5, A and B), PsbP and PsbQ protein subunitsof PSII, and light-harvesting complex I and LHCII(Velikova et al., 2014). When the thylakoid membraneprotein complexes were resolved by blue native-PAGE,the protein patterns of the two groups of poplar lineslooked quite similar in content and intensity of theindividual bands (supplemental figure S3 in Velikovaet al., 2014). However, semiquantitative analysis of theindividual protein bands showed that the levels of PSI,the PSII dimer, ATP synthase, the PSII monomer, andthe cytochrome b6 f complex were slightly reduced in NEcompared with IE chloroplasts (supplemental figure S3in Velikova et al., 2014).

Decreased lipid and protein levels were associatedwith changes in the ultrastructure of the chloroplastsfrom NE plants, suggesting a role for isoprene bio-synthesis in the structural organization of plastidicmembranes. These results are consistent with previousstudies that indicated a role for MGDGs and DGDGsin the structure of thylakoid membranes (Dörmannet al., 1995; Jarvis et al., 2000). In this study, we observeda significant reduction of grana stacks per chloroplast inNE compared with IE poplar lines (Fig. 4, E and F),which was related to an important decrease in PSI andPSII proteins (Fig. 5, A and B). Alterations in proteinstoichiometry could exert a direct influence on the

Figure 5. Protein abundance of PSI-RCI (four peptides; for protein ac-cession nos., see “Materials and Methods”; A), PSII-RCII (10 peptides; forprotein accession nos., see “Materials andMethods”; B), and chlorophyllcontent (C) in IE (wild type [WT] and empty vector [EV]) and NE (RA1and RA2) poplar plants. Protein abundance represents the sum of MSdata extracted from our proteome study (Velikova et al., 2014). Error barsdisplay the SE (n = 4). Asterisks indicate significant differences from thewild type: **, P , 0.01; and ***, P , 0.001.




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thylakoid membrane ultrastructure (Pribil et al., 2014),in particular, the assembly of PSII and LHCII intosupercomplexes and megacomplexes (Kou�ril et al., 2012).It was demonstrated (Labate et al., 2004) that the con-stitutive expression of the pea (Pisum sativum) Lhcb1 genein transgenic tobacco (Nicotiana tabacum) plants leadsto increased grana stacking, indicating that increasedconcentrations of LHCII result in more stacking.

Typically, chloroplast membranes have a uniquelipid composition characterized by a high proportionof galactolipids containing a large portion of triunsa-turated fatty acids (C16 or C18; Joyard et al., 2010). Thehigh content of triunsaturated fatty acids guaranteesthe high fluidity of the thylakoid membranes and theprecise allocation of the photosynthetic machinery toefficiently acquire light energy (Gounaris and Barber,1983). The level of membrane viscosity is an importantfactor for photosynthetic performance (e.g. providingoptimal conditions for the diffusion of hydrophobicmolecules, such as plastoquinol; Kirchhoff et al., 2000,2002) or membrane intrinsic protein complexes (e.g.during state transitions; Allen and Forsberg, 2001;Tikkanen et al., 2008). The low linolenic acid (18:3)content in all lipid fractions from NE chloroplasts in-dicates that, in the absence of isoprene, the thylakoidmembrane fluidity is reduced, which in turn nega-tively affects the efficiency of PSII photochemistry (Fig.6). A low level of unsaturation in thylakoid membranesmakes PSII extremely susceptible to photoinhibitionand causes a significant reduction in the content of theD1 protein (the reaction center protein) at high irradi-ance (Kanervo et al., 1995), suggesting that membranefluidity is a critical factor for PSII D1 protein turn-over. Moreover, we detected in NE chloroplast lower

amounts of phosphatidic acid (18:1), an importantintermediate in lipid biosynthesis (Joyard et al., 2010)with functions as a signaling lipid (Testerink andMunnik, 2005, 2011; Horváth et al., 2012; McLoughlinand Testerink, 2013).

We observed that the lower level of linolenic acid(18:3) detected in NE chloroplasts was associated withsignificantly lower MDA chloroplast content in NEcompared with IE poplar (Fig. 2; Table I). Previousstudies have reported that MDA is primarily derivedfrom triunsaturated fatty acids in chloroplasts (Yamauchiet al., 2008; Schmid-Siegert et al., 2012). MDA can beused as an oxidative stress marker when plants are ex-posed to unfavorable conditions (Esterbauer et al., 1991)but is also present in healthy plants (Weber et al., 2004;Mène-Saffrané et al., 2007, 2009). At the whole-leaf ex-tract level, MDA levels were higher in NE poplar(Behnke et al., 2010b), which agrees with their higherconcentrations of linolenic acid (Way et al., 2013). Theproduction of MDA from triunsaturated fatty acidsserves to adsorb a portion of the reactive oxygen species(Mène-Saffrané et al., 2009); therefore, MDA is a by-product in the mechanism of cell protection.

Another remarkable observation in this study wasthe increased abundance of plastoglobules in NE com-pared with IE chloroplasts. Plastoglobules are lipopro-tein particles containing isoprenoid-driven metabolites(primarily prenylquinones, including plastoquinone andphylloquinone), tocopherols (Vidi et al., 2006), and struc-tural proteins (plastoglobulins; Bréhélin et al., 2007). Theincreased number of plastoglobules in NE comparedwith IE chloroplasts might reflect the higher levels ofa-tocopherol in leaves of these lines, as demonstratedpreviously (Behnke et al., 2010b).

Figure 6. FPSII (A), redox state of PSII (qL; B),NPQ (C), and qE (D) of IE (wild type [WT] andempty vector [EV]) and NE (RA1 and RA2)poplar plants at growth conditions. Valuesrepresent means of five to seven different plantsout of three independent experiments (n = 15–21; SE is given). Photosynthetic parameters aredescribed in “Materials and Methods.” Aster-isks indicate significant differences from thewild type: *, P , 0.05; **, P , 0.01; and ***,P , 0.001.


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Electron tomography revealed that plastoglobulesare physically coupled to thylakoid membranes via ahalf-lipid bilayer, providing a direct lipid conduit formetabolite channeling between plastoglobules and thy-lakoid membranes (Austin et al., 2006). Moreover,plastoglobules are involved in different secondary me-tabolism pathways, stress responses, and the develop-ment of thylakoids (Bréhélin et al., 2007). In a previousstudy, we showed that the lipid-associated proteinfibrillin content, comprising lipid-associated proteinsand fibrillins, is negatively correlated with the NEplants (Velikova et al., 2014). This observation suggeststhe involvement of isoprene in the maintenance ofthylakoid membranes.

Interestingly, we observed larger associative zonesbetween chloroplasts and mitochondria in NE plants.Mitochondria are instrumental for the generation ofmetabolic energy in eukaryotic cells, and these organ-elles deliver intermediates to support different meta-bolic pathways, including photosynthesis (Jacoby et al.,2012). One of the important benefits of mitochondria-chloroplast interactions is the optimization of photo-synthetic carbon assimilation through the coordinatedproduction and utilization of ATP and NADPH, theinduction of photosynthesis, the activation of enzymes,and the maintenance of metabolite levels (Raghavendraand Padmasree, 2003). We propose that the larger as-sociative zones between chloroplasts and mitochondriain NE plants reflect a higher demand for assimilatorypower (ATP and NADPH) compared with IE plants.Indeed, the down-regulation of the cytochrome b6 fcomplex in NE chloroplasts indicates the inhibition ofATP production, associated with the down-regulationof extrinsic subunits of ATP synthase in isoprene-suppressed lines (Velikova et al., 2014). Because isoprene

Figure 7. Score (A), loading (B), and correlation coefficient plots (C) oforthogonal partial least squares (OPLS) of lipid classes, fatty acidcomposition, and MDA contents in isolated chloroplasts, chlorophyllfluorescence parameters measured in intact leaves (NPQ, FPSII, qE, andqL), and chloroplast proteins related to photosynthesis and proteinswith structural activity. A, IE plants (wild type [WT] and empty vector[EV]), gray circles; NE plants (RA1 and RA2), white triangles. B, Eachparameter is indicated with a different symbol: dark-gray circles,MGDG; dark-gray squares, DGDG; gray triangles, PL; dark-gray cir-cles with a dot, MGDG fatty acids; dark-gray squares with a dot,DGDG fatty acids; gray triangles with a dot, PL fatty acids; green di-amonds, MDA; red stars, NPQ; red down triangles, qE; blue squares,qL; blue stars, proteins with structural activity; and green stars, proteinsrelated to photosynthesis. C, Parameter colors match those of thesymbols in B. Only discriminant data with variable of importance forthe projection (VIP) . 1 (for all except proteins) and VIP . 0.5 (pro-teins) are presented. Model fitness: Q2(Y) = 84%, R2(X) = 44%, r2 =93%, and R2(Y) = 100% using one principal component; P = 0.00061,cross-validated ANOVA.

Figure 8. Schematic overview of the changes in chloroplast ultrastruc-ture, lipid composition, protein abundance, and PSII photochemicalefficiency triggered by the suppression of isoprene biosynthesis andemission in poplar plants. VDE, Violaxanthin deepoxidase.





functions as a protective molecule against oxidativestress (Loreto and Schnitzler, 2010), the isoprene sup-pression in NE plants might be balanced by enhancingother compensatory protective mechanisms such as pho-torespiration and oxidative electron transport, which areboth mediated by mitochondria.

Recent analyses demonstrated that the suppression ofisoprene biosynthesis dramatically reduces carbonfluxes throughout the methylerythritol 4-phosphatepathway (Ghirardo et al., 2014), followed by the reallo-cation of carbon to other pathways, which in turn in-duces profound metabolic changes, particularly in lipidbiosynthesis (Way et al., 2013; Kaling et al., 2015). Thus,at the cellular level, lipid metabolism is up-regulated inNE leaves, whereas at the subcellular level, as shownhere, low levels of galactolipids and PLs comprise thestructure of NE chloroplasts. These results suggest thatthe crucial needs of NE plants to maintain the cor-rect fluidity of thylakoid membranes induces theup-regulation of lipid metabolism, including lipid in-termediates, likely compensating for the low levels ofgalactolipids and PLs packed into chloroplast mem-branes. Thus, isoprene might (1) directly improve thefluidity of thylakoid membranes in synergy with gal-actolipids or (2) indirectly affect lipid biosynthesis ortrafficking into the chloroplast. Whether the lack ofisoprene function or the alteration of the plastidic iso-prenoid pathway itself induces changes in the chloro-plast lipid levels, thereby affecting membrane fluidity,should be examined in future studies.

Functional Changes Relate to Structural Alterations inNE Chloroplasts

We measured light- and dark-adapted states ofchlorophyll fluorescence in IE and NE poplar plantsgrown under unstressed conditions in order to assesswhether the structural changes have functional signifi-cance with regard to the differences in the ability to emitisoprene. Our results showed thatFPSII was lower in NEplants than in IE plants, consistent with previous ob-servations that the proteins involved in photosyntheticprocesses are down-regulated in NE plants, potentiallydecreasing the efficiency of the photochemistry ofphotosynthesis (Fig. 5; Velikova et al., 2014). The lowerFPSII values in NE plants were negatively correlated toNPQ, a protective mechanism for the removal of excessexcitation energy within pigment complexes and theinhibition of the formation of free radicals (Demmig-Adams and Adams, 2006).

Higher NPQ levels in concert with restricted electrontransport rate between both photosystems and a re-duced plastoquinone pool have been shown (Härtelet al., 1998) to be accompanied by DGDG modificationsin the Arabidopsis mutant (dgd1). Moreover, in thismutant, PSI showed an increased capacity for cyclicelectron transfer and a reduced capacity for state tran-sitions (Ivanov et al., 2006). Similar to the dgd1 mutant(Dörmann et al., 1995), the NE poplar plants showed a

lower DGDG content, modified chloroplastic ultra-structure, increased NPQ, restricted electron trans-port rate (Behnke et al., 2007), and decreased totalchlorophyll content (Fig. 5C; Behnke et al., 2013; Wayet al., 2013; Ghirardo et al., 2014).

The NPQ comprises qE (i.e. dependent on the en-ergization of thylakoid membranes), state transitions(Minagawa, 2011), photoinhibition quenching (Mülleret al., 2001), and zeaxanthin-dependent quenching(Nilkens et al., 2010). qE is the most important andwell-characterized component of NPQ. This transitionis triggered through the acidification of the thylakoidlumen (Ruban et al., 2012), which in turn leads to theprotonation of violaxanthin deepoxidase, for the con-version of violaxanthin to zeaxanthin, and PsbS, a poly-peptide of the PSII-associated light-harvesting complex(Kiss et al., 2008; Murchie and Niyogi, 2011).

Here, we showed higher values of qE in NE plants,which might reflect a particular conformation of theLHCII complex resulting from chlorophyll and/orxanthophyll-protein interactions (Horton et al., 2005).Indeed, we observed that many proteins associated withphotosynthesis are less abundant in NE chloroplasts(Figs. 5 and 7; Velikova et al., 2014). This lack of pho-tosynthetic proteins could lead to specific conforma-tional changes, which in turn could determine the higherqE in NE poplar. However, the supramolecular organi-zation of the PSII antenna involves numerous interac-tions between proteins, suggesting that the changes inthese interactions (Garab and Mustardy, 1999; Hortonet al., 2005) could be responsible for the increase in NPQwe observed in NE plants. Indeed, with circular di-chroism spectroscopy, it has been shown that isoprenedeficiency inhibits the formation of the chirally orga-nized macrodomains. This effect in turn decreases thethermal stability of thylakoid membranes (Velikovaet al., 2011). We also observed the significant down-regulation of the cytochrome b6f complex in NE lines(Velikova et al., 2014), which might inhibit the produc-tion of ATP in isoprene-suppressed plants. The increaseof qE in NE lines might reflect the optimization of elec-tron transport and ATP synthesis through the modula-tion of the cyclic electron transfer around PSI, theactivation state of ATP synthase, and the partitioning ofthe proton-motive force between DpH and the mem-brane electrical potential (Horton et al., 2005).

CONCLUSION

The proposed biological functions of isoprene in plantshave been associated with the ability of this molecule toaffect thylakoid membrane organization and reduce theformation of reactive oxygen species, conferring toler-ance to heat and oxidative stress. It has been hypothe-sized that isoprene improves the thermal stability ofthylakoid membranes by affecting the membrane lipidcomposition (Velikova et al., 2011). Here, we provideddirect evidence of the relationship between isopreneemission and the level of main lipid classes and their


Velikova et al.



fatty acid composition, and we characterized the struc-tural organization of the photosynthetic machinery in IEand NE poplar genotypes. The suppressed isoprene pro-duction in NE plastids was associated with the reducedamount of galactolipids and PLs, the lower level of themajor fatty acid (18:3), and the altered chloroplastultrastructure (Fig. 8). The suppression of isoprenebiosynthesis causes considerable metabolic changes,particularly in lipid biosynthesis (Way et al., 2013;Kaling et al., 2015), and significant alterations in thechloroplast proteome (Velikova et al., 2014). The ma-jority of the plastidic and mitochondrial proteome isencoded in the nuclear genome, and there is a continu-ous exchange of forward information from nucleus toorganelle (anterograde) and of backward informationfrom organelle to nucleus (retrograde; Pfannschmidt,2010). According to the retrograde signaling concept,based on the available experimental data, signals origi-nating in chloroplasts and/or mitochondria modulatenuclear gene expression (Leister, 2012). These signalsoriginate from carotenoid biosynthesis, reactive oxygenspecies, photosynthetic redox processes, and changes inthe pool of metabolites (Pfannschmidt, 2010; Leister,2012). The plastidic signals identified so far have beenassociated with specific stress conditions. It is likely thatthe comprehensive changes in the metabolome (Wayet al., 2013; Kaling et al., 2015), liposome, proteome(Velikova et al., 2014), and ultrastructure of the chloro-plasts in NE poplar (Fig. 8), as well as the distinctphysiological behavior of these plants, reflect finelytuned retrograde signaling. The precise mechanisms forthe transmission of the changes in chloroplast to thenucleus in NE plant cells remain elusive.

MATERIALS AND METHODS

Plant Material

In this study, we used the same gray poplar (Populus 3 canescens; syn. Populustremula 3 Populus alba) genotypes as utilized in previous chloroplast proteomeresearch (Velikova et al., 2014), namely, two IE lines (WT and EV) and two NElines (RA1 and RA2). The empty vector line was used to ensure that the differencesbetween NE and IE plants reflected specific alterations in the isoprene synthasegene and not a more general genetic manipulation effect. The plants were grownin a greenhouse as described previously (Velikova et al., 2014). Briefly, the ambienttemperature was 25°C/20°C with a relative humidity of 50%/60% and a photo-period of 16 h of day/8 h of night. The plants were fertilized weekly with Triabon(Compo) and Osmocote (Scotts Miracle-Gro; 1:1 [v/v]; 10 g L21 soil).

Four-month-old plants were used for the experiments. Fully expandedleaves (ninth node from the apical meristem) from six to seven different plants,considered as biological replicates, were used for physiological, biochemical,and structural studies. The chloroplasts were isolated as described previously(Velikova et al., 2014) and used for lipid and MDA analyses.

Lipid Extraction Procedure

The total lipids from chloroplasts were extracted according to Bligh and Dyer(1959). All procedures were performed in dim light using chilled solvents(containing 0.01% [w/v] butylated hydroxytoluene) and glassware. The chloro-plast samples (0.5 mL) were mixed with chloroform:methanol (1:2 [v/v]; 1.9 mL)for approximately 2 min; subsequently, 0.625 mL of chloroform and 0.625mL of distilled water were added. The lower chloroform phase, containing thelipids, was removed, and aliquots were transferred into vials and exsiccatedunder N2. The residues were weighed and calculated for total lipids.

Gas Chromatography-MS Analysis of PL FattyAcid Composition

PL fatty acids were analyzed as described previously (Behnke et al., 2013;Way et al., 2013). Briefly, the PL fatty acids were separated from other lipidsusing a silica-bonded phase column (MEGA-BE-SI, 2 g 12 mL21, 20/PK, BondELUT; Agilent Technologies). Fatty acid methyl esters were obtained aftermild alkaline hydrolysis. Myristic acid was used as an internal standard forgas chromatography analysis. Unsubstituted fatty acid methyl esters weremeasured using the 5973MSD gas chromatograph-mass spectrometer (AgilentTechnologies) coupled with a combustion unit to an isotope ratio massspectrometer (DeltaPlus; Thermo Electron) and identified using the estab-lished fatty acid libraries and characteristic retention times of pure standards.The fatty acids were named according to the total number of carbon atomsand double bonds. Saturated straight-chain fatty acids are indicated with an n.

Ultraperformance Liquid Chromatography ElectrosprayTime-of-Flight MS of Galactolipids

Lipids were dissolved in 1 mL of liquid chromatography-MS-grademethanol (Fluka). MGDG and DGDG contents were analyzed using the ultra-performance liquid chromatography electrospray time-of-flight MS system(maXis; Bruker). Aliquots of 2.5 mL of each sample were analyzed in threetechnical replicates in randomized order.

The chromatographic separation was achieved on a C18 ACQUITYUPLCBEH column, 50 mm, 2.1 mm, and 1.7 mm (Waters), using a gradient elution.The composition was changed from 50% to 92% B for 10 min and maintainedfor an additional 10 min, then changed to 100% B for 1 min and maintained for5 min. The flow was set to 0.4 mL h21. Mobile phase A comprised water:isopropyl alcohol (95:5, v/v), and mobile phase B comprised acetonitrile:iso-propyl alcohol (95:5, v/v). A format of 0.001 mM sodium (Sigma-Aldrich) wasadded to both mobile phases. This method has been published previously forprofiling photosynthetic glycerol lipids (Xu et al., 2010).

MGDGs and DGDGs were detected as sodium adducts through positiveelectrospray ionization. The instrument was calibrated with ESI Tune Mix(Agilent Technologies). Acquired spectra were internally calibrated andexported to GENEDATA software for chromatographic alignment and peakpicking. MGDGs and DGDGs were identified based on the retention timesand detected exact masses (mass error , 0.01 D).

MGDG and DGDG standards (Larodan) were used to evaluate the ana-lytical performance and determine the quality control, which was injected 10times in the beginning for column conditioning and after every 10th sample tovalidate the measuring performance.

MDA Content

The lipid peroxidation level in extracted chloroplast samples was quantifiedafter measuring the MDA content using the thiobarbituric acid-reactive sub-stances assay according to Hodges et al. (1999). The chloroplast sample (0.100mL) was mixed with 1.2 mL of 80% (w/w) ethanol (containing 0.01% [w/v]butylated hydroxytoluene) and sonicated in a water bath sonicator for 3 min,followed by centrifugation at 5,000g for 10 min at 4°C. An aliquot of theobtained supernatant (0.5 mL) was mixed with the same volume of 0.65% (w/v)thiobarbituric acid solution containing 20% (w/v) TCA. Another aliquot of thesupernatant (0.5 mL) was mixed with 0.5 mL of 20% (w/v) TCA, representingthe zero control. The mixture was heated at 95°C for 30 min. The reaction wasterminated after incubation in an ice bath. The cooled mixture was centrifuged at10,000g for 10 min at 4°C, and the absorbance of the supernatant was measuredat 532, 600, and 440 nm (Perkin Elmer). MDA equivalents were calculatedaccording to Hodges et al. (1999):

½A532þTBA 2A600þTBA 2 ðA532-TBA 2A600þTBAÞ� ¼ A ð1Þ

½ðA440þTBA 2A600þTBAÞ3 0:0571� ¼ B ð2Þ

MDA equivalents�nmol mL–1

� ¼ ðA2BÞ=157000Þ3 106 ð3Þ

Protein and Chlorophyll Analyses

For the calculation of the abundance of reaction center proteins in PSI andPSII, we used the chloroplast proteome data published by Velikova et al.





(2014). Peak intensities of peptides identified as RCI (protein accessionnos. POPTR_0008s15100.1, POPTR_0006s27030.1, POPTR_0003s14870.1, andPOPTR_0002s25510.2) and RCII (protein accession nos. POPTR_0011s03390.1,POPTR_0004s03160.1, POPTR_0005s22780.1, POPTR_0002s05660.1, POPTR_0005s01430.1,POPTR_0005s27800.3, POPTR_0002s05720.1, POPTR_0002s25810.1, POPTR_0001s44210.1,and POPTR_0006s26270.1) were summed and expressed per mg of chlorophyll.

The chlorophyll content was measured in isolated chloroplast suspensionafter extraction with 80% (v/v) ice-cold acetone. Absorbance at 663 and 646 nmwas detected to determine chlorophyll a and b concentrations, calculatedaccording to Porra et al. (1989).

Chlorophyll Fluorescence Measurements

The chlorophyll fluorescence parameters were measured on intact leavesusing a MINI-PAM Photosynthesis Yield Analyzer (Heinz-Walz). The leaveswere dark adapted for 15 min prior to the determination of the minimal (Fo) andmaximal (Fm) chlorophyll fluorescence, and subsequently, the leaves were ex-posed to actinic light (430 mmol m22 s21). After steady-state fluorescence wasobtained, a saturating pulse was applied to determine the maximum fluores-cence in the light (Fm9). TheFPSII was calculated from (Fm9 – F9)/Fm9 (Genty et al.,1989). The redox state of PSII was assessed based on the parameter qL = (Fq9/Fv9)/(Fo9/F9), where F9 is the fluorescence emission from the light-adapted leaf,Fv9 is variable fluorescence from the light-adapted leaf, and Fq9 is the differencein fluorescence between Fm9 and F9 (Baker, 2008). Fo9 was estimated using thefollowing equation: Fo9 = Fo/[(Fv/Fm) + Fo/Fm9] (Oxborough and Baker, 1997).The NPQ was calculated as NPQ = (Fm 2 Fm9)/Fm9 (Bilger and Björkman, 1991).The NPQ relaxation kinetics in the dark was used to calculate qE. qE wasassigned as a fast-relaxing component (within the first 2 min of dark relaxationafter switching off the actinic light), calculated as qE = (Fm99 – Fm9)/F99m, 2min dark(Zaks et al., 2013).

TEM

Leaf segments (1 mm2) were cut from the middle of the leaves for TEManalyses. The segments were fixed in 2.5% (v/v) glutaraldehyde (electron mi-croscopy grade) in 0.1 M sodium cacodylate buffer, pH 7.4 (Science Services),postfixed in 2% (v/v) aqueous osmium tetraoxide (Dalton, 1955), dehydrated inan ethanol gradient (30%–70%), stained with uranyl acetate (2% in 70% ethanol),dehydrated in an ethanol gradient (70%–100%) and propylene oxide (100%),embedded in Epon (Merck), and cured for 24 h at 60°C. Semithin sections (300nm) were cut and stained with Toluidine Blue. Ultrathin sections of 50 nm werecollected onto 200-mesh copper grids before examination using TEM (ZeissLibra 120 Plus; Carl Zeiss). The images were acquired using a Slow Scan CCDcamera and iTEM software (Olympus Soft Imaging Solutions).

Statistical Analyses

Correlation analyses between different data sets of PL and galactolipid(MGDG and DGDG) contents, fatty acid compositions, chlorophyll fluorescenceparameters, MDA content, the data groups, and IE or NE genotypes wereperformed using PCA and OPLS from the software package SIMCA-P (version13.0.0.0; Umetrics). In addition, we included the chloroplastic protein contentsassociated with photosynthesis and structure (Velikova et al., 2014) to correlatelipids to proteins. Because the proteomic data originated from three samples foreach plant genotype (containing six to seven different leaves each of the threesamples), multivariate analyses were performed using only the data matchingthe same three samples used for both proteomic and PL analyses. Galactolipids,MDA, and chlorophyll fluorescence measurements were obtained from moreand different extracts; therefore, only the data from three samples were takenrandomly and used for these analyses. We added the means of all biologicalreplicates to examine the correlations between genotypes using data originatingfrom different leaf extracts. The resulting matrix size, therefore, was 78 3 16(variables 3 observations). Thus, our analyses could correlate any data valuewith an isoprene emission trait (IE and NE plant genotype), but correlationsbetween/within variables could be achieved only using data from the same leafmaterial (i.e. within PL and proteins, MGDG, DGDG, and MDA, and withinchlorophyll fluorescence data).

The multivariate data analyses followed the established procedures toanalyze MS data as described previously (Ghirardo et al., 2005, 2012;Kreuzwieser et al., 2014; Vanzo et al., 2014; Velikova et al., 2014). The isopreneemission trait was selected as the Y variable for the OPLS analysis by settingNE = 1 and IE = 0. The X variables were centered, and each type of data was

block-wise scaled with 1 SD21, considering the different number of X variablesin each group of data. Each calculated significant principal component wasvalidated using full cross validation, with 95% confidence level on parameters.The regression model OPLS was further tested for significance using cross-validated ANOVA (Eriksson et al., 2008). Variables showing VIP valuesgreater than 1 and jack-knifing method uncertainty bars smaller than the re-spective VIP values were defined as discriminant variables to distinguish IEfrom NE samples. For the proteomics data, containing a much higher numberof variables, the VIP threshold was set to 0.5. The statistical significance of thedifferences between the means of discriminant variables and the functionaland structural parameters measured in NE and IE plants was additionallyevaluated using Student’s t test and at an a level of 0.05, unless otherwisestated.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Lipid content and fatty acid composition in iso-lated chloroplasts of IE (WT and EV) and NE (RA1 and RA2) poplar.

Supplemental Figure S2. Score and loading plots of all PCA parametersanalyzed (lipid and fatty acid composition, MDA, NPQ, FPSII, qE, qL),proteins related to photosynthesis, and proteins with structural activity.

Received April 27, 2015; accepted May 13, 2015; published May 14, 2015.

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