Providing an additional electron sink by the introduction of cyanobacterial flavodiirons enhances the growth of Arabidopsis thaliana in varying light

The ability of plants to maintain photosynthesis in a dynamically changing environment is of central importance for their growth. As their photosynthetic machinery typically cannot adapt rapidly to fluctuations in the intensity of radiation, the level of photosynthetic efficiency is not always optimal. Cyanobacteria, algae, non-vascular plants (mosses and liverworts) and gymnosperms all produce flavodiirons (Flvs), a class of proteins not represented in the angiosperms; these proteins act to mitigate the photoinhibition of photosystem I. Here, genes specifying two cyanobacterial Flvs have been expressed in the chloroplasts of Arabidopsis thaliana in an attempt to improve the robustness of Photosystem I (PSI). The expression of Flv1 and Flv3 together shown to enhance the efficiency of the utilization of light and to boost the plant’s capacity to accumulate biomass. Based on an assessment of the chlorophyll fluorescence in the transgenic plants, the implication was that photosynthetic activity (including electron transport flow and non-photochemical quenching during a dark-to-light transition) was initiated earlier in the transgenic than in wild type plants. The improved photosynthetic performance of the transgenics was accompanied by an increased production of ATP, an acceleration of carbohydrate metabolism and a more pronounced partitioning of sucrose into starch. The indications are that Flvs are able to establish an efficient electron sink downstream of PSI, thereby ensuring that the photosynthetic electron transport chain remains in a more oxidized state. The expression of Flvs in a plant acts to both protect photosynthesis and to control the ATP/NADPH ratio; together, their presence is beneficial for the plant’s growth potential.


Introduction
The growth and development of plants, fueled by photosynthesis, depends on the capture of light energy (Stitt et al., 2010), a process which is carried out by the chloroplast. Photosynthesis can be inhibited by either an inadequate pool of ATP or an imbalance between the quantity of ATP and NADPH present (Avenson et al., 2005b;Cruz et al., 2005;Amthor, 2010). Adjustments of ATP in chloroplasts through CET pathway results in the non-photochemical quenching (NPQ) of Photosystem II (PSII), resulting in a significant loss in the fixation of CO 2 (Long et al., 1994, Werner et al., 2001, Zhu et al., 2004. Fine tuning of NPQ in tobacco has enabled an enhanced level of photosynthetic efficiency to be attained, leading to a measurable increase in biomass yield (Kromdijk et al., 2016). One of the consequences of reducing photosynthetic efficiency is the accumulation of surplus electrons in the photosynthetic electron transport chain (PETC), which promotes the production of reactive oxygen species (ROS). Since the functioning of both PSI and PSII is compromised by the presence of ROS (Miyake, 2010), the result is a diminution in the plant's capacity to assimilate CO 2 (Zivcak et al., 2015a,b;Takagi et al., 2016). According to Gollan et al. (2017), the inhibition of PSI experienced by the Arabidopsis thaliana pgr5 mutant when exposed to a high light intensity limits both the light and dark reactions associated with photosynthesis, as well as chloroplast signaling and nuclear gene expression. Thus, avoiding PSI electron acceptor limitation or introducing additional electron dissipating pathways into the chloroplast have the potential to improve photosynthetic efficiency and hence increase the plant's productivity.
Analysis of the genome of the cyanobacterium Synechocystis sp. PCC6803 has identified the presence of four Flv genes. Their products, in the form of the heterodimers Flv1/Flv3 and Flv2/Flv4 drive oxygen-dependent electron flow under low levels of CO 2 availability and fluctuating light conditions (Zhang et al., 2012;Allahverdiyeva et al., 2013;Hayashi et al., 2014;Shimakawa et al., 2015). The Flv1/ Flv3 heterodimer generates an electron sink downstream of PSI and directs the electron flow to reduce O 2 to H 2 O, thereby protecting PSI (Allahverdiyeva et al., 2013). Cyanobacterial Flv3 can also form a functional homodimer, which in Chlamydomonas reinhardtii acts in conjunction with ferredoxin 9 to transfer electrons into a glutathione-cysteine peroxidase pathway (Cassier-Chauvat et al., 2014). According to Yamamoto et al. (2016), inducing the expression of the Physcomitrella patens Flv1/Flv3 heterodimer in the A. thaliana pgr5 mutant provides partial compensation for the impairment of the cyclic electron transport pathway, an observation which demonstrates that Flvs can be functionally beneficial in an angiosperm. Shimakawa et al. (2017) have shown that in the liverwort Marchantia polymorpha, Flv1 contributes to P700 oxidation and hence protects PSI against photoinhibition. When Gómez et al. (2018) expressed the cyanobacterial genes Flv1 and Flv3 in tobacco chloroplasts, the photosynthetic performance under steady-state illumination of the resulting transgenic plants proved to be comparable to that of wild type (WT) leaves, while the recovery of electron transport and NPQ during a dark-to-light transition was more rapid. The joint expression Flv1 and Flv3 isolated from a moss species in either the rice mutants pgr5 or NDH has been shown to rescue biomass accumulation (Wada et al., 2017). Meanwhile, the enhancement to ATP synthesis resulting from the over-expression of Flv3 in Synechocystis results in an accumulation of glycogen and a consequent increase in cell dry weight (Hasunuma et al., 2014).
The purpose of the present investigation was to determine the phenotypic effect of expressing Synechocystis Flv genes in A. thaliana. The focus was to establish whether heterologously expressed Flv could mediate electron transfer to oxygen within the PETC, and if so, whether this capacity had the potential to boost the plant's productivity.

Materials and methods
The heterologous expression of cyanobacterial Flvs and the localization of transgene products A. thaliana Col-0 lines constitutively expressing either Flv1/Flv3 were generated using the floral dip method (Clough et al., 1998), using the pCHF3-derived vectors described by Gómez et al. (2018). Transgene expression was driven by the cauliflower mosaic virus (CaMV) 35S promoter, and a pea sequence encoding a ferredoxin-NADP + reductase transit peptide was fused in-frame to the 5' end of the transgene to direct its expression to the chloroplast ( Figure S1a). Five independent transformants were chosen for each construct, and the presence of the transgene in the host's nuclear genome was confirmed by PCR analysis. Of the five transformants selected per construct, three were developed into fixed transgenic lines: their ability to accumulate Flv transcript was monitored using a quantitative real time PCR (qRT-PCR) assay (see below). The level of transcription of the various transgenes is shown in Figure S1b. The chloroplast targeting 5 of the transgenes was validated by fusing the GFP sequence (encoding green fluorescent protein) to the C terminus of the Flv transgene, taking advantage of PGBW5 Gateway binary vectors driven by the 35S CaMV promoter ( Figure S2). The binary vector containing the Flv transgene was transferred into Agrobacterium tumefaciens strain EHA 105 using the Dower et al. (1988) electroporation method, and from thence into leaves of Nicotiana benthamiana using the agroinfiltration method described by Sainsbury and Lomonossoff (2008). Leaves sampled 48 h after infiltration was subjected to confocal scanning microscopy to monitor GFP activity ( Figure   S2).

Experimental growing conditions for A. thaliana transgenics
Seed of each of the Flv transgenic lines and WT were surface-sterilized by immersion for 15 min in 70% (v/v) ethanol and 0.05% (v/v) Tween 20, then rinsed for 30 s in 96% (v/v) ethanol. After air drying, the seed was held at 4°C for 48 h, plated on vertically-orientated agar containing half strength Murashige and Skoog (1962) medium and held under an 8 h photoperiod (160 µmol photons m -2 s -1 ) at 22°C. After two weeks, the seedlings were potted into a mixture of 70 L substrate 1 (Duesseldorf, Germany), 23 L vermiculite, 372 g plantacote depot 4m and held at 22°C, 80% relative humidity under an 8 h photoperiod provided by Master HPI-T Plus 250 W fluorescent lights (Philips, Netherlands) involving four different light intensities: low (50 µmol photons m -2 s -1 ), moderate (160 µmol photons m -2 s -1 ), moderately high (300 µmol photons m -2 s -1 ) and high (600 µmol photons m -2 s -1 ). The CO 2 level was maintained at 400 ppm and plants were kept fully hydrated. Shoot dry weights were derived from six week old plants. For the biochemical analysis of leaf, rosettes of six week old plants exposed to 160 µmol photons m -2 s -1 harvested at various time points during the diurnal cycle (0,4,8,16,20 and 24 h) were snapfrozen in liquid nitrogen and ground to a powder. A number of plants were also grown under long day conditions (16 h photoperiod, 160 µmol photons m -2 s -1 ), with all the other environmental parameters identical with those used for the short day grown plants.

Determination of the leaf content of carbohydrates, amino acids and metabolites
For the determination of the content of soluble sugars (glucose, fructose and sucrose) and amino acids, a 50 mg aliquot of powdered frozen leaf was extracted in 0.7 mL 80% (v/v) ethanol at 80°C for 1 h. Following centrifugation (14,000 rpm, 10 min), the supernatant was evaporated under vacuum at 40°C, and the residue dissolved in 0.2 mL deionized water. Sugar contents were 6 quantified using the enzymatic method described by Ahkami et al. (2013), while those of the individual amino acids were obtained following Mayta et al. (2018). The pelleted material was used to assess the leaf's starch content: the pellet was rinsed twice in 80% (v/v) ethanol, air-dried at 80°C for 1 h and resuspended in 0.2 M KOH. The resulting suspension was held at 80°C for 1 h, the pH adjusted to neutrality using 1 M acetic acid, then incubated overnight at 37°C in 50 mM NaAc (pH 5.2) containing 7 units mg -1 amyloglucosidase. The glucose thereby released was quantified as above. The methods used for the quantification of primary metabolites followed Ghaffari et al. (2016).

Determination of the leaf content of adenine phosphate and redox equivalents
Adenine phosphates were quantified employing a UPLC-based method developed from that described by Haink and Deussen (2003). Prior to the HPLC separation step, a 20 µL aliquot of the sample used for the quantification of metabolites (as well as a mixture of ATP, ADP, AMP and ADPGlc) were derivatized by the addition of 45 µL 10% (v/v) chloracetaldehyde and 435 µL 62 mM sodium citrate/76 mM KH 2 PO 4 (pH 5.2), followed by a 40 min hold at 80°C, cooling on ice, and a centrifugation (20,000 g, 1 min). Reverse phase UPLC separations were achieved using an Infinity 1200 device (Agilent, Waldbronn, Germany). The gradient was established using eluents A [TBAS/KH 2 PO 4 (5.7 mM tetrabutylammonium bisulfate/30.5 mM KH 2 PO 4 , pH 5.8)] and B (a 2:1 mixture of acetonitrile and TBAS/KH 2 PO 4 ); the Roti C Solv HPLC reagents were purchased from Roth (Karlsruhe, Germany). The 1.8 µm, 2.1x50 mm separation column was an Eclipse plus C18. The column was pre-equilibrated for at least 30 min in a 9:1 mixture of eluents A and B; during the first two minutes of the run, the column contained 9:1 A:B, changed thereafter to 2:3 A:B for 2.3 minutes followed by a change to 1:9 A:B for 3.1 minutes and set to initial values of 1:9 for 2.6 minutes. The flow rate was 0.6 mL min -1 and the column temperature was maintained at 37°C. The excitation and emission wavelengths were, respectively 280 nm and 410 nm.
For the quantification of the redox equivalents, a 0.5 g sample of powdered leaf was suspended in 1 ml 0.1 M KOH dissolved in 50% (v/v) ethanol (NADPH, NADH) or in 1 mL 0.1 M HCl (NADP + , NAD + ). The entire extraction procedure was carried out under green light to minimize degradation of the analytes. The extractions were held on ice for 5 min, then centrifuged (14,000 7 rpm, 10 min, 4°C) and the supernatants subjected to ion chromatography coupled MS/MS following Ghaffari et al. (2016). However, the recovery experiment of NADPH and NADH in the samples was 50%. The results after light for 4 h could not be reliably calculated since the values were bellow detection limit and the results after light for 8 h did not show significant differences between the WT and different transgenic plants ( Figure S3).

Determination of the leaf content of glutathione
The method used to extract glutathione from the leaf material followed Davey et al. (2003). The separation and detection methods were established as follow. Approximately 100 mg of fresh leaf material was ground to fine powder using tissue homogenizer with 1 mM EDTA and 0.1% (v/v) formic acid at 4°C under green safe light and centrifuged at maximum speed (35,280 g) for 10 min. Measurements of oxidized and reduced glutathione were carried out immediately in freshly prepared extracts. Separation and analysis of the desired compounds was performed out on a C18 column (HSS T3, 1.8 µm, 2.1x150 mm, Waters Germany) and an UPLC/MS-MS (Infinity ll, 6490 Triple Quadrupole LC/MS, Agilent, Waldbronn, Germany), respectively. Two µL of extracts and the corresponding standards were injected in the mobile phase consisting of purest water plus 0.1% (v/v) formic acid and pure methanol plus 0.1% (v/v) formic acid. The temperatures of the auto sampler and column were maintained at 8°C and 37°C, respectively. Separated compounds were eluted at a flow rate of 0.5 mL min -1 . The evaluation and quantification of the compounds were performed using the software MassHunter, release B.04.00 (Agilent, Waldbronn, Germany).

Determination of photosynthetic parameters
The quantification of chlorophyll a was based on fluorescence measurements obtained using a multispeQ v1.0 device (photosynq.org) in conjunction with photosynQ software (Kuhlgert et al., 2016). Measurements were taken from the fully expanded leaves of six week old plants held in the dark for 4 h. The minimal fluorescence parameter F 0 was measured before exposing the leaf to a pulse of red (650 nm) light (500 ms, 3,000 μmol photon m −2 s −1 ), from which the maximal fluorescence parameter (F m ) was obtained. F m ′ and F s were determined during exposure to 160 μmol photon m −2 s −1 actinic light. Chlorophyll transient induction measurements on darkadapted leaves were carried out following Gómez et al. (2018). PSII parameters were calculated 8 based on the suggestion of Baker (2008), namely: The parameters Φ PSII, Φ NPQ, Φ No and qL were calculated according to Kramer et al. (2004).

Transmission electron and confocal laser scanning microscopy
Transmission electron microscopy was performed following Mayta et al. (2018). A 2 mm 2 piece cut from the center of the tobacco leaf was taken from both WT and Flv transgenics, and processed according to Kraner et al. (2017). For confocal laser scanning microscopy, the leaf samples were degassed to remove any entrapped air, and GFP activity was detected using an LSM780 device (Carl Zeiss, Jena Germany), with the excitation wavelength set to 488 nm; fluorescence was detected over the wavelength range 491-535 nm.

RNA isolation, cDNA synthesis and transcription analysis
Total RNA was extracted from young leaves following the protocol given by Logemann et al. (1987) after which it was subjected to DNase treatment (Life Technologies, Darmstadt, Germany) and converted to ss cDNA using a RevertAid first strand cDNA synthesis kit (Life Technologies, Darmstadt, Germany) provided with a template of 1 μg total RNA and oligo dT primer: the reaction was run at 42°C for 60 min. The primers used for qRT-PCR analysis of Flv transgenes are given in Table S4. The assays were performed in a CFX384 touch real-time system using the SYBR Green Master Mix Kit (Bio-Rad, Feldkirchen, Germany). The primer pairs employed to amplify Flv1 (Flv1-RT F/R) and Flv3 (Flv3-RT F/R), along with those amplifying the reference sequence Ubi10 (GenBank accession number At4g05320) are listed in Table S4. Relative transcript abundances were determined using the method Schmittgen and Livak (2008) method.

Statistical analysis
Means and standard errors (SE) were calculated using SigmaPlot software (www.sigmaplot.co.uk/products/sigmaplot/sigmaplot-details.php). The Students' t-test was used to test for the statistical significance of differences between means. 9

The growth response of Flv transgenics to variation in the light intensity
The development of biomass in both WT and Flv transgenic plants grown at varying light intensities is illustrated in Figure 1. When illuminated with 50 µmol photons m -2 s -1 , the performance of the transgenic plants was not distinguishable from that of the WT ones. However, when the intensity was increased to either 160 or 300 µmol photons m -2 s -1 , the transgenic plants were clearly larger (Figure 1a), as confirmed by comparisons of their shoot dry weight in transgenic plants harbored Flv1/Flv3, out-performed WT by 10-30% (Figure 1b). Among the plants exposed to the highest intensity (600 µmol photons m -2 s -1 ), the only lines which were superior to WT were those expressing Flv1/Flv3.

The effect of expressing Flv transgenes on the biomass accumulation of plants exposed to long days
Under a long day regime, the Flv transgenics reached flowering earlier than WT plants (data not shown), and were more bushy, with increased inflorescences (Figure 2a). Their shoots dry weight was up to 1.8 fold greater (Figure 2b). Seed size was unaffected by the presence of the transgene (data not shown), but seed yield was 1.2-1.8 fold greater (Figure 2c).

Chlorophyll fluorescence in the leaf of Flv transgenics
Chlorophyll fluorescence measurements were taken from the fully expanded leaves of darkadapted six week old plants. The F v /F m ratio (a measure of the maximum quantum yield of PSII) was somewhat higher in the leaves of the Flv1/Flv3 transgenic than in those of WT (Figure 3a). In leaves exposed to 160 µmol photons m -2 s -1 , Φ PSII (representing the quantum yield of PSII) in the Flv1/Flv3 transgenics showed higher than in WT leaves (Figure 3b). Similarly, qL (an estimate of the fraction of open reaction centers) was greater in the leaves of the Flv1/Flv3 transgenics than in WT leaves (Figure 3c). Φ NPQ (the quantum yield of quenching due to lightinduced processes) was significantly lower during the measurement in the Flv1/Flv3 trangenics than in WT (Figure 4a). In contrast, Φ NO (the quantum yield of non-light-induced quenching processes) in Flv1/Flv3 trangenics clearly shows no difference to the WT level ( Figure 4b).
Finally, NPQ (non-photochemical quenching) was up to two fold times higher than the WT level in the Flv1/Flv3 transgenics (Figure 4c).

The effect of expressing Flv transgenes on leaf sugar, starch and amino acid content
The diurnal variation in the content of both soluble and insoluble sugars was determined in the leaves of WT and Flv transgenics (Figs 5 and S4). The leaves of plants harboring either Flv1/Flv3 accumulated a significantly higher concentration of sucrose than did those of WT plants; in each line, the sucrose content increased gradually during the light period and fell during the dark period ( Figure 5a). Leaf starch contents at the beginning of the light period did not vary between the genotypes, but the accumulation of starch during the day increased more rapidly (by as much as 1.7-fold) in the transgenics than in WT (Figure 5b). Glucose and fructose contents varied rather inconsistently ( Figure S4). No clear genotypic differences were observed with respect to the accumulation of any of the amino acids following the plants' exposure to 4 h of light (Table   S1), but by the end of the light period (8 h of light), the increased pool of asparagine, aspartate, glutamine and alanine was observed in Flv1/Flv3 transgenics with respect to WT (Table S2).

The effect of expressing Flv transgenes on starch granule size and number
Based on micrographs generated by transmission electron microscopy, it was apparent that while starch granule size was unaffected by the expression of Flv, their number was enhanced in the leaves of plants expressing Flv1/Flv3 (Figure 5c).

The effect of expressing Flv transgenes on the ATP levels in the leaf
The leaf content of ATP, ADP and AMP was measured both during the light period (after exposure to both 4 h and 8 h) and during the dark period (16 h). The ATP content of the Flv1/Flv3 transgenics' leaves was up to 1.5 fold higher than that of WT leaves, whether the leaves were sampled during the light or the dark period ( Figure 6a). The ratio of ATP/ADP is higher at 4 h and 16 h in all the trangenics lines (Figure 6b). The leaf content of neither ADP nor AMP differed significantly between the Flv1/Flv3 transgenics and WT (Table S3). The total adenylate content was not changed at all time points except L1 than that of WT plants (Figure 6c).

The effect of expressing Flv transgenes on the leaf metabolome
The content of a number of metabolites was affected by the expression of Flv transgenes. After a 4 h exposure to light, the concentration of hexose phosphates was raised in all of the transgenic plants to a level significantly higher than that obtaining in WT leaves ( Figure 7a

The effect of expressing Flv transgenes on the content of antioxidants
The content of both the reduced and oxidized forms of glutathione were measured and their ratio was calculated. There was a significant shift towards the reduced form in L3 whereas oxidized form is statistically higher in line L2 of the transgenic leaves ( Figure S6a,b), resulting in a reduction in the GSH/GSSG ratio of up to 2.5 fold ( Figure S6c).

Discussion
Photosynthesis is essential for the growth and development of plants, but the process is relatively inefficient since just 8-10% of the overall spectrum of solar radiation is used to convert CO 2 to sugar, while only 2-4% of incident light energy is channeled into growth (Long et al., 2006;Zhu et al. 2010). The rest harvested solar energy is released as heat and fluorescence. Tapping the lost solar energy and avoiding uncontrolled ROS production potentially enhance photosynthesis and overall plant growth by establishing electron sinks in chloroplasts.
Here, the intention was to establish an additional electron sink downstream of PSI in A. thaliana by heterologously expressing either Flv1 and/or Flv3. The observation was that plants harboring Flv1/Flv3 were able to maintain PETC in a more oxidized state prior to the induction of the dark reaction of photosynthesis (Figure 3a). Furthermore, plants harboring Flv1/Flv3 exhibited an improved performance with respect to ɸ PSII , qL (Figure 3b,c) and NPQ (Figure 4a,c); these outcomes imply that the transgenic plants were capable to enhancing the flow of electrons from PSII to PSI, as well as having an improved capacity to dissipate electron pressure in the PETC during the dark-to-light transition, and the ability to activate protective non-photochemical quenching. The implication is that Flvs can act as regulators of photosynthesis like CET and Mehler reaction, specifically avoiding the over-reduction of the PETC (Figure 3). Gómez et al. (2018)  In phototrophic organisms, AEF pathways are induced shortly after their exposure to light, thereby generating ATP to supply the Calvin-Benson cycle and to support photorespiration during the dark-to-light transition. As reported by Shikanai and Yamamoto (2017), under steady-state conditions, Flvs have poor access to either NADPH or Fd, due to activation of the Calvin-Benson cycle, but may regain functionality under highly reduced stromal conditions. Here, the growth of A. thaliana plants heterologously expressing Flv responded differentially to the intensity of light provided (Figure 1). Under low light intensity conditions, electron transport is typically limited by the availability of photons, so that there is no need of relief with respect to the electron pressure on the PETC. The latter becomes important as the light intensity increases, and the Flv system is able to dissipate surplus reducing equivalents as long as the light intensity does not become excessive (Figure 1). Indeed, plants look stressed at this irradiation levels, as revealed by the color of the leaves (presumably due to anthocyanin accumulation, a typical response to high light). Wild type plants exhibited light stress symptoms during early exposure but acclimatized later, a similar phenomenon observed in Flv-expressing plants. When exposed to a long day (16 h photoperiod), the presence of the Flv transgenes accelerated flowering (data not shown) and the plants biomass and seed yield potential were boosted (Figure 2b,c), illustrating the benefit of Flvs in conditions where surplus electrons are produced.
In chloroplasts, ATP is generated via the linear electron transport and cyclic electron transport pathways (DalCorso et al., 2008), while in the mitochondria; the respiratory electron transport 13 chain makes an additional contribution for ATP during daylight hours (Bailleul et al., 2015). The level of ATP was higher in the leaves of the Flv transgenics than in those of WT after the plants' exposure to either 4 h or 8 h of light, indicating that the Flvs were able to dissipate electrons at PSI, enhance linear electron flow and thereby establish the pH gradient required for ATP synthesis (Figure 6a). Here, we cannot exclude the possibility that heterologous expression of the Flvs provided an AEF able to function independently of the cyclic electron transport pathway and to contribute to the ATP demand, consequently enhancing growth. The adenylate pool is also an important regulator of plant metabolism (Geigenberger et al., 2010). In the Flv transgenics, an increased ATP level served to boost the level of activity in the Calvin-Benson cycle, which in turn helped to maintain a high level of hexose phosphate during the middle of the light period ( Figure   7a,b). The hexose phosphates and ADPGlc (Figure 7a (Figure 5a,b). The absence of any effect of Flv expression on the level of the TCA cycle intermediates citrate and malate implies that these organic acids most likely play at best a minor role in determining biomass production ( Figure S5a-d).
The manipulation of plastidial levels of adenylate kinase was shown in both potato -where it has succeeded in raising starch and amino acid content (Regierer et al., 2002) -and in A. thalianawhere it has been shown to boost the accumulation of amino acids and promoted plant growth (Carrari et al., 2005). In the Flv transgenics exposed to 4 h of light, however, there was no evidence for any significant alteration in the leaf's amino acid content, suggesting that illuminated leaves converted most of their photoassimilate into starch (Table S1). In contrast, the content of each of aspartate, asparagine, glutamine and alanine was raised by the plant's exposure to 8 h of light (Table S2), consistent with the observation that an increase in carbon availability enhances the assimilation of the nitrogen needed for protein synthesis and hence for the continuation of growth in the absence of light (Lawlor, 2002). Furthermore, the level of both sucrose and ATP were higher in the Flv transgenics than in WT, not only during the light period, but also during the dark period. According to Sharkey et al. (2004), the levels of both sucrose and ATP are highly dependent on carbon metabolism during the night, while both Sulpice et al. (2009) and Graf and Smith (2009) have shown that these levels constitute important determinants of biomass accumulation. It seems probable therefore that the growth advantage enjoyed by the Flv transgenics reflects their superior capacity to generate photoassimilate and ATP.
14 The ratio between the reduced and oxidized forms of glutathione was significantly lower in the leaf of all of the Flv transgenics than in WT leaf ( Figure S6c). Glutathione makes a major contribution to balancing the redox status of the mitochondria and chloroplasts. Considering the pool of glutathione in all the organelles, the ratio GSH to GSSG was estimated to be 20:1 in control conditions in average (Mhamdi et al., 2010a). However, in the Flv transgenics, the level of GSH was lower than in WT, while that of GSSG was higher ( Figure S6a,b). The presence of the Flvs likely inhibits the leakage of electrons from the PETC, thereby damping down the production of ROS. No convincing experimental evidence was obtained to show that level of ROS (H 2 O 2 ) was lower in the leaves of the Flv transgenics than in WT leaves (data not shown).
Overall, the effect of heterologously expressing Flv in A. thaliana appeared to impact the cellular redox homeostasis; the Flvs are unlikely to promote oxidative stress because surplus electrons are diverted to the alternative Flv sink (Figure 8).

Conclusion
The present data have demonstrated that electron sinks are essential for the efficient functioning of the PETC and that they can be provided to an angiosperm such as A. thaliana by introducing genes encoding additional electron carriers such as cyanobacterial Flvs, either by expressing one Flv or by co-expressing two. The postulation is that the Flvs mediate the flow of electrons through PSI and PSII by delivering them to oxygen, which is converted to water without the production of ROS. In this way, the production of redox equivalents in the form of NADPH is maintained, allowing carbon to be recycled through the Calvin-Benson cycle. The latter generates the phosphorylated metabolites required for starch synthesis. The energy needed for this reaction is provided by ATP, which is produced via Flv-enabled electron flow and lumen acidification.
ATP is also used for the conversion of Glc1P to ADPGlc, mediated by ADPGlc pyrophosphorylase activity. The promotion of ADPGlc finally results in an enhanced level of starch synthesis in leaves exposed to light, and the accumulated starch is metabolized during the dark phase, allowing for a continuous growth of the plant (Figure 8).         balances the surplus of the electron flow through PSI and PSII by delivering these electrons to oxygen, which is converted to water. The production of redox equivalents such as NADPH is maintained, resulting in the recycling of carbon through the Calvin-Benson cycle, which in turn generates an increased supply of the phosphorylated metabolites needed for starch synthesis. The energy required for this reaction is provided by ATP, which is synthesized in the PETC. The continuous flow of electrons results in the acidification of the lumen, which is the driving force for ATP synthesis. ATP is used for the conversion of Glc1P to ADPGlc via ADPGlc pyrophosphorylase. An increased availability of ADPGlc supports a higher level of starch synthesis; the starch accumulates when the leaf is exposed to light and is degraded during the dark phase.       Table S1. The contents of individual amino acids in WT and transgenic plants expressing Flv1/Flv3. Measurements taken from six week old plants which had been exposed to 4 h of light.

Supplementary Material
Data shown in the form mean±SE (n=5). Table S2. The content of amino acids in the leaf of WT and transgenic plants expressing Flv1/Flv3. Measurements taken from six week old plants which had been exposed to 8 h of light.
Data shown in the form mean±SE (n=5). *,**: means differ from the performance of WT at, respectively, P≤0.05 and ≤0.01.