Knocking Down of Isoprene Emission Modifies the Lipid Matrix of Thylakoid Membranes and Influences the Chloroplast Ultrastructure in Poplar

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene-emitting (IE) and nonisoprene-emitting (NE) poplar (Populus × 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 amount of unsaturated fatty acids, particularly linolenic acid in NE chloroplasts, was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects photosystem II photochemical efficiency. The low photosystem II photochemical efficiency in NE plants was negatively correlated with nonphotochemical quenching and the energy-dependent component of nonphotochemical quenching. Transmission electron microscopy revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained fewer grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in IE species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.Isoprene is globally the most abundant biogenic hydrocarbon constitutively emitted from many plant species (Guenther et al., 2012). It has been proposed that leaf isoprene emission is an important adaptation for plants, conferring tolerance to different environmental constraints (Vickers et al., 2009; Loreto and Schnitzler, 2010; Loreto and Fineschi, 2014). However, biogenic isoprene emission represents a nontrivial carbon loss in plants, 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 isoprene are still ambiguous, and the true role of isoprene emission remains elusive. Different approaches and techniques have been used to determine whether and how the cost of this expensive carbon emission is matched by the accomplishment of the physiological function in planta. It has been shown that isoprene might quench and/or regulate reactive oxygen and nitrogen species formation (Behnke et al., 2010a; Velikova et al., 2012), thereby indirectly providing a general antioxidant action (for review, see Vickers et al., 2009; Loreto and Schnitzler, 2010) and stabilizing thylakoid membrane structures due to the lipophilic properties of this molecule (Sharkey et al., 2001; Velikova et al., 2011).Protein and pigment-protein complexes are assembled and embedded in a lipid matrix, which has a unique lipid composition. The thylakoid lipid bilayer of chloroplasts is characterized by a high proportion of galactolipids with one (monogalactosyldiacylglycerol [MGDG]) or two (digalactosyldiacylglycerol [DGDG]) Gal molecules (Joyard et al., 2010). MGDGs are the primary constituents (approximately 50%) of thylakoid membrane glycerolipids, followed by DGDGs (approximately 30%), sulfoquinovosyldiacylglycerol (approximately 5%–12%), and phosphatidylglycerol (approximately 5%–12%; Kirchhoff et al., 2002). Galactolipids contain a large proportion of polyunsaturated fatty acids, and consequently, the thylakoid membrane is a relatively fluid system (Gounaris and Barber, 1983) compared with other biological membranes. The fluidity of the thylakoid membrane is essential for photosynthetic processes.The thylakoid membranes are highly organized internal membrane chloroplast systems that conduct the light reactions of photosynthesis. These membranes comprise pigments and proteins organized in complexes. Thylakoid membranes are arranged into stacked and unstacked regions called grana and stroma thylakoids, respectively, differentially enriched in PSI and PSII complexes (Mustárdy et al., 2008). The spatial separation of the PSI and PSII complexes in the stacked and unstacked membrane regions and the macromolecular organization of PSII in stacked grana thylakoids are self-organizing processes and important features to maintain the functional integrity of the photosynthetic apparatus (Kirchhoff et al., 2007).It is not known how changes in the lipid matrix affect lipid-protein interactions and vice versa, and how membrane macroorganization ensures the efficient diffusion of protein complexes associated with plant adaptation to the changing environment remains elusive. The isoprene impact on thylakoid intactness and functionality has been assessed using different biophysical techniques (Velikova et al., 2011). Thermoluminescence data demonstrated that the position of the main peak (Qb peak; Sane, 2004) was upshifted approximately 10° in isoprene-emitting (IE) plants, suggesting modifications in the lipid environment due to the presence of isoprene in heterologous Arabidopsis (Arabidopsis thaliana) plants expressing the isoprene synthase gene from poplar (Populus spp.). It was also shown that isoprene improves the stability of PSII light-harvesting complex II (LHCII) through the modification of pigment-protein complex organization in thylakoid membranes (Velikova et al., 2011). Moreover, we recently showed that knocking down isoprene emission in poplar remodels the chloroplast proteome (Velikova et al., 2014). The lack of isoprene resulted in the down-regulation of proteins associated with the light reactions of photosynthesis, redox regulation, and oxidative stress defenses and several proteins responsible for lipid metabolism (Velikova et al., 2014).In this study, we focused on the lipid composition of thylakoid membranes in IE and nonisoprene-emitting (NE) poplar (Populus × canescens) leaves. Specifically, we determined whether the translational suppression of isoprene synthase in NE leaves influences the lipid matrix of thylakoids and how this phenomenon affects membrane structure and function. Here, we provide evidence that the suppression of isoprene biosynthesis in poplar (1) reduced total galactolipids, phospholipids (PLs), and linolenic fatty acid (18:3), (2) altered the chloroplast ultrastructure, and (3) stimulated photoprotective mechanisms.