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these findings point to a possible link between photosynthesis and germination ..... as well as the heavy metabolic cost for the plant to build up and degrade this.
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Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana Guillaume Allorent1,2,3,4, Sonia Osorio5, Joseph Ly Vu6, Denis Falconet1,2,3,4, Juliette Jouhet1,2,3,4, Marcel Kuntz1,2,3,4, Alisdair R. Fernie5, Silva Lerbs-Mache1,2,3,4, David Macherel6,7,8, Florence Courtois1,2,3,4 and Giovanni Finazzi1,2,3,4 1

Laboratoire de Physiologie Cellulaire & Vegetale, Unite Mixte de Recherche 5168, Centre National de la Recherche Scientifique, F-38054 Grenoble, France; 2Universite Grenoble-Alpes,

F-38054 Grenoble, France; 3Commissariat a l’Energie Atomique et Energies Alternatives, Institut de Recherches en Technologies et Sciences pour le Vivant, F-38054 Grenoble, France; 4Unite Sous Contrat 1359, Institut National Recherche Agronomique, F-38054 Grenoble, France; 5Max-Planck Institut f€ur Molekulare Pflanzenphysiologie, Am M€ uhlenberg 1, 14476 Golm-Potsdam, Germany; 6Institut National Recherche Agronomique, Institut de Recherche en Horticulture et Semences, F-49045 Angers, France; 7Agrocampus Ouest, F-49045 Angers, France; 8Universite d’Angers, SFR 4207 QUASAV, F-49045 Angers, France

Summary Author for correspondence: Florence Courtois Tel: +33 438783753 Email: [email protected] Received: 27 June 2014 Accepted: 10 August 2014

New Phytologist (2015) 205: 707–719 doi: 10.1111/nph.13044

Key words: Arabidopsis thaliana, cyclic electron flow, development, embryo, germination, photosynthesis, seed.

 In this work, we dissect the physiological role of the transient photosynthetic stage

observed in developing seeds of Arabidopsis thaliana.  By combining biochemical and biophysical approaches, we demonstrate that despite similar features of the photosynthetic apparatus, light absorption, chloroplast morphology and electron transport are modified in green developing seeds, as a possible response to the peculiar light environment experienced by them as a result of sunlight filtration by the pericarp. In particular, enhanced exposure to far-red light, which mainly excites photosystem I, largely enhances cyclic electron flow around this complex at the expenses of oxygen evolution.  Using pharmacological, genetic and metabolic analyses, we show that both linear and cyclic electron flows are important during seed formation for proper germination timing. Linear flow provides specific metabolites related to oxygen and water stress responses. Cyclic electron flow possibly adjusts the ATP to NADPH ratio to cope with the specific energy demand of developing seeds.  By providing a comprehensive scenario of the characteristics, function and consequences of embryonic photosynthesis on seed vigour, our data provide a rationale for the transient building up of a photosynthetic machinery in seeds.

Introduction In the angiosperm life cycle, seeds allow the mature embryos to await favorable conditions for germination and growth, thus increasing the potential of successful establishment of a new generation. Seed formation starts immediately following the double fertilization event. In Arabidopsis thaliana, three stages have been described, which are classified based on the numbers of days after fertilization (DAF; note that in this paper 5 DAF is written DAF5) and are completed within 18–21 d depending on the growth conditions (Allorent et al., 2013a). The first stage, termed embryogenesis (DAF0–5), is characterized by multiple cell divisions in the embryo until the heart stage is reached. The second stage, maturation (DAF6–12), involves accumulation of storage compounds and embryo differentiation (Baud et al., 2002). During the third stage, late maturation (DAF13–18), acquisition of desiccation tolerance is achieved (Ooms et al., 1993). In A. thaliana seeds (as well as in other oil-seed plants), the maturation phase is characterized by the differentiation of a Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

photosynthetic stage (Eastmond et al., 1996). Given that seeds are sink organs supplied with carbohydrates and other nutrients from the maternal tissues (Hua et al., 2012), the role of this photosynthesis phase is intriguing. Several functions for seed photosynthesis have been proposed so far. Based on biochemical and physiological studies mostly in Brassica napus, photosynthesis has been shown to play a direct role in lipid storage accumulation (Eastmond et al., 1996; Ruuska et al., 2004; Goffman et al., 2005). Moreover, in situ imaging with O2 sensors in photosynthetic crop seeds has shown that the O2 concentration decreases from the seed coat towards the centre, leading to hypoxic conditions within the mature seed (Borisjuk & Rolletschek, 2009). It has been proposed therefore that release of photosynthetic O2 inside the seed should counteract O2 consumption, fuelling respiration and reducing the NO : O2 ratio, to properly regulate developmental stress responses (Borisjuk & Rolletschek, 2009). Finally, analysis of Arabidopsis mutants (Inaba & Ito-Inaba, 2010) and in silico gene expression studies in B. napus (Hsu et al., 2010) have suggested an additional role for photosynthesis, that New Phytologist (2015) 205: 707–719 707 www.newphytologist.com

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is, proper embryogenesis, likely linked to plastid metabolic activities. Here, we investigated the molecular adaptation of the photosynthetic apparatus in Arabidopsis seeds to their prevailing environmental settings, and examined the metabolic and physiological impacts of seed photosynthesis. We found that the overall organization of the photosynthetic complexes was similar in seeds and in leaves. However, specific features of photosynthesis were affected in seeds, probably reflecting an adaptation to the particular illumination conditions caused by sunlight filtration by the pericarp (the silique wall). In particular, enrichment in farred light, which is mainly absorbed by photosystem I (PSI), correlates with changes in the thylakoid structure and in enhanced cyclic electron flow around PSI. We also demonstrate that inhibition of photosynthesis in the embryo is detrimental for seed germination timing and storability. This does not stem from impaired accumulation of main storage compounds, but rather from changes in the concentration of specific metabolites related to both oxygen and water responses. Eventually mutant analysis indicates a correlation between cyclic flow activity and seed lot germination rate. Overall this study underlines several important functions of plastid photosynthesis, which ultimately affect seed quality and germination timing, thereby unravelling the important consequences of this process at the ecological and agronomic level.

Materials and Methods Plant material and growing conditions Arabidopsis thaliana L. Heynh., ecotype Col0 (WT) and mutant lines were used in this study. To probe the effect of an impaired electron flow on seed germination, we used mutants lacking a functional cytochrome b6f complex: prfB3 affected in the stability of the petB gene encoding for the cytochrome b6 (Stoppel et al., 2011) and ccb2 affected in the assembly of the c0 heme of the complex (Lezhneva et al., 2008; independent alleles ccb2-5 and ccb2-14). To pinpoint the role of the cyclic electron flow pathway in seed germination, we used the crr2 mutant affected in the expression of a protein essential to translate ndhB mRNA (therefore, the NADPH dehydrogenase (NDH) complex, which catalyses one of the putative cyclic flow pathways is not assembled; Hashimoto et al., 2003), and the pgrl1-ab mutant (DalCorso et al., 2008; Leister & Shikanai, 2013) affected in the expression of a protein essential for the second cyclic electron flow pathway in plants. Seeds were sown on soil : vermiculite (4 : 1) substrate and grown for 2 months at 80 lmol photons m2 s1 (23°C, 16 : 8 h, light : dark photoperiod). Plastid RNA and total protein analyses RNA purification, cDNA synthesis, macroarray hybridization were carried out as previously described (Allorent et al., 2013a). Total proteins were extracted from developing seeds or from rosette leaves in extraction buffer (62.5 mM Tris/HCl pH 6.8, 2.5% (w/v) SDS, 2% (w/v) dithiothreitol (DTT), 10% (v/v) New Phytologist (2015) 205: 707–719 www.newphytologist.com

glycerol). Polypeptides were separated by 12.5% polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, blotted on nitrocellulose and analysed by immunodetection as previously described (Allorent et al., 2013a). Antisera were obtained from Agrisera (http://www.agrisera.com, Sweden) for detection of ATPB, CP43, PSBA, RBCL, LHCA2 and LHCB2 and from the Institut de Biologie Physico Chimique (Paris, France) for PSAD and CYTF. Pigment analysis Chlorophyll content was measured upon extraction in buffered aqueous 80% acetone (Porra, 2002). Triacylglycerol content analysis and metabolic profiling Lipids were extracted from 100 mg of dry seeds ground in liquid nitrogen and fatty acids were quantified by surface peak method using 21 : 0 as internal standard for calibration as previously described (Simionato et al., 2013). For metabolite analysis, 50 mg of dry seeds per biological replicate were used. Metabolite extraction and derivatization for GC-MS were performed as previously described (Lisec et al., 2006). Metabolites were identified by comparison to database entries of authentic standards and relative quantification was performed. Full documentation of metabolite profiling, data acquisition and interpretation is provided in the Supporting Information (Table S1) following recommended guidelines (Fernie et al., 2011). Confocal laser and transmission electron microscopy Confocal laser microscopy micrographs were taken using a Leica TCS-SP2 operating system (Leica, Heidelberg, Germany). Polyphenolic compounds and Chl were excited at 405 nm and 633 nm, respectively. Fluorescence emission spectra were recorded from 420 to 620 nm and 644 to 719 nm, respectively. Mature seeds used for transmission electron microscopy imaging were prepared as described previously (El-Kafafi et al., 2008). Sections were observed at 80 kV in an electron microscope (1200EX; JEOL, Peabody, MA, USA). Spectroscopy, fluorescence and oxygen evolution measurements Seed and leaf absorption spectra were measured with a spectrophotometer (JBeamBio; Bio-Logic Science Instruments, Claix, France), based on a detecting diode array (USB 2000; Ocean Optics, Dunedin, FL, USA). To measure absorption changes, samples were illuminated with a white LED and absorption was measured with a light guide placed directly on the sample, to minimize artefacts related to light diffusion. Fluorescence imaging was performed using a Speedzen MX setup (JBeamBio) as previously described (Allorent et al., 2013b). Fv/Fm was calculated as (FmFo)/Fm, where Fm and Fo are the maximum and minimum amounts of fluorescence emission, respectively. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist In vivo spectroscopic measurements were performed with a JTS10 spectrophotometer (Bio-Logic). Changes in the amount of functional photosynthetic complexes were evaluated measuring the electrochromic shift (ECS) spectral change, a shift in the pigment absorption bands that is linearly correlated to the number of light-induced charge separations within the photosynthetic complexes (Bailleul et al., 2010). Photosystem I : II (PSI : PSII) ratios were estimated from changes in the amplitude of the fast (500 ls) phase of the ECS signal (at 520–546 nm) upon excitation with a saturating laser flash (520 nm; 5 ns duration). PSII contribution was evaluated from the decrease in the amplitude of the signal upon poisoning samples with 40 lM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and hydroxylamine (HA, 2 mM) to irreversibly block PSII charge separation. PSI was estimated as the fraction of the signal that was insensitive to these inhibitors (Bailleul et al., 2010). Cytochrome f and P700 were quantified by assessing the maximum absorption change in samples incubated with 10 lM 2,5-dibromo-6-isopropyl-3-methyl-1,4–benzoquinone (DBMIB). DBMIB blocks plastoquinol oxidation via the cytochrome b6f complex, and allows their relative content to be assessed after deconvoluting their concentration from the absorption spectra. Cytochrome f was quantified as the difference between absorption at 554 nm and a baseline drawn between 546 and 573 nm, respectively (Finazzi et al., 1997), using the extinction coefficient provided by Metzger et al. (1997). P700 concentration was measured at 705 nm using an extinction calculated from Ke (1972). Samples were illuminated with continuous red (630 nm) light, which was transiently switched off to measure P700 absorption changes at 705 nm. Linear and cyclic electron flows (LEF and CEF, respectively) were calculated from the relaxation kinetics of the ECS signal in the dark (Sacksteder et al., 2000; Joliot & Joliot, 2002). Briefly, under steady-state illumination, the ECS signal results from concomitant transmembrane potential generation by PSII, the cytochrome b6f complex and PSI and from transmembrane potential dissipation by the ATP synthase CF0-F1. When light is switched off, reaction center activity stops immediately, while ATPase and the cytochrome b6f complex activities remain (transiently) unchanged. Therefore, the difference between the slopes of the ECS signal measured in the light and after light switch off (SL – SD; see Fig. 3a,b) is proportional to the rate of PSI and PSII photochemistry (i.e. to the rate of ‘total’ electron flow). This can be calculated by dividing (SL – SD) by the amplitude of the absorption changes induced by the transfer of one charge across the membrane (e.g. one PSI turnover). The latter is estimated as the amplitude of the ECS signal upon exposure to a saturating single turnover laser flash under conditions where PSII is inactive (see earlier). The rate of CEF can be evaluated using the same approach under conditions where PSII activity is inhibited by DCMU. Here, the SL – SD slope divided by PSI charge separation only reflects CEF. Note that this procedure may underestimate CEF contribution, as inhibition of linear electron flow is known also to diminish the CEF efficiency (Joliot & Joliot, 2002). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Photosystem II-dependent oxygen evolution was quantified using a Clark-type electrode (Hansatech, Kings Lynn, UK) connected to a PAM fluorometer (Walz, Effeltrich, Germany) to measure oxygen evolution and fluorescence parameters simultaneously. Maximum electron transport rate (ETR) was deduced from fluorescence traces as 0.5 9 I 9 ΦPSII, where I is the intensity of the incident light and ΦPSII is the quantum efficiency of PSII in the light (Genty et al., 1990). Samples (developing seeds or mature rosette leaves) were measured for O2 exchanges following vacuum infiltration in a 10 mM K-phosphate buffer, pH 7.2, 10 mM KCl, 10 mM NaHCO3 at 20°C. To facilitate O2 exchanges, leaves were cut in small pieces before measurements. Seed treatments Developing stages were identified as described by Allorent et al. (2013a). After harvesting, dry seeds were stored at 16°C in a box containing silica gel and developing seeds were kept frozen at 80°C for biochemical and macroarray analyses. Germination assays were performed with c. 100 dry seeds, which were sown on three filter paper layers covered with a 0.2 lm nitrocellulose membrane (Whatman, Versailles, France) and imbibed with 1.4 ml water in Petri dishes. Plates were kept at 23°C under continuous light (90 lmol photons m2 s1) for 5 d to allow germination. A seed is considered as germinated when its radicle had pierced the endosperm and seed coat. For tests on cytochrome b6f assembly mutants, germinated seeds were arranged on agarose-MS medium plates in order of germination and further grown for 4 d to allow seedling phenotype identification. For artificial ageing experiments, seeds were incubated at 40°C and 75% relative humidity up to 9 d and then dehydrated to their original water content by incubation in a box with silica gel (Tesnier et al., 2002). They were then sown on filter papers and germination percentages were calculated after 7 d at 23°C under continuous light with three biological replicates. To prepare DCMU poisoned seeds, siliques of wild-type (WT) Col0 plants were painted twice during seed photosynthetic stage (at DAF5 and DAF8) with 1 mM DCMU in 0.1% Tween 20 (DCMUtreated samples) or with 0.1% Tween 20 (control treatment). The detergent enhances inhibitor diffusion through the cuticule. Treated seeds were allowed to complete their development cycle until the mature stage, and were then used for germination assays or metabolic profiling as described.

Results Architecture of photosynthesis in green embryos Fig. 1(a) displays the morphological traits of developing A. thaliana seeds collected from fruits harvested at DAF3–15. As revealed by the appearance of Chl fluorescence at DAF5 (heart stage), a transient photosynthetic phase is observed in the whole embryos, including cotyledons and radicle, which lasts until their full development (at DAF13). Conversely, Chl was mostly undetectable in both the seed coat and the subepidermal cell layers New Phytologist (2015) 205: 707–719 www.newphytologist.com

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throughout the whole developmental stage (see Fig. 1b for a magnified picture at DAF9). This indicates that these tissues never become photosynthetic. During the transient greening, many chloroplasts become visible in the embryo cells amongst the different storage structures – starch granules (which transiently accumulate in Arabidopsis seeds), oil bodies and vacuoles (Fig. 1c,d). We therefore investigated the features of photosynthesis that was likely performed by seed chloroplasts. Owing to sunlight filtration by both the seed coat and the pericarp (a functionally modified leaf), the light spectrum available to the photosynthetic membranes of embryos and leaves differs from that of sunlight (Fig. 1e). Light available to embryo chloroplasts is enriched in the green and far-red part of the spectrum (Fig. 1f). This prompted us to further investigate possible adaptations of the photosynthetic machinery of embryo chloroplasts. We first observed that the organization of thylakoids in seed chloroplasts was different from that usually observed in leaves: seed thylakoids contain less grana with fewer stacks (Fig. 1d) than do leaf thylakoids (El-Kafafi et al., 2008). The membrane organization of seed chloroplasts recalls the one previously observed in pea leaves developed under far-red light (De Greef et al., 1971). This suggests that the morphological changes observed in seeds could have been triggered by constitutive exposure to far-red enriched light. Furthermore, we found that light capture in the green region of the spectrum is enhanced in seeds in agreement with earlier reports (Allen et al., 2009), suggesting a possible New Phytologist (2015) 205: 707–719 www.newphytologist.com

Fig. 1 Arabidopsis thaliana developing seeds. (a) Developmental stages of Arabidopsis thaliana developing seeds (ecotype Col). Confocal micrographs using Chl (excitation, 663 nm; emission, 644– 719 nm, red) and polyphenol (excitation, 405 nm; emission, 420–620 nm, green) fluorescence. Seeds were collected from siliques at the indicated days after fertilization (DAF) before measuring their optical features. (b) DAF9 developing Arabidopsis seed: (1), polyphenol fluorescence; (2), Chl fluorescence; (3) merged. (c) Electron micrographs of an embryo in a developing seed at DAF9 stage. V, vacuole; O, oil body; M, mitochondria; S, starch. (d) Higher magnification showing thylakoid membranes (star). (e) Absorption spectra of seeds (DAF9, orange line), pericarp (silique wall, DAF9, green line) and 15-d-old rosette leaves (blue line). Spectra were normalized to the 675 nm peak and are representative of four biological replicates. (f) Developing seeds show enhanced absorption of green light. Green line, spectrum of light transmission through the pericarp; orange line, difference between the absorption spectrum of DAF9 seeds and pericarp (silique wall). Traces are representative of three biological replicates.

adaptation of the photosynthetic apparatus of embryos to their chromatic environment. Based on these observations, we looked for possible molecular changes involved in these adaptive responses. Previous reports in leaves have suggested that exposure to PSI-enriched light leads to significant modification of the relative PSII : PSI ratio (Pfannschmidt et al., 1999). To test this possibility, we first used a plastidspecific macroarray (Allorent et al., 2013a) to evaluate changes in the accumulation of mRNAs of photosynthesis-related genes between green seeds (DAF6–11) and leaves. We found that plastid-encoded mRNAs followed a similar trend in both organs (Fig. 2a; Table S2), in agreement with recent results concerning the expression of nuclear-encoded genes in these organs (Belmonte et al., 2013). Immunoblot analyses using antibodies raised against polypeptides (PSBA and CP43, CYTF, PSAD, ATPB, LHCB2, LHCA2) representative of the major membrane embedded complexes (PSII, cytochrome b6f, PSI, ATP synthase, LHCII and LHCI, respectively) further demonstrate that all these complexes accumulate in embryo thylakoids in similar amounts (Fig. 2b) when compared with the Chl content. The latter was c. two times lower in developing seeds than in leaves, on a FW basis (Table 1). Biochemical data were corroborated by measurements of the relative abundance of photosynthetic complexes using an in vivo spectroscopic approach. We first determined the cytochrome b6f and PSI ratios in seeds and leaves from the extent of P700 (the primary electron donor to PSI) and cytochrome f (the Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Research 711 Table 1 Comparative analysis of photosynthetic parameters of green seeds and leaves of Arabidopsis thaliana

Total Chl (lg mg FW1) Chla/Chlb Fv/Fm Maximum ETR (lmol photons m2 s1) Maximum net photosynthesis (lmol O2 min1 mg Chl1) Respiration (lmol O2 min1 mg Chl1)

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Leaves

Seeds

1.52  0.17 3.41  0.30 0.82  0.01 73  10 0.98  0.07

0.75  0.04 3.40  0.16 0.77  0.01 51  13 0.66  0.07

0.14  0.08

0.30  0.03

Leaves, 15-d-old rosette leaves; seeds, green seeds (6–11 d after fertilization). Values represent means  SEM (n = 3–7 biological replicates). Significant differences between leaves and seeds (t-test, P < 0.05) are indicated in bold. Fv/Fm, maximum quantum efficiency of photosynthesis. Chl content was determined as described in the Materials and Methods section. Respiration and net O2 evolution were measured with a Clark-type electrode as O2 consumption in the dark and O2 evolution (minus respiration) at 500 lmol photons m2 s1, respectively. Maximum electron transport rate (ETR) was deduced from fluorescence measurements performed at 400 lmol photons m2 s1 as explained in the Materials and Methods section.

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Fig. 2 Architecture of photosynthetic apparatus in green developing seeds of Arabidopsis thaliana. (a) Plastid gene expression in photosynthetic tissues. Total RNAs were purified from green seeds (6–11 d after fertilization (DAF6–11), grey bars) and from 6-d-old rosette leaves (black bars) and analyzed using a plastid-specific macroarray as previously described (Allorent et al., 2013a). Values (see Table S2 for a complete dataset) represent means  SEM (n = 4 biological replicates) and are expressed as a percentage of total signal recovered. mRNA families are grouped based on the function of the encoded proteins: photosynthesis; gene expression; other functions (Allorent et al., 2013a). (b) Accumulation of the major photosynthetic complexes. L, 15-d-old rosette leaves; S, green seeds (DAF6–11). Lines were normalized on equal quantity of Chl (90 ng; corresponding to 3 lg (L) and 15 lg (S) of total protein). Data are representative of five biological replicates. (c) Functional photosystem I : II : cytf (PSII : PSI: cytf) ratios in seeds vs leaves. Ratios were measured as explained in the text. Bars indicate  SEM. Data are representative of three biological replicates.

electron donor to plastocyanin) that could be oxidized in the presence of DBMIB. This inhibitor specifically blocks the cytochrome complex and leads to full oxidation of cyt f and P700 in the light. Based on this approach, we confirmed that the PSI and the cytochrome contents are similar in seeds and leaves, after normalization for the Chl content (Fig. 2c). We also evaluated the PSII : PSI functional stoichiometry ratio from the amplitude of the light-induced ECS (Fig. 2c). This technique has been already successfully employed to assess the PSI : PSII ratio in Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

photosynthetic tissues (Bailleul et al., 2010; see also the Materials and Methods section). A similar PSII : PSI ratio was found in both leaves and green embryos, suggesting that the relative PSII content was not different in seed and leaf chloroplasts. A similar light-harvesting capacity was found in the case of PSII (Fig. S1). Overall, these data confirmed that the major photosynthetic complexes are active and present in similar stoichiometry in leaves and green seeds. Thus, the overall photosynthetic architecture is maintained in leaves and green developing seeds, despite the light conditions experienced by embryos at the maturation stage. Cyclic electron flow around PSI is enhanced in green seeds As mentioned earlier, increasing the number of photons available in the near far-red part of the spectrum as a result of sunlight filtration by the pericarp should lead to a constitutive unbalance between PSI (which is preferentially excited at these wavelengths) and PSII. As this unbalance is not compensated for by adjustments in the PSI : PSII ratio, we hypothesize that photosynthetic electron transfer adjustments could occur in seed embryos. In the dark, the oxygen consumption rate was increased in seeds (Table 1), suggesting a higher respiration activity, while the photochemical capacity of PSII (Fv/Fm) was similar (Table 1), indicating a comparable photosynthetic capacity. Otherwise, differences were seen in the light, where the PSII-driven electron flow, measured as the fluorescence-derived ETR and ΦPSII parameters (Fig. S2) and as oxygen evolution, turned out to be lower in green embryos than in leaves. This suggests that, although PSII and PSI are present in similar amounts in green embryos (Fig. 2c), PSI activity is enhanced in the light at the expense of PSII. This finding can be interpreted assuming the existence of alternative electron sinks competing with NADP reduction. PSII-generated electrons can be delivered to molecular oxygen in the Mehler reaction (occurring at the PSII acceptor New Phytologist (2015) 205: 707–719 www.newphytologist.com

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side via the plastid terminal oxidase PTOX, or at the PSI acceptor side; Ort & Baker, 2002). However, this reaction should decrease the yield of O2 evolution without affecting the PSII activity (i.e. the ETR parameter). Similarly, a limitation of oxygen diffusion through the seed coat should lead to an apparent decrease of O2 evolution, but should not modify the ETR parameter. Therefore, an involvement of these processes in the reduced O2 and ETR activities observed in seeds is unlikely. In oxygenic photosynthesis, PSI is involved in LEF, working in series with PSII, but also in CEF around PSI, working independently of PSII. When working in CEF, PSI does not consume PSII-generated electrons, thus leading to a reduction of the PSII soluble acceptor pool, and therefore to a decrease in PSII activity. Enhanced CEF in seeds could thus explain a concomitant decrease of O2 evolution and ETR. We assessed separately the linear and cyclic flow activities using the ECS signal (see the Materials and Methods section; Bailleul et al., 2010). Similar values of total electron flow were found in seeds and in leaves (Fig. 3). However, the relative weight of CEF, evaluated as the fraction of electron flow that was insensitive to addition of the PSII inhibitor DCMU, turned out to be significantly higher in seeds than in leaves (Fig. 3a,b). While CEF in leaves represents only c. 10% of electron flow, in agreement with previous reports (Avenson et al., 2005), this process becomes more prominent in green seeds (c. 30%, Fig. 3c). This suggests that the main consequence of the PSI/PSII imbalance in embryos is the enhancement of PSI activity in CEF. Impaired photosynthetic activity alters seed metabolism and germination The occurrence of a functional thylakoid system with stronger CEF capacity in Arabidopsis seeds raises the question of its putative impact on seed germination and vigour. To test the link between photosynthesis and germination, we employed previously used approaches in which siliques were covered with aluminium foil after fertilization and the consequences for seeds and germination were tested. At variance with B. napus (Borisjuk

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et al., 2013), A. thaliana silique development was blocked by this treatment (Fig. S3), suggesting a stronger effect of light in this plant. Therefore, the protocol was modified and siliques were covered with an aluminium foil only after embryogenesis (from DAF5). In this case, pale green seeds were observed at DAF10, which display impaired germination compared with the control after complete development and dessication (Fig. S3). Although these findings point to a possible link between photosynthesis and germination, no clear-cut conclusion could be reached because maintaining siliques in the dark for several days could also prevent photoreceptor-mediated responses, which are involved in seed germination (Strasser et al., 2010). To overcome this difficulty, we decided to selectively block photosynthesis in developing seeds and siliques by treating siliques with DCMU, a specific inhibitor of PSII. Inhibition of photosynthesis in seeds by DCMU was confirmed by measuring fluorescence induction kinetics in isolated seeds and pericarp (Fig. S4), while no effect was found in rosette leaves from the same plants (Fig. S5). This indicated that inhibition of photosynthesis by DCMU was restricted to siliques and developing seeds. Upon maturation, seeds were harvested from DCMU-treated and control plants and their germination kinetics were examined. When compared with the control, DCMU-poisoned seeds showed a significant delay in germination, with no effects on the final germination percentage (Fig. 4a). This result confirms that embryonic photosynthesis has an impact on seed germination timing. However, it does not allow discrimination between photosynthesis in developing seeds and in siliques, as photosynthesis was blocked by DCMU in both organs. Therefore, a genetic approach was used to selectively block photosynthesis in the embryo, taking advantage of two independent heterozygous Arabidopsis lines bearing a deficiency in the assembly of the cytochrome b6f complex. Following autofertilization, 25% of seeds lack the cytochrome complex and should therefore be unable to perform photosynthesis during the green phase, while the remaining organs (including siliques and integuments of these seeds) are either WT or heterozygous, and therefore maintain their photosynthetic capacity.

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Fig. 3 Linear and cyclic electron flow in leaves and developing seeds of Arabidopsis thaliana. (a, b) Changes in the electrochromic shift (ECS) signal during a transition from light to dark in Arabidopsis thaliana mature leaves (a) and green seeds (b). Leaves, 15-d-old rosette leaves; seeds, green seeds (6–11 d after fertilization (DAF6–11)). Leaves and seeds were subjected to 2 min of illumination and then transferred to the dark. Data represent changes in the ECS signal measured at 520–546 nm upon normalization of the last data point in the light to one, to allow for a better comparison. To quantify linear and cyclic flow activity, the difference between the slopes of the ECS changes in the light (SL) and in the dark (SD) was normalized to the amplitude of the one charge separation by PSI, that is, the ECS measured in samples poisoned with 20 lM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and hydroxylamine (1 mM) to block PSII photochemistry. Open circles, control; closed circles, DCMU (20 lM). Measurements were performed four times and representative traces are shown. (c) Quantification of cyclic and linear electron flow of green seeds and leaves. Values represent means  SEM (n = 3–7 biological replicates). Significant differences between samples (t-test): *, P < 0.05. Total and cyclic electron flow rates were derived from ECS measurements as explained in the Materials and Methods section. New Phytologist (2015) 205: 707–719 www.newphytologist.com

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Homozygous seeds were discriminated from heterozygous and WT seeds because they displayed an albino phenotype following germination (Fig. 4c; Lezhneva et al., 2008). As in the case of DCMU-treated seeds, germination of mutant seeds was significantly delayed when compared with WT and heterozygous seeds, which could not be distinguished in the assay (Fig. 4a). Altogether, these data demonstrate that embryonic photosynthesis in the seeds per se plays an important role in their germination rate. Embryo photosynthesis and seed metabolism Within a given species, the accumulation of storage compounds for germination is positively correlated with seedling fitness (Westoby et al., 1992). Thus, the slower germination observed in seeds where photosynthesis was impaired could stem from lack of accumulation of key metabolites. This possibility was investigated by comparing the features and metabolic profiles of untreated and DCMU-poisoned seeds, because homozygous mutant seeds lacking the cytochrome b6f complex could not be selected before germination. As shown in Table 2, DCMU-treated mature seeds turned out to be heavier with a reduced water content. Because the water content of mature seeds is a key parameter for seed storability (Walters, 1998), an accelerated ageing treatment was performed on control and DCMU-treated seeds to test their longevity. Seeds poisoned with DCMU during their development displayed a reduced capacity to germinate after accelerated ageing when compared with the control, indicating that (a)

embryonic photosynthesis has indeed played an important role with respect to seed storability (Fig. 4b). However, this trait could not be linked to changes in the major storage compounds in Arabidopsis seeds (Baud et al., 2002) as neither triacylglycerides (TAGs) nor storage proteins (cruciferins and globulins) were affected in DCMU-poisoned seeds (Fig. 5a–c). We cannot exclude the possibility that seed photosynthesis contribution to seed filling is compensated in the case of impairment by DCMU treatment by silique or leaf photosynthesis. Nevertheless, a wider metabolic analysis indicates that the overall content of the most abundant primary metabolites is not different between control and DCMU-treated seeds (Table S3). Otherwise, a few key Table 2 Morphology of Arabidopsis thaliana 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU)-poisoned mature seeds

FW (mg per 100 seeds) DW (mg per 100 seeds) Water content (mg H2O mg DW1) Water content (% of FW)

Control

DCMU

2.010  0.062 1.860  0.062 0.081  0.06 7.478  0.499

2.157  0.088 2.057  0.092 0.049  0.004 4.651  0.373

Developing seeds were treated twice with 0.1 mM DCMU in 0.1% Tween 20 (DCMU) or 0.1% Tween 20 (control) at DAF5 and DAF9 and harvested after complete drying (see the Materials and Methods section). Values are obtained by weighting 100 seeds and are expressed as means  SEM (n = 3 technical replicates). Significant differences between control and DCMU-treated seeds (t test, P < 0.05) are indicated in bold.

(b)

(c)

Fig. 4 Electron flow influences seed germination capacity and longevity in Arabidopsis thaliana. (a) Germination tests. Tests were performed under continuous light (90 lmol photons m2 s1) at 23°C as indicated in the Materials and Methods section. The germination percentage of cytochrome b6f mutant seeds (Lezhneva et al., 2008) is calculated as the number of germinated homozygous seeds (identified by the phenotype of the seedlings) divided by the total amount of germinated seeds as described in the Materials and Methods section. Data are representative of at least three technical and two biological repetitions. DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated siliques (see the Materials and Methods section). (b) Accelerated ageing. Accelerated ageing of seeds was performed as described in the Materials and Methods section at 40°C and 75% relative humidity. The germination percentage is calculated after 10 d germination at 23°C under continuous light. Values represent means  SEM (n = 3 biological replicates). Open circles, control seeds; closed circles, DCMU-treated seeds. (c) Albino phenotype of mutants impaired in cytochrome b6f assembly. Seeds obtained from heterozygous ccb2-5, ccb2-14 and prfB3 mutants plants were sown on agarose 1% MS medium and grown under continuous light (90 lmol m2 s1) at 23°C during 6 d. Homozygous seedlings (white star) were identified form their albino phenotype. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(b)

(c)

Fig. 5 Storage compound content in Arabidopsis thaliana mature dry seeds. Developing seeds were treated twice with 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU) as described in the Materials and Methods section. (a) Tricacylglycerol (TAG) total content. Values represent means  SEM (n = 3 biological replicates) and are expressed in nmol for 10 mg of dry seeds. (b) TAG fatty acid composition analysis. Values represent means  SEM (n = 3 biological replicates) and are expressed as mol% of total fatty acid analysed (see also in the Materials and Methods section). Black bars, control; grey bars, DCMU-treated seeds. (c) Polypeptide profile analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. Total proteins were extracted from exactly 15 dry seeds (control or DCMU-treated) in 15 ll of sample buffer as indicated in the Materials and Methods section and the total volume of the protein extract was loaded in each well on the gel. The major bands where identified by their apparent molecular weight and correspond to the major storage protein in Arabidopsis thaliana seeds (a and b subunits of 12S cruciferins; Withana-Gamage et al., 2013). Using ImageJ software (http://rsbweb.nih.gov/ij/), the total amounts of polypeptides in control and DCMU-treated samples were estimated as 69 203 and 66 222 counts, respectively, indicating that the difference between the two samples was c. 5%. MW, molecular weight markers.

metabolites were affected by this treatment (Table 3). In particular, proline and galactinol (one of the major sugar derivatives stored in mature seeds) were significantly decreased in DCMUtreated seeds, while the content of the nonproteinogenic amino acid GABA was increased (Table 3). These data suggest that embryonic photosynthesis does not impact seed storability and germination timing by affecting the synthesis of major metabolic storage compounds, but because it impacts several pools of metabolites that could specifically be linked to seed physiological quality.

Cyclic electron flow during seed development is correlated to seed germination A careful analysis of the results of Fig. 4 indicates that inhibition of cytochrome b6f turnover in the ccb2 and prfB3 mutants has a more severe impact on seed germination than the inhibition of PSII by DCMU. Because PSII can only contribute electrons to LEF while the cytochrome complex is involved in both LEF and CEF, these data suggest that CEF activity during embryonic photosynthesis could also play a role in seed germination timing.

Table 3 Different metabolic content of Arabidopsis thaliana control and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-poisoned mature seeds

Galactinol Inositol, myoinositol Proline Aspartate Citric acid Thryptophane Isoleucine Phenylalanine Xylose Valine GABA Mannitol Ornithine Erythritol Glycerol.3P Asparagine

v-test

Mean in control seeds

Overall mean (control and DCMU-treated seeds)

2.9554 2.7212 2.7087 2.3430 2.2737 2.2168 2.2066 2.2276 2.2840 2.3041 2.4567 2.5009 2.6400 2.6504 2.6553 2.7259

8.808 2.452 10.382 3.802 0.914 4.124 2.322 1.954 0.132 4.93 1.168 0.072 0.144 0.126 0.232 0.056

5.548 2.008 7.165 3.278 0.78 3.549 2.911 2.435 0.168 6.136 1.765 0.099 0.205 0.169 0.324 0.122

SD in control category

Overall SD (control and DCMU-treated seeds)

P-value

0.78670 0.25230 1.72248 0.43545 0.10092 0.61425 0.54916 0.45811 0.02638 0.88812 0.33996 0.01166 0.04079 0.02245 0.03250 0.02498

3.30920 0.48949 3.56297 0.67094 0.17680 0.77813 0.80079 0.64778 0.04729 1.57022 0.72902 0.03239 0.06932 0.04867 0.10394 0.07264

0.00312 0.00650 0.00675 0.01913 0.02298 0.02663 0.02734 0.02591 0.02237 0.02121 0.01402 0.01239 0.00829 0.00804 0.00792 0.00641

Control and DCMU seeds were treated as described in the Materials and Methods section. Relative metabolite content of dry mature seeds harvested after complete drying is quantified as described in the Materials and Methods section. The complete set of data is listed in Table S3. Differences between the metabolite abundance in control and DCMU-treated samples were tested using the v-Kuiper’s test. Differences between control and DCMU-treated seeds are significant when |v| > 2 and the P-value < 0.05. Metabolites quantified from Table S3 having significant v statistics are ranked based on v-value (n = 5 in each group). New Phytologist (2015) 205: 707–719 www.newphytologist.com

Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 6 Cyclic electron flow impacts Arabidopsis thaliana seed germination. (a) Evaluation of cyclic electron flow (CEF) contribution to photosynthesis in leaves and green seeds. The contribution of cyclic electron flow was deduced from electrochromic shift (ECS) measurements, as indicated in the legend of Fig 3. Bars represent + SE. Significant differences between wild-type (WT) and mutants are indicated (t-test; *, P < 0.05; **, P < 0.01). Grey bars, seeds; black bars, leaves. (b) Germination tests. Tests were performed under continuous light (90 lmol photons m2 s1) at 23°C as indicated in the legend of Fig 4. Data are representative of three technical and two biological repetitions. LEF, linear electron flow.

This hypothesis was tested by comparing germination in seeds issued from WT and CEF-deficient mutants. Two main pathways have been identified to mediate CEF in photosynthesis: the NDH and the PGRL1-PGR5 pathways (Leister & Shikanai, 2013). We first measured the extent of CEF in seeds and leaves of WT and mutant plants lacking the NDH (crr2, Hashimoto et al., 2003) or the PGRL1-PGR5 activity (pgrl1a-b, DalCorso et al., 2008). We found that CEF was more reduced in the leaves of pgrl1a-b plants than in crr2 leaves (Fig. 6a), consistent with previous assessments of the relative contribution of the two pathways in plants (Munekage et al., 2004; Johnson, 2011). Conversely, an opposite trend was observed in seeds, where the role of the NDH complex-mediated CEF turned out to be more relevant, as indicated by the stronger consequences of the crr2 mutation. Next the correlation between CEF and seed germination was examined by looking at the germination timing in seeds produced by mutants affecting the two CEF pathways. We found that germination was slowed down in both genotypes and the effect was stronger in crr2 than in pgrl1a-b seeds (Fig. 6b). This indicates a strong correlation between CEF activity in developing seeds and subsequent germination. Consequences of photosynthesis on seed vigour Inhibiting LEF (with DCMU), blocking CEF in the pgrl1a-b and crr2 genotypes, or impairing both electron flow pathways in mutants affected in the assembly of the cytochrome b6f complex, affects seed germination. However, environmental conditions (e.g. temperature) experienced during seed maturation are known to strongly affect the germination capacity and seedling vigour (Donohue et al., 2008; de Casas et al., 2012). Thus, we assessed possible consequences of impairing seed photosynthesis on the vigour of seedlings. This was done by relating seed germination to seedling morphology and cotyledon photosynthetic performances at an early stage of development (4 d after imbibition), which were assessed measuring their Fv/Fm using fluorescence imaging (Fig. S6). Seedlings from DCMU-treated seeds or Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

mutants affecting CEF displayed altered seed morphology and a lower Fv/Fm value when compared with untreated plants, suggesting a weaker vigor. After 7 d, differences were no longer visible (data not shown), indicating that the effect of seed photosynthesis was lost, most likely because photosynthesis that developed in leaves was compensated for by the initial defects in cotyledons. Overall, these data confirm the conclusion that embryonic seed photosynthesis is important for germination and the vigour of seedlings.

Discussion This study unambiguously demonstrates that A. thaliana embryos develop a fully active photosynthetic apparatus during seed development. The overall structure of this machinery is similar to that of mature leaves. However, differences are seen at the level of light capture (with an enhanced seed absorption capacity in the green part of the spectrum) and electron flow (CEF being more efficient in seeds than in leaves). The finding of specific acclimation responses of photosynthesis in seeds, as well as the heavy metabolic cost for the plant to build up and degrade this transient photosynthetic apparatus, suggests that photosynthesis in green seeds performs a relevant function for the seed development and vigour. This hypothesis is corroborated by our results showing that embryo photosynthesis is important for efficient germination timing. Seeds harvested from siliques poisoned with DCMU, or collected from mutant plants lacking the cytochrome b6f complex displayed delayed germination under standard laboratory conditions. Light availability in developing seeds Owing to filtration by the pericarp, the spectrum of the light perceived by the developing seeds is different from that of sunlight, being enriched in the green and far-red bands of the spectrum. Light enrichment in the green is paralleled by an increased absorption capacity of the seed in this particular part of the spectrum (Fig. 1e,f). In principle, this enhanced absorption ability in developing seeds could stem from changes in the photosynthetic machinery (e.g. in the light-harvesting apparatus). However, all the major complexes (including cytochrome b6f, the ATPaseATPsynthase CF0-Fi, PSII, PSI and their antenna complexes) are present in similar stoichiometries in embryos and leaves and are fully active in embryos (Fig. 2; Table 1). The observed changes in the absorption features could also reflect a different flattening distortion of the absorption spectrum, because of the modified thylakoid membrane organization in the embryo chloroplasts (Fig. 1c) and a reduced Chl content. As an alternative hypothesis, we propose that the enhanced absorption capacity of seeds in the green region of the spectrum reflects a true adaptation of green seeds to optimize their capture of available photons. Seeds are characterized by the presence of a network of void spaces (Cloetens et al., 2006), the function of which is still debated. These spaces could provide room to store oxygen within the seeds to avoid anaerobic metabolism or merely facilitate gas diffusion as has already been suggested (Cloetens et al., 2006; Borisjuk & New Phytologist (2015) 205: 707–719 www.newphytologist.com

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Rolletschek, 2009; Verboven et al., 2013). However, these void spaces could also enhance light diffusion within the seeds, thereby increasing photon capture at poorly absorbed wavelengths as previously observed in the case of the spongy mesophyll in leaves (Evans, 1999). Cyclic electron flow is enhanced in developing seeds As mentioned earlier, the second major difference in the light environment experienced by seeds is the enrichment in the near far-red part of the spectrum (Fig. 1f), which leads to an imbalance between PSI and PSII activity, because of the better PSI absorption in this part of the spectrum. At variance with previous reports in leaves (Pfannschmidt et al., 1999), exposure to PSIenriched light does not lead to any modification of the relative PSII : PSI ratio in seeds (Fig. 1). However, structural and functional modifications take place in developing seeds as a response to their peculiar light environment. First of all, the chloroplast structure of green seeds, where the grana apparently contain fewer stacks (Fig. 1c) than in leaves, is reminiscent of that observed in chloroplasts from pea leaves developed under far-red light (De Greef et al., 1971). Moreover, cyclic electron flow around PSI is higher in green embryos than in leaves, possibly to compensate for the increased number of photons available to this complex. Based on the comparative analysis of the phenotype of crr2 and pgrl1a-b mutant leaves and seeds, it appears that while the PGL1PGR5 CEF path should be predominant in leaves, the NDH path seems to be prevailing in seeds, being responsible for the overall increase in CEF observed in these organs. Consistent with this hypothesis, a study in A. thaliana has pinpointed the NDH pathway as the possible target for increasing cyclic flow activity in plants (Livingston et al., 2010). Effects of embryonic photosynthesis impairment on seed germination Inhibiting photosynthesis in seeds results in delayed germination and decreased seed storability, as well as lower seedling vigour. This suggests that photosynthesis in developing seeds plays an important role in their maturation. The metabolic analysis of DCMU-treated mature seeds allows two possible functions of embryonic photosynthesis in seed germination to be pinpointed: avoiding hypoxia within the developing seed, that is, an organ where oxygen consumption by respiration is enhanced (Table 1; Tschiersch et al., 2012); and improving desiccation tolerance through influencing the final water content of mature seeds. Such mechanisms are known to contribute to seed physiological quality, along with the hormonal balance (Yamaguchi et al., 2007) and the amount of storage compounds (Westoby et al., 1992). The metabolic profiling of DCMU-treated mature seeds reveals overaccumulation of GABA compared with control seeds. GABA concentration increase could be related to the decreased oxygen concentrations resulting from the inhibition of photosynthesis and increased respiration (Table 1). Moreover, increasing the GABA concentration also prevents cell death during dehydration (Fait et al., 2005) and is involved in the defence mechanisms New Phytologist (2015) 205: 707–719 www.newphytologist.com

New Phytologist against anoxia by scavenging reactive oxygen species (ROS). Such GABA increase is not necessarily indicative of a general up-regulation of the GABA shunt, as a recent study in which flux through the GABA shunt was transgenically up-regulated yielded different results (Fait et al., 2011). Up-regulation of the GABA shunt by the overexpression of an unregulated glutamate decarboxylase resulted not only in enhanced GABA concentrations but also in smaller seed size and an altered protein to lipid balance, features that were not induced by the DCMU treatment of green siliques. However, it is important to note that the change in GABA concentrations following DCMU treatment is probably a consequence of the altered energy metabolism rather than a primary effect of the experimental treatment that is the case for the genetic perturbation. Indeed, an increase of the GABA concentration is a reliable metabolic marker for exposure to low oxygen (Obata & Fernie, 2012). Thus, these findings, combined with the O2 exchange measurements, provide molecular features that support the hypothesized role of embryonic photosynthesis: avoiding anoxia during the daytime in green developing seeds, where the low gas permeability of the integuments limits O2 diffusion from the outside (Borisjuk & Rolletschek, 2009) and oxygen consumption by respiration is high (Tschiersch et al., 2012). Metabolic profiling in DCMU-treated seeds also reveals a depletion of galactinol and proline when compared with controls. Proline (Verbruggen & Hermans, 2008) and galactinol (Nishizawa et al., 2008) accumulate in leaves and seeds in response to a wide variety of abiotic stresses, mainly related to drought. As osmoprotectants, proline and galactinol (Nishizawa et al., 2008) are expected to act during the dehydration process as molecular chaperones and ROS scavengers, contributing to the protection of proteins and subcellular structures. This indicates that, besides improving the O2 seed content in the light, embryonic photosynthesis also participates in modulating the osmoprotectant balance. Consistent with this, mature seeds issued from DCMUtreated siliques displayed a lower water content (Table 2) and showed decreased germination efficiency and storability in artificial ageing experiments. These findings point to an intimate relationship between light-driven electron flow and control of final seed water content. This link could be provided by changes in the ABA concentrations. Indeed, galactinol activates the synthesis of ABA (Taji et al., 2002), which in turn modulates the proline biosynthetic pathway (Verbruggen & Hermans, 2008). The link between photosynthesis and water content could also account for previous findings that degreening and dehydration prevent unregulated precocious germination (Ruppel et al., 2011), and that chlb degradation during late maturation of seeds is important for their storability (Nakajima et al., 2012). However, the ABA concentration estimated in control seeds was consistent with previous estimates (Kanno et al., 2010), but not significantly different in DCMU-poisoned seeds (Table S4). Thus, we tend to exclude a role of ABA in the reduced germination rate observed in DCMU-poisoned seeds. Eventually, the analysis of mutants impaired in CEF during the embryonic photosynthetic stage supports an active role of this process in the building of seed quality, with effects that extend up Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist to germination. The major metabolic activity of developing seeds is the accumulation of storage compounds (specifically oil and proteins in Brassicaceae). While the oil content in mature B. napus seeds is primarily controlled by the maternal genotype (Hua et al., 2012), its production requires light and embryonic photosynthesis at least in B. napus, (Ruuska et al., 2004) and soybean (Allen et al., 2009). Thus, although lipid production for storage could be the major activity in developing embryos of crop plants (canola, B. napus, sunflower, pea, soybean) as a result of extensive selection, other functions could be relevant in plants like A. thaliana. Oil accumulation in A. thaliana seeds strongly depends on the light intensity experienced by the mother plant during growth (Li et al., 2006). Other differences could explain the differences observed between B. napus and A. thaliana. In the former, the green phase lasts until full maturation of seeds (Borisjuk et al., 2013), while it is clearly a transient phenomenon in A. thaliana. In Arabidopsis, light seems to be mandatory for proper silique maturation, while the opposite is true in Brassica (Borisjuk et al., 2013). In general, plastid metabolism is mainly photoheterotrophic in green seeds, meaning that, at variance with leaves, seeds are less efficient assimilators of CO2. Rubisco is highly active in green seeds (Ruuska et al., 2004), but its activity is partially disconnected from the Calvin cycle and drives carbohydrate fluxes through the nonoxidative pentose phosphate pathway to produce mainly NADPH (Schwender et al., 2004). In vitro analyses in isolated soybean embryo demonstrate that nonphotosynthetic metabolism provides enough reductant for biosyntheses (Allen et al., 2009). It has been argued that the high amounts of ATP needed for fatty acid and particularly for amino acid biosynthesis (for reserve protein biosynthesis) could not be produced solely by mitochondria because of the largely hypoxic environment of green developing embryos (Schwender et al., 2006). Conversely, an elevated CEF activity in A. thaliana green developing seeds, which would represent a physiological response of seed photosynthesis to their light environment, could provide additional ATP supply. Consistent with this hypothesis, we found that a genetic deficiency in CEF has a larger impact than the inhibition of LEF on the germination efficiency of seeds. Overall, this study provides a unified view about the role of chloroplast during seed development in A. thaliana. Previous studies have demonstrated that gene expression in the plastid is crucial for seed germination, even during the early steps of maturation when plastids are not yet green (Demarsy et al., 2012; Allorent et al., 2013a). This work reveals another key role for the chloroplast that goes beyond this initial phase of seed maturation, showing that green plastids provide energy and metabolites important for germination timing and seedling photosynthesis, and therefore also exert a control on seed development and plant fitness.

Acknowledgements We thank Didier Gr€ unwald (CEA) for access and help with the confocal microscope; Catherine de Vitry and Lina Lezhneva (IPBC Paris) for providing the cytochrome b6f mutants; Paolo Pesaresi (Milano University) and Dario Leister Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(Munich University) for pgrl1a-b mutant seeds; Sandrine Longis from Anastats (www.anastats.fr) for help in statistical analysis; Francßois Parcy (CNRS) for valuable suggestions; and Francßois Perreau (Plant Observatory, INRA Versailles) for the determination of ABA concentration. Financial support was obtained from the ANR (ANR-07-GPLA-013-001, Phytadapt NT09_567009, ANR-2010-GENOM-BTV-002-01), CNRS, Univ. Grenoble Alpes, CEA, the Labex ‘ GRAL ’ (ANR-10-LABEX-04), and the Marie Curie ITN Accliphot (FP7-PEPOPLE-2012-ITN; 316427). G.A. also acknowledges a PhD grant from the Ministere de l’Enseignement Superieur et de la Recherche.

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Fig. S4 Fluorescence rise in green seeds and siliques of Arabidopsis thaliana in the absence and presence of DCMU.

Supporting Information

Table S3 Analysis of metabolite content in mature seeds of Arabidopsis thaliana

Additional supporting information may be found in the online version of this article. Fig. S1 Light-harvesting capacity of PSII in seeds and in isolated thylakoids of Arabidopsis thaliana. Fig. S2 Light dependency of the PSII charge separation yield in green seeds and leaves of Arabidopsis thaliana. Fig. S3 Arabidopsis thaliana seed and silique development in the dark.

Fig. S5 Fluorescence rise in rosette leaves and siliques of Arabidopsis thaliana in the absence and presence of DCMU. Fig. S6 Effect of seed photosynthesis on seedling vigour in Arabidopsis thaliana. Table S1 Overview of the metabolite reporting list and technical checklist Table S2 RNA accumulation quantified by plastid specific macroarray profiling in Arabidopsis thaliana

Table S4 ABA content of Arabidopsis thaliana DCMU treated seeds

Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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