Journal of Experimental Botany, Vol. 72, No. 13 pp. 4949–4964, 2021 doi:10.1093/jxb/erab193 Advance Access Publication 8 May, 2021 This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) RESEARCH PAPER Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco María Ancín1, , Luis Larraya1, , Igor Florez-Sarasa2,3, , Camille Bénard4, Alicia Fernández-San Millán1, Jon Veramendi1, , Yves Gibon4, , Alisdair R. Fernie2, , Iker Aranjuelo5, , and Inmaculada Farran1,*, 1 Institute for Multidisciplinary Applied Biology (IMAB), Dpto. Agronomía, Biotecnología y Alimentación, Universidad Publica de Navarra (UPNA), Campus Arrosadia, 31006 Pamplona, Spain 2 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany 3 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, 08193 Barcelona, Spain 4 UMR 1332 Biologie du Fruit et Pathologie and Plateforme Metabolome Bordeaux, INRA, Bordeaux University, 33882 Villenave d’Ornon, France 5 Instituto de Agrobiotecnología, CSIC-Gobierno de Navarra, Avda. Pamplona 123, 31192 Mutilva, Spain * Correspondence: farran@unavarra.es Received 19 January 2021; Editorial decision 23 April 2021; Accepted 27 April 2021 Editor: Rodrigo Gutiérrez, Pontificia Universidad Católica de Chile, Chile Abstract In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is funda- mental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered me- tabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and sol- uble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These re- sults showed an up-regulation of the glutamine synthetase–glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespir- ation and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts. Keywords: Carbon metabolism, chloroplast, glutamine synthetase, GS-GOGAT pathway, nitrogen metabolism, photorespiration, thioredoxin. Introduction The carbon (C) and nitrogen (N) metabolisms of higher plants and C skeletons are available for plant metabolic requirements, are tightly coordinated to maintain optimal growth and de- which entails that photosynthetic products must be parti- velopment. This strong connection ensures that amino acids tioned between carbohydrate and amino acid biosynthesis. © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 4950 | Ancín et al. This partitioning is controlled by an extremely complex net- reassimilation of ammonia released from a variety of metabolic work involving signals from various metabolites, proteins, and pathways such as photorespiration, catabolism of amino acids, hormones. Moreover, the redox status of the cell has been pro- and metabolism of phenylpropanoids (Foyer et al., 2006). The posed as an important factor that might impact the integration two enzymes constitute the GS-GOGAT cycle, which is es- of plant C and N assimilation (Nunes-Nesi et al., 2010). sential for N assimilation and photorespiration in chloroplasts. Within chloroplasts, two thiol-based redox regulatory sys- In vitro experiments have also provided evidence that these two tems function in parallel: (i) the ferredoxin-thioredoxin enzymes may be subjected to redox regulation in higher plants: (Fd-Trx) system, which depends on photosynthetically reduced (i) spinach and soybean Fd-GOGAT enzymes were reported Fd to supply electrons to the Fd-Trx reductase, which in turn to be preferentially activated by Trx m (Lichter and Häberlein, reduces Trxs; and (ii) the NADPH-dependent Trx reductase 1998); and (ii) the activity of GS2 has been suggested to be type C (NTRC), which contains both Trx-reductase and Trx directly redox modulated in chloroplasts (Choi et al., 1999). domains in a single polypeptide, and is reduced by NADPH. Moreover, both enzymes were identified as Trx targets in dif- Plastid classical Trxs transfer reducing power to target enzymes ferent proteomic studies (Montrichard et al., 2009), and a redox through the action of Fd-Trx reductase, modulating their ac- regulation of the GS-GOGAT cycle in vivo was suggested to tivity related to various metabolic pathways. Five Trx types, take place in Arabidopsis leaves treated with the reductant termed f, m, x, y, and z, have been found within the chloroplast. DTT (Kolbe et al.,2006). Originally, f- and m-type Trxs were identified as regulators of The present study aims to shed light on the Trx-mediated thiol enzymes involved in photosynthetic C assimilation in the redox regulation of N metabolism in plant leaves. Previous work chloroplast (Wolosiuk and Buchanan, 1977; Buchanan, 1980), has indicated that the altered redox status of tobacco chloro- whereas x-, y-, and z-type Trxs have been more closely linked plasts achieved by the overexpression of Trx m might impair to the regulation of antioxidant enzymes (Meyer et al., 2012). the cell C status (Sanz-Barrio et al., 2013), involving processes However, proteomic approaches revealed a great number of such as photosynthesis (Courteille et al., 2013) and chloroplast chloroplast-localized Trx targets that basically cover all the redox homeostasis (Rey et al., 2013). However, the effect of main chloroplast functions (Montrichard et al., 2009). More Trx m overexpression on C and N metabolism still remains to recently, genetic studies have been used to dissect specific be analyzed. Here, we conducted a wide metabolomic charac- roles for plastid Trxs in vivo (Geigenberger and Fernie, 2014; terization of Trx m-overexpressing tobacco plants, concluding Geigenberger et al., 2017; Nikkanen et al., 2017). According to that Trx m alters C and N partitioning by promoting N instead previous studies,Trx f is mainly associated with the redox regu- of C metabolism. Moreover, a putative role of Trx m in GS2 lation of C reactions, including enzymes of both the Calvin– redox protection, linked to its effect on the photorespiratory N Benson cycle (CBC) and starch metabolism (Collin et al., 2003; cycle, is discussed. Taken together, our results suggest a role for Marri et al., 2009; Sanz-Barrio et al., 2013; Thormählen et al., Trx m in controlling N metabolism in plant chloroplasts. 2013;Yoshida et al., 2015; Naranjo et al., 2016). By contrast,Trx m is proposed to control a wider range of processes, such as the biogenesis of Cytb6f and PSII (Motohashi and Hisabori, 2010; Materials and methods Wang et al., 2013), cyclic electron flow around PSI (Courteille Plant material and growth conditions et al., 2013), tetrapyrrole synthesis (Luo et al., 2012), export of The study was carried out with tobacco (Nicotiana tabacum cv. Petit Havana reducing power via the malate valve (Thormählen et al., 2017), SR1) wild-type (Wt) plants and transplastomic plants overexpressing the non-photochemical quenching-dependent photoprotection mature Trxm coding sequence (hereafter termed the o/exTrxm geno- (Da et al., 2018), and the activation of antioxidant (Collin et al., type) from the chloroplast genome under the control of the rrnG10L 2003; Rey et al., 2013) and CBC (Okegawa and Motohashi, promoter (Sanz-Barrio et al., 2013). Because insertion of transgenes into 2015) enzymes. However, the involvement of plastid Trxs in plastid DNA occurs via homologous recombination at a specific inser- tion site, all transplastomic lines are identical (Bock, 2015) and therefore the regulation of N metabolism and, more precisely, N assimi- a single transplastomic line was used. Two-week-old seedlings, germin- lation and partitioning has been poorly studied. ated in vitro, were transferred to 3 litre pots filled with soil and grown Previous studies have suggested a role of Trxs in the regula- in a phytotron under a 16 h light/8 h dark photoperiod with 150 µmol tion of N assimilation via redox regulation of chloroplast glu- m–2 s–1 photosynthetic photon flux density, at 28 °C. Plants were watered tamine synthetase (GS2) and Fd-dependent glutamate synthase twice a week with 0.5× Hoagland nutrient solution. To test the behavior of o/exTrxm plants under N-limiting conditions, a second experiment (Fd-GOGAT) in algae (Schmidt, 1981; Tischner and Schmidt, was conducted. In this case, plants were grown under hydroponic condi- 1982; Florencio et al., 1993) and cyanobacteria (Papen and tions in 3 litre pots filled with an inert substrate such as perlite. Plants were Bothe, 1984; Lindahl and Florencio, 2003). Fd-GOGAT cata- irrigated daily with nutrient solution [3 mM KH2PO4, 1 mM MgSO4, lyzes the conversion of glutamine (Gln) and 2-oxoglutarate to 1 mM CaCl2 and 2 g l–1 Nutrel C commercial micronutrient mixture two molecules of glutamate (Glu), whereas GS2 catalyzes the (Yara Iberian, Madrid, Spain), pH 5.8–6.2] containing 1 mM or 2 mM KNO3 (Geiger et al., 1999). For the 1 mM KNO3 treatment, 1 mM KCl ATP-dependent formation of Gln from Glu and ammonia, was added to supplement potassium in line with the 2 mM KNO3 treat- thus participating in primary N assimilation as well as in the ment. Pots were washed every week with distilled water to prevent salt Impact of thioredoxin m on N metabolism in chloroplasts | 4951 accumulation. All analyses were carried out on the youngest fully ex- St. Joseph, MI, USA). Metabolites were identified by comparison with panded leaves of 6-week-old plants.Whole plants, including leaves, stems, database entries of authentic standards (Kopka et al., 2005; Schauer et al., and roots, were harvested and dried in an oven at 60 °C for 72 hours, and 2005) using TagFinder software (Luedemann et al., 2012). The peak in- total biomass [g dry weight (DW)] was determined. tensity of a representative fragment was normalized with that of the in- ternal standard ribitol and sample fresh weight (FW) and referred to as relative abundance.The parameters used for the peak annotation are listed Trx m immunoblot analysis in Supplementary Table S1 according to Fernie et al. (2011). For quantification of Trx m, proteins from leaf samples were extracted and blotted using specific anti-Trx m antibody (Sanz-Barrio et al., 2013). Total protein was extracted with extraction buffer (125 mM Tris–HCl, Enzyme assays pH 6.8, 4% SDS, and 20% glycerol), and quantified using the RC-DC Leaves harvested after 4 h illumination were immediately frozen and protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin ground in liquid N2.Aliquots of ~20 mg FW were extracted with 500 µl of as a standard, according to the manufacturer’s instructions. Overexpressed extraction buffer according to Gibon et al. (2004). Phosphoglycerokinase Trx m in leaf extracts was quantified by western blotting using serial (PGK), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase dilution of Trx m protein produced in Escherichia coli as a standard. (NADP-GAPDH), fructose-bisphosphatase (FBPase), GS, Fd-GOGAT, Immunoblots were quantified using the GeneTools analyzer software NAD-dependent glutamate dehydrogenase (NAD-GDH), and (SynGene, Cambridge, UK). phosphoenolpyruvate carboxylase (PEPC) were assayed following Gibon Determination of the Trx m redox state in vivo was performed in leaves et al. (2004), and triose-phosphate isomerase (TPI) was assayed according of plants overexpressing Trx m in light and dark conditions. Leaves were to Biais et al. (2014). All assays were performed on microplates using ground in liquid N2, and protein was extracted and incubated with or 96-head pipetting robots (Hamilton, Villebon-sur-Yvette, France) and without 50 mM DTT (reducing agent) or 50mM diamide or H2O2 (oxi- UV-visible readers (SAFAS, Monaco, France). Assay optimization and dant agents). Extracts were precipitated with 10% (w/v) trichloroacetic calculations were conducted as described in Bénard and Gibon (2016). acid and washed twice with 100% ice-cold acetone before the protein pellet was resuspended in 2% SDS and 150 mM Tris–HCl, pH 7.5, buffer. One portion of the samples was incubated with 10 mM 4-acetamido-4′- RNA extraction and RT–qPCR experiments maleimidylstilbene-2,2′-disulfonate (AMS) (Invitrogen, ThermoFisher A 1 µg sample of DNAse-treated total RNA, extracted from tobacco Scientific) for 1 h at room temperature in the dark, to alkylate cysteine leaves with Trizol® reagent (Invitrogen), was used for cDNA synthesis (Cys) residues. Both AMS-treated and untreated samples were separated by reverse transcription (RT) with a PrimeScript™ RT–PCR Kit by 15% non-reducing SDS-PAGE and immunoblotted with anti-Trx m (Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions. specific antibody (1:5000). The in vivo reduced form becomes labelled Quantitative PCR (qPCR) analysis was carried out using gene-specific with AMS and migrates more slowly in SDS-PAGE. primers listed in Supplementary Fig. S1, and was conducted in three bio- logical replicates. The analysis was performed with SYBR® Premix Ex Taq™ (Takara Bio), and the comparative threshold cycles (Ct) method Determination of starch, soluble sugars, protein, and (ΔΔCt) was applied for relative quantification of gene expression using an amino acids AriaMx real-time PCR System (Agilent Technologies). Relative expres- The concentrations of soluble sugars and starch were determined after 4 h sion levels were normalized against 16s rRNA and actin as reference genes. light and at the end of the light and dark periods in tobacco leaf ethanol extracts and pellets by using, respectively, HPLC and an amyloglucosidase- based test kit (R-Biopharm AG, Darmstadt, Germany) as previously Chlorophyll, nitrate, and ammonium quantification described (Sanz-Barrio et al., 2013). For determination of protein con- Photosynthetic pigments were extracted from leaf disks collected from centrations, leaf samples (100 mg) harvested after 4 h illumination were fully expanded leaves and crushed in 5 ml of 80% acetone. After centrifu- ground in liquid N2, resuspended in 200 μl of soluble protein extraction gation at 10 000 g for 5 min at 4 °C, the amount of Chl a and b was meas- buffer (100 mM Tricine–NaOH, pH 8, 8 mM MgCl2, and 2 mM EDTA) ured spectrophotometrically and calculated according to Lichtenthaler and incubated for 15 min on ice. The supernatant was obtained after (1987). 5 min of centrifugation at 10 000 g at 4 °C and the concentration of Aliquots of ~30 mg of frozen leaf material were homogenized with soluble protein was measured with an RC-DC protein assay (Bio-Rad) 600 μl of ultrapure water. Samples were then incubated at 80 °C for using bovine serum albumin as a standard. For amino acid quantifica- 5 min and centrifuged at 20 000 g and 4 °C for 20 min. The supernatant tion, leaf samples harvested after 4 h illumination (50 mg) were extracted was recovered and used for nitrate (Cataldo et al., 1975) and ammonium with ethanol, derivatized, and analyzed on a reverse-phase HPLC column (Sarasketa et al., 2014) determination. using the fluorescing reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (Loiret et al., 2009). Gas exchange and chlorophyll fluorescence determinations Gas exchange measurements were carried out on fully expanded Metabolomics leaves with the LI6400XT gas exchange portable photosynthesis Frozen leaf samples harvested after 4 h illumination were ground in 2 ml system (LI-COR, Lincoln, NE, USA). Determinations were con- microcentrifuge tubes using stainless steel beads and a Retsch Mixer Mill ducted between 4 h and 7 h after the onset of illumination. The light- MM 400 (Retsch, Haan, Germany) under cryogenic conditions. From saturated rate of CO2 assimilation was measured at 1000 µmol m–2 s–1 these samples, metabolites were extracted, derivatized, and analyzed via photosynthetic photon flux density, with 400 μmol s−1 air flow rate, gas chromatography–time-of-flight mass spectrometry (GC-TOF-MS) a temperature of 25 °C, and 60% relative humidity. Photosynthetic analyses as described before (Lisec et al., 2006). The GC-TOF-MS parameters were obtained using the equations of von Caemmerer and system consisted of a CTC CombiPAL autosampler (CTC Analytics, Farquhar (1981). After performing the net photosynthesis determin- Zwingen, Switzerland), an Agilent 6890N gas chromatograph (Agilent ations under ambient CO2 and O2 conditions, leaves were kept in the Technologies, Santa Clara, CA, USA) and a LECO Pegasus III time- chamber and N2 from a tank (Air Liquide, Paris, France) was plugged of-flight mass spectrometer running in EI+ mode (Leco Instruments, into the inlet of the Li-6400XT to remove O2 from the air entering 4952 | Ancín et al. the leaf chamber, to allow photorespiration determinations (AN2%). phenotype similar to that previously reported (Rey et al., 2013; Net photosynthesis at high O2 concentration (AN40%) was measured Sanz-Barrio et al., 2013; Ancín et al., 2019). In the described when a tank containing 40% O2 was plugged into the inlet of the Li-6400XT. Photorespiration was expressed as the percentage re- growing conditions, o/exTrxm plants accumulated Trx m pro- duction in the net photosynthetic rate by photorespiration using the tein at levels around 12-fold higher than that of Wt plants (Fig. equation: (AN2%–AN21%)/AN2%×100 (Florez-Sarasa et al., 2019). The 1B). A significant portion of the overexpressed protein was re- measurements of dark respiration (Rd) were performed at least 20 min duced in the o/exTrxm genotype in the light (Fig. 1C), and after the lights were switched off to avoid light-enhanced dark respir- presumably was active and able to transfer reducing equivalents ation, and thus to obtain a proper Rd measurement. to its target enzymes. We then investigated the accumulation of C and N-derived Redox-dependent structure and activity of glutamine synthetase metabolites in the o/exTrxm genotype. Concerning C-derived Protein complexes were analyzed from intact chloroplasts solubilized metabolites, starch concentrations were significantly decreased with 1.5% digitonin by Blue Native (BN)-PAGE using a 4–16% Native in o/exTrxm leaves relative to the Wt, by ~40% after both 4 h PAGE Novex Bis-Tris gel (Invitrogen) according to the manufacturer’s and 16 h of light, while an 80% decrease was observed at the instructions. After electrophoresis, protein complexes were transferred to a polyvinylidene fluoride (PVDF) membrane and probed with anti-GS2 end of the 8 h dark period (Fig. 2A). Likewise, the sucrose antibody (AS08 296, Agrisera,Vännäs, Sweden). Protein samples were in- concentration was significantly lower in the transplastomic cubated for 30 min on ice in the presence or absence of 10 mM DTT or line, showing a quite similar pattern to that of starch (Fig. 2B). 1 mM CuCl2 before loading. Furthermore, o/exTrxm leaves showed a dramatic decrease in Determination of GS redox-dependent activity was performed, as the glucose and fructose concentrations compared with the previously described (Scheible et al., 1997), on protein extracts from Wt plants incubated with or without 5 mM DTT or 100 µM CuCl2 for Wt, with the concentrations of these metabolites being very 30 min on ice. Assessment of the recovery of GS activity by reduction was similar in the light and dark (Fig. 2C, D). These results indi- performed by incubating oxidized samples (after treatment with 100 µM cate that, as suspected from previous works (Sanz-Barrio et al., CuCl2) with 5 mM DTT for 30 min on ice. 2013), the leaf carbohydrate metabolism was impaired by the overexpression of Trx m in tobacco chloroplasts. Quantification and stability of GS2 protein By contrast, the amounts of soluble protein and total amino For quantification of GS2, ground leaf samples were homogenized in 2 acids were higher in o/exTrxm leaves (~40%) compared with volumes of total protein extraction buffer (125 mM Tris–HCl, pH 6.8, the Wt (Fig. 3, Supplementary Table S2). To further analyze 4% SDS, and 20% glycerol). Samples were incubated on ice for 15 min the impact of Trx m overexpression on N metabolism, the leaf and then centrifuged at 10 000 g for 5 min at 4 °C, and the supernatant free amino acid profile was analyzed. The concentrations of 10 was recovered. Protein extracts were quantified by a RC-DC protein amino acids were significantly higher in the o/exTrxm geno- assay (Bio-Rad), and 2.5 µg samples were separated by SDS–PAGE on 10% polyacrylamide gels and transferred to a PVDF membrane for im- type: alanine, asparagine, aspartic acid, γ-aminobutyric acid, Glu, munoblotting with anti-GS2 antibody. Immunoblots were quantified Gln, isoleucine, leucine, serine (Ser), and valine. In contrast, only using the GeneTools analyzer software (SynGene). phenylalanine (Phe) showed a significantly lower concentration, Determination of the stability of GS2 protein was performed essen- by ~14%, in the transplastomic plants compared with the Wt tially as described in Ortega et al. (1999) with some modifications. Leaves (Fig. 3, Supplementary Table S2). Therefore, both the observed of Wt and o/exTrxm plants were ground in liquid N2 and homogenized with three volumes of extraction buffer [50 mM Tris–HCl, pH 8.0, 20% decrease in starch and sugars and the increase in soluble protein (v/v) glycerol, 1 mM MgCl2, 1 mM DTT, and protease inhibitor cock- and amino acids suggest that the overexpression of Trx m in tail].The homogenate was centrifuged at 20 000 g for 15 min at 4 °C.The tobacco chloroplasts induces an imbalance between C and N. supernatant was incubated in a reaction containing 50 mM imidazole, pH 7.4, 0.125 mM FeCl3 and 5 mM DTT, for 24 h at 30 °C. Aliquots were taken at several times and analyzed by western blot. The experi- Metabolomic analysis supports a C and N imbalance ment was repeated with eight biological replicates, and a representative in the o/exTrxm genotype immunoblot is shown. GC-TOF-MS-based metabolite profiling was performed to investigate the global effect of Trx m overexpression on Results plant metabolism. A total of 44 metabolites were annotated, including amino acids, sugars, organic acids and other metabol- Overexpression of Trx m alters the balance of C and N ites (Supplementary Table S3). Only few metabolites displayed in tobacco plants statistically significant differences between o/exTrxm and the We first characterized the tobacco plants overexpressing Wt in the GC-MS analysis due to high variation of the data the endogenous Trxm gene from the plastid genome (o/ (Supplementary Table S3). Nevertheless, several metabolites exTrxm plants) grown in a phytotron under a 16 h light/8 h exhibited the same general trend as that shown in Figs 2 and dark regime with 150 µmol m–2 s–1 photosynthetic photon 3. Sugars displayed the same general trend to decrease in both flux density, at 28 °C. Plants of this genotype showed slower analyses, and the concentrations of alanine and Ser were both growth than the Wt and pale-green colored leaves (Fig. 1A), a significantly increased while Phe was significantly decreased, as in the HPLC analysis (Figs 2, 3, Supplementary Table S2, Impact of thioredoxin m on N metabolism in chloroplasts | 4953 the overexpression of Trx m in chloroplasts alters C and N partitioning in tobacco plants, promoting N (rather than C) me- tabolites and also decreasing the contents of phenylpropanoid precursors. Activity of C and N metabolism enzymes We further analyzed the activity of several enzymes involved in primary C and N metabolism in Wt and o/exTrxm leaves harvested after a 4 h light period. Consistent with the proposal that Trx m plays an important role in the activation of CBC en- zymes in vivo (Okegawa and Motohashi, 2015), a significant in- crease in PGK, NADP-GAPDH, and stromal FBPase activities was observed in the o/exTrxm genotype compared with the Wt, whereas the activity of TPI was not significantly different between the genotypes (Fig. 4). When the activity of N cycle- related enzymes was analyzed, the o/exTrxm genotype also showed a general increase in the activity of the GS-GOGAT cycle enzymes. GS and Fd-GOGAT activities were higher in o/exTrxm leaves, although this was statistically significant only for GS (Fig. 4). NAD-GDH and PEPC activities were also measured because of their central position at the interface between C and N metabolism. The o/exTrxm genotype dis- played higher extractable activities for both of these enzymes (Fig. 4). It is also worth mentioning that all these enzyme ac- tivities were still increased when expressed on a protein basis Fig. 1. Trx m overexpression and redox state in tobacco plants. (Supplementary Fig. S1). With the exception of FBPase ac- (A) Phenotype of transplastomic o/exTrxm and Wt plants. (B) Trx m accumulation in transplastomic tobacco plants. Quantitation of Trx tivity, the observed increase in enzyme activity also seemed to m protein was done by densitometric analysis of a dilution series of be specific to Trx m because tobacco plants overexpressing its recombinant Trx m from E. coli and leaf extracts from o/exTrxm and Wt counterpart, Trx f (Sanz-Barrio et al., 2013), did not show the plants. Representative western blot analyses of Trx m expression are same effect (Supplementary Table S4). shown. The amount of plant extract loaded, indicated over the blots, was To assess whether the significant and specific increase ob- adjusted to be comparable to the signal of the recombinant Trx m used as a standard TP, total protein. Results are presented as the fold increase served in the enzyme activities of Trx m-overexpressing plants of Trx m content in o/exTrxm genotype relative to the Wt (=1). Results resulted from a transcriptional or post-transcriptional regula- are the mean ±SE of three independent plants per genotype. Statistical tion, the levels of mRNA transcripts of genes encoding PGK, significance compared with the Wt is indicated by an asterisk (*P<0.05, NADP-GAPDH, and GS2 were analyzed using RT–qPCR. Student’s t-test). (C) Redox status of overexpressed Trx m in tobacco The transcript levels of all these genes were unchanged in the chloroplasts. AMS alkylation and western blot analysis after non-reducing SDS-PAGE of Trx m (specific antibody was used). Protein leaf extracts o/exTrxm genotype compared with the Wt (Supplementary incubated with DTT, H2O2, or diamide (Diam) before treatment with Fig. S2), revealing that their increased activity likely resulted AMS were used as reduced and oxidized controls. Protein extracts not from a post-transcriptional regulation specifically promoted by incubated with AMS were used as non-alkylated controls. The mobility the overexpression of Trx m in chloroplasts. of the alkylated (reduced; red) and non-alkylated (oxidized; ox) forms is indicated. Impact of Trx m overexpression on oligomerization, activity, and stability of GS2 Supplementary Table S3). Metabolites not overlapping with those detected by HPLC are shown in Table 1 as relative levels We further investigated the oligomerization, activity, and stability to Wt. Among the sugars, a general downward trend was ob- of GS2 in o/exTrxm and Wt plants (Fig. 5). Since GS2 polypep- served in the o/exTrxm metabolome, with fucose being sig- tides must be assembled into decameric complexes to be active nificantly decreased and maltose and trehalose being below (Unno et al., 2006), we analyzed whether the overexpression of the level of detection. Some organic acids, most of them re- Trx m in the chloroplast might affect the formation of GS2 deca- lated to the phenylpropanoid pathway, were also significantly mers (~420 kDa). BN-PAGE analyses, followed by immuno- lower in the o/exTrxm genotype, including threonic acid, cis- blotting, were performed on protein extracts from o/exTrxm 3-caffeoylquinic acid, trans-3-caffeoylquinic acid, and quinic and Wt chloroplasts. Our results showed that the oligomeric acid. Altogether, the metabolomic results corroborate that GS2 enzyme migrated with a size compatible with a decameric 4954 | Ancín et al. Fig. 2. Starch and sugar concentrations in Wt and o/exTrxm tobacco leaves. (A) Starch, (B) sucrose, (C) glucose, and (D) fructose accumulation in young fully expanded leaves of phytotron-grown plants harvested after 4 h light, 16 h light, and 8 h dark periods. Results are the mean ±SE of six individual plants. Statistical significance compared with Wt plants is indicated by asterisks (*P<0.05, Student’s t-test). Fig. 3. Log2 fold change (FC) of soluble protein and amino acid contents in o/exTrxm plants compared with the Wt. Leaf samples were taken after 4 h illumination. Significant differences between Wt and o/exTrxm are indicated by asterisks (*P<0.05, Student’s t-test). structure in both Wt and o/exTrxm plants (Fig. 5A). Similarly, genotype was increased by DTT treatment and decreased when GS2 decamers were not affected by DTT treatment, while sig- protein extracts were incubated with the oxidant CuCl2 (Fig. nificant structural changes (aggregation) were observed when 5B). In addition, GS activity was restored when protein extracts chloroplast proteins were oxidized with CuCl2 (Fig. 5A). As in- were incubated with 5 mM DTT after an oxidation treatment dicated before (Fig. 4), GS activity was higher in o/exTrxm than (100 µM CuCl2 for 30 min) (Fig. 5B). in Wt plants, but we also noticed that the GS activity in the Wt Impact of thioredoxin m on N metabolism in chloroplasts | 4955 Table 1. Changes in leaf metabolite levels after 4 h illumination in and N metabolisms. Gas exchange analyses showed that net o/exTrxm plants relative to Wt photosynthesis under ambient conditions (AN, determined at 21% O2) decreased significantly (by ~30%) in o/exTrxm plants Wt Trx m relative to the Wt, whereas no significant differences in CO2 Sugars fixation rates were observed between the genotypes when ana- 1,6-anhydro-beta-d-glucose 1±0.15 0.96±0.07 lyzed at ~2% O2 (AN2%; Fig. 6A). Moreover, the reduction of net dl-rhamnose 1±0.13 0.90±0.06 photosynthesis in the o/exTrxm genotype compared with the dl-fucose 1±0.18 0.53±0.04 Wt (by ~60%) was greatly intensified at the higher O2 concen- Gentiobiose 1±0.24 0.48±0.08 d-xylose 1±0.30 0.32±0.05 tration (AN40%; Fig. 6A), indicating a higher flux through photo- d-maltose 1±0.03 n.d. respiration. Therefore, the reduction of net photosynthetic rate α-α′-d-trehalose 1±0.31 n.d. by photorespiration in the o/exTrxm genotype (36±2%) was Organic acids twice as high as that observed in the Wt (19±2%; Fig. 6B). trans-Caffeic acid 1±0.25 1.30±0.09 These results indicate that photorespiration most likely contrib- Nicotinic acid 1±0.09 1.18±0.20 utes to the decrease in net photosynthesis observed in the o/ Citric acid 1±0.21 1.13±0.40 exTrxm genotype. However, no significant differences in dark Fumaric acid 1±0.18 1.12±0.18 respiration (Rd) were detected between the genotypes (Fig. n-Nonanoic acid 1±0.22 0.93±0.14 6C). Because photorespiration is intimately associated with N 4-Aminobutyric acid 1±0.11 0.87±0.16 assimilation, the concentrations of nitrate and ammonium were Pyruvic acid 1±0.15 0.86±0.25 also analyzed in these plants.The concentrations of ammonium Succinic acid 1±0.16 0.86±0.07 dl-malic acid 1±0.18 0.85±0.17 and, more importantly, nitrate were significantly higher in the Phosphoric acid 1±0.32 0.60±0.19 o/exTrxm genotype than in the Wt (Fig. 6D).These results col- 2-Oxoglutaric acid 1±0.21 0.41±0.13 lectively indicate that the overexpression of Trx m in chloro- Threonic acid 1±0.15 0.39±0.09 plasts also alters the so-called photorespiratory N cycle. cis-3-Caffeoylquinic acid 1±0.18 0.35±0.06 trans-3-Caffeoylquinic acid 1±0.19 0.25±0.06 Influence of Trx m overexpression in plant development dl-glyceric acid 1±0.41 0.15±0.03 under N-limiting conditions d-(–)-quinic acid 1±0.37 0.08±0.02 Others Given that overexpression of Trx m in chloroplasts affected Putrescine 1±0.13 1.60±0.24 the N status of tobacco plants, we next analyzed the influence Myo-inositol 1±0.23 0.55±0.11 of nitrate supply on plant growth. Wt and o/exTrxm plants Tyramine 1±0.31 0.42±0.06 were grown in perlite and irrigated with nutrient solution Metabolite profiling was performed using GC-TOF-MS analysis. Results containing low concentrations of N (1 or 2 mM KNO3). are means ±SE (n=4). Values that are significantly different from the Wt according to Student’s t-test are indicated in bold (P< 0.05). n.d., Not In all assayed conditions, the o/exTrxm genotype exhibited detected. slower growth and lower chlorophyll content compared with the Wt (Fig. 7A–D), as previously reported (Rey et al., 2013; Sanz-Barrio et al., 2013). However, we observed a signifi- The amount of GS2 protein was clearly increased in o/exTrxm cant decrease in the biomass of Wt plants grown at 1 mM plants compared with the Wt (by 40%; Fig. 5C).As shown before nitrate compared with 2 mM nitrate, which did not occur (Supplementary Fig. S2), GS2 transcripts were unaltered in the in the o/exTrxm plants (Fig. 7A–C). The same phenomenon o/exTrxm genotype, suggesting that the overexpression of Trx was observed when chlorophyll content was measured (Fig. m might have an influence on maintaining GS2 stability. The 7D), indicating that low N supply has a greater impact on stability of GS2 was evaluated in the presence of the oxidizing plant performance in Wt than in o/exTrxm plants. It should agent FeCl3 (Fig. 5D). In the Wt genotype, the intensity of the be noted that, under N-limiting conditions, o/exTrxm leaves immunoreactive GS2 band decreased by 45% and 62% after 8 h also accumulated more nitrate and ammonium than Wt leaves and 24 h of incubation, respectively, relative to 0 h, whereas in (Fig. 7E, F). In fact, Wt leaf nitrate concentrations were much the o/exTrxm genotype, a decrease of ~20% was visible only lower than in plants irrigated with full nutrient solution (Fig. after 24 h. These results indicate that the overexpression of Trx 6D, Fig. 7E), whereas they were still high in the o/exTrxm m led to increased activity and stability of GS2. genotype grown under these conditions. Conversely, leaf ammonium concentrations appeared to be less sensitive to Integrating C and N metabolisms with photorespiration N deficiency because only Wt plants grown at 1 mM ni- and respiration trate showed a significant decrease in ammonium concen- tration (Fig. 6D, Fig. 7F). Together, these results indicate that We further analyzed the photorespiration in the o/exTrxm the o/exTrxm phenotype was relatively unaffected by the genotype, given its influence over the interaction between C N-limiting conditions. 4956 | Ancín et al. Fig. 4. C and N metabolism enzyme activities in o/exTrxm tobacco leaves. Samples were harvested after 4 h illumination from the youngest fully expanded leaves of plants grown in a phytotron under a 16 h light photoperiod and assayed for phosphoglycerokinase (PGK), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), triose-phosphate isomerase (TPI), glutamine synthetase (GS), ferredoxin-dependent glutamate synthase (Fd-GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and phosphoenolpyruvate carboxylase (PEPC). Results are the mean ±SE of five individual plants. Statistical significance compared with Wt plants is indicated by asterisks (*P<0.05, Student’s t-test). Discussion advances are providing more evidence that modulation of the redox status of the plant might be an important signaling mech- C and N assimilation in leaves requires the partitioning of reduc- anism for the coordination of C and N assimilation (Chaput et al., tant and photosynthates to sustain the demands of amino acid 2020), with chloroplast Trxs serving as crucial components of the and carbohydrate biosynthesis. These processes are regulated at redox network (Schürmann and Buchanan, 2008). In the current different levels according to the prevailing environmental condi- study, we characterized the C and N metabolism of tobacco plants tions. The ability of plants to coordinate C and N metabolism overexpressing Trx m from the chloroplast genome, and we found consists of both a complex sensory system and an intricate as- that Trx m could be related to the regulation of N metabolism. sociated signaling network (Nunes-Nesi et al., 2010). Recent Impact of thioredoxin m on N metabolism in chloroplasts | 4957 Fig. 5. Determination of GS2 oligomerization, activity, and stability under oxidative conditions. (A) GS2 protein complexes. Proteins from Wt and o/ exTrxm chloroplasts were extracted under non-reducing conditions, separated by BN-PAGE, and analyzed by Western blot with anti-GS2 antibody. Photosynthetic thylakoid complexes are shown on the right and the molecular weight marker on the left. (B) Redox-sensitive GS activity. Protein extracts from Wt plants were incubated with or without 5 mM DTT or 100 µM CuCl2 for 30 min on ice, and the activity was then measured. Recovery of GS activity by reduction was performed by incubating with 5 mM DTT on ice after sample oxidation treatment (100 µM CuCl2). The activity of protein extracts from o/exTrxm plants was also measured. Results are expressed as means ±SE (n=3). (C) Quantification of GS2 in the o/exTrxm genotype. Total protein extracts (2.5 µg) were separated by SDS-PAGE and analyzed by western blot. Three different plants per line are shown in the blot. Immunoblots were analyzed with Gene Tools Analyzer software (SynGene). Data are given as means ±SE with the Wt relativized to 1. Statistical significance relative to the Wt is indicated by an asterisk (*P<0.05, Student’s t-test). (D) Degradation of GS2 protein in Wt and o/exTrxm plants. Leaves were incubated at 30 °C for up to 24 h in the presence of FeCl3, and the content of GS2 protein was analyzed by immunodetection. Similar results were obtained in eight independent biological replicates, and a representative blot is shown (upper panel). Immunoblots were analyzed with Gene Tools Analyzer software. Protein levels in plants treated for 4, 8, and 24 h are shown relative to the levels in plants treated for 0 h. Data are given as means ±SE. Statistical significance relative to Wt plants is indicated by asterisks (*P<0.05, Student’s t-test). C and N imbalance is associated with Trx m being redox activated by Trxs (Skryhan et al., 2018). However, overexpression in tobacco chloroplasts this is an unlikely explanation for the low starch content re- ported in this genotype, because the major starch breakdown Tobacco plants overexpressing Trx m exhibited altered C and products (maltose and glucose; Zeeman et al., 2010) did not N partitioning, promoting the accumulation of N-related accumulate in the o/exTrxm genotype (Fig. 2, Table 1). The metabolites at the cost of C-related ones (Figs 2, 3, Table 1). lower photosynthetic rates of o/exTrxm (Fig. 6A) could, This effect seems to be specific to the m-type Trx because it furthermore, have contributed to the observed reduction in does not occur when Trx f is overexpressed at the same levels carbohydrates. To identify candidates that might explain the (Sanz-Barrio et al., 2013; Aranjuelo et al., 2015). Several en- down-regulation of photosynthesis, the activities of some CBC zymes of the starch degradation pathway have been reported as 4958 | Ancín et al. Fig. 6. Net photosynthesis, photorespiration, respiration, and N status in o/exTrxm tobacco plants. (A) Gas exchange measurements at different oxygen concentrations (2%, 21%, and 40% O2). AN, net CO2 uptake rate. (B) Percentage of reduction in the net photosynthetic rate by photorespiration. The asterisk indicates a statistically significant difference (*P<0.05, Student’s t-test). (C) Dark respiration rates in Wt and o/exTrxm tobacco plants grown under a 16 h light photoperiod. (D) Nitrate and ammonium concentrations in Wt and o/exTrxm plants harvested after 4 h illumination. Results are the mean ±SE of six individual plants. Asterisks indicate statistically significant differences (*P<0.05, Student’s t-test). enzymes were determined (Fig. 4). Curiously, these determin- form in this genotype (Fig. 1C), a direct Trx m-dependent ations revealed that the activities of PGK and NADP-GAPDH regulation of some of these chloroplastic enzymes cannot be were higher in o/exTrxm than in Wt and o/exTrxf plants (Fig. ruled out. Moreover, the general increase in amino acids and, 4, Supplementary Table S4). However, the higher activities of in particular, the higher Glu and Gln pools demonstrated in PGK and NADP-GAPDH were not in line with the lower the o/exTrxm genotype (Fig. 3, Supplementary Table S2) may photosynthetic rates. These results suggest that while Trx m be indicative of an up-regulation of the GS-GOGAT cycle via seems to redox activate CBC enzymes, as previously reported post-translational reductive activation, as shown by Kolbe et al. (Okegawa and Motohashi, 2015), the limitation in net photo- (2006) in Arabidopsis leaves treated with DTT. In fact, our results synthesis in the o/exTrxm genotype could rather be associated revealed a significant increase in GS activity in the o/exTrxm with an impairment of the photosynthetic machinery (Ancín genotype relative to the Wt (Figs 4, 5B). Hence, the increased et al., 2019) that produces the reducing power and energy ne- amino acid concentrations observed in the present study could cessary for CO2 fixation. be caused by a reductive activation of the GS-GOGAT cycle While C metabolism was impaired, our study showed that in vivo, exerted by the overexpressed Trx m. The mitochondrial overexpression of Trx m favored N metabolism. Indeed, in GDH, which operates at the interface of C and N metabolism addition to amino acids, the soluble protein concentration was and participates in the balancing of the cellular levels of three increased in the o/exTrxm genotype relative to the Wt (Fig. 3, major components (ammonium, 2-oxoglutarate, and Glu), also Supplementary Table S2). Amino acid concentrations basically exhibited higher activity in the o/exTrxm genotype (Fig. 4). depend on their relative rates of production and consumption Assuming that in leaves, GDH, which is coupled to respiration, and/or transport (Nunes-Nesi et al., 2010). Several enzymes mostly operates in the deaminating direction (Fontaine et al., involved in amino acid biosynthesis have been postulated as 2012), one hypothesis is that the flux through the futile cycle potential Trx targets in the spinach chloroplast (Balmer et al., involving GS, Fd-GOGAT, and GDH could be increased in the 2003) or in Chlamydomonas reinhardtii (Lemaire et al., 2004), o/exTrxm genotype. Such a cycle would be in line with sugar and some of these enzymes are involved in the synthesis of the depletion (energy is mostly used by the reactions catalyzed by amino acids that were found to be increased in concentration GS and Fd-GOGAT) and with the increased concentrations in the o/exTrxm genotype (Fig. 3, Supplementary Table S2). of nitrate and ammonia, as well as increased photorespiration. Therefore, given that the overexpressed Trx m is in the reduced Indeed, more N would be cycled and less N would be used for Impact of thioredoxin m on N metabolism in chloroplasts | 4959 mutations in one of these Cys residues still retained its redox activation. In agreement with these observations, the activity and stability of GS2 from Lotus japonicus, in which only one of the two conserved Cys residues is preserved, were consider- ably improved in the presence of thiol compounds (Betti et al., 2006). It should be noted that GS2 from tobacco also con- serves only one of these Cys residues (Supplementary Fig. S3), indicating that this unique conserved Cys should be sufficient for the redox activation of GS2, as occurs in L. japonicus plants. However, we have not been able to demonstrate a direct redox activation of GS2 in the o/exTrxm genotype. The analysis of the quaternary structure revealed that the o/ exTrxm genotype maintained the ability to form GS2 deca- mers (Fig. 5A), the protein complex known to be catalytic- ally active in chloroplasts (Unno et al., 2006; Kimata-Ariga and Hase, 2014). However, the complex formation, activity, and protein stability of GS2 seemed to be affected by oxi- dants (Fig. 5A, B, D). In fact, it was previously demonstrated that GS2 displays an extreme sensitivity to oxidants in plants, resulting in its aggregation and cleavage (Palatnik et al., 1999; Ishida et al., 2002; Castro-Rodríguez et al., 2015). In this line, it is possible that an indirect protection of GS2 against cel- lular oxidants exists in the o/exTrxm genotype. On the one hand, peroxiredoxins (Prxs) have been proposed as principal actors in the mechanism protecting GS from oxidative inacti- vation (Cheong et al., 1999; Rahlfs and Becker, 2001; Jeon and Ishikawa, 2003; Liao et al., 2017). In this way, Trx m may Fig. 7. Response to nitrate supply. Plants were grown under a 16 h light be activating this protection system through Prxs, given that photoperiod and watered daily with 1 mM or 2 mM KNO3. After 30 days, multiple Prxs have been reported as Trx targets in chloroplasts plants were photographed (A, B), harvested after 4 h illumination, and (Montrichard et al., 2009). In fact, we saw that 2-Cys Prx was analyzed for (C) whole plant biomass, (D) total chlorophyll, (E) nitrate concentration, and (F) ammonium concentration. Results are the mean more efficiently reduced in o/exTrxm than in Wt plants in ±SE of four individual plants. Statistical significance between treatments is both dark and light conditions (Supplementary Fig. S4), ac- indicated by asterisks (*P<0.05, Student’s t-test). companied by a reduced H2O2 content (Ancín et al., 2019). On the other hand, Trxs provide reducing power to methio- growth, even if the amino acid and protein contents increased. nine sulfoxide reductases (MSRs), enzymes that repair oxidized Altogether, our results suggest that the overexpression of Trx m proteins (Vieira Dos Santos et al., 2007), which could likewise is involved in the control of the cellular C/N balance. function to up-regulate the biological activity of GS protein (Levine et al., 2000). Interestingly, we have previously reported a 2-fold higher MSR enzymatic capacity in o/exTrxm plants Trx m overexpression leads to an increase in GS2 compared with the Wt (Rey et al., 2013). These findings sug- activity and stability gest that an antioxidant protective mechanism might be af- Our findings showed that the overexpression of Trx m in to- fecting the activity and stability of GS2 in o/exTrxm plants. bacco chloroplasts caused an increase in both the activity and content of GS2 (Fig. 4, Fig. 5B, C), while transcript levels were Overexpression of Trx m in chloroplasts favors unaltered (Supplementary Fig. S2). The observed increase in the photorespiratory N cycle rather than nitrate GS2 protein content could mainly contribute to the increase assimilation in GS activity, but a post-translational regulation of GS2 in the o/exTrxm genotype, improving its stability or activity (or We have recently demonstrated that a reduction in the electron both), cannot be ruled out. Supporting this hypothesis, Choi transport rate in the o/exTrxm genotype may be related to the et al. (1999) showed that the addition of reducing agents, es- reduced photosynthetic capacity in this genotype (Ancín et al., pecially DTT, increases the activity of recombinant GS2 from 2019). However, our current results support that changes in Canavalia lineata. These authors demonstrated that the redox photorespiration also contributed to the decreased net photo- sensitivity of GS2 is due to two conserved Cys residues found synthesis. In fact, the net photosynthesis in the o/exTrxm exclusively in the chloroplast isoform, although GS2 with genotype was Wt-like at 2% O2 but greatly decreased at higher 4960 | Ancín et al. O2 concentrations (21% and 40%; Fig. 6A). Accordingly, we the o/exTrxm genotype accumulated extremely large amounts observed a much higher percentage of AN reduction by photo- of nitrate in leaves (Fig. 6D), a phenotype that resembles that of respiration in the o/exTrxm genotype than in the Wt (Fig. nitrate reductase (NR) mutant plants (Scheible et al., 1997). In 6B). Photorespiration is known to interact with several pri- addition, we showed that the o/exTrxm genotype was less sen- mary metabolic pathways (Hodges et al., 2016), including the sitive than the Wt to nitrate deprivation (Fig. 7). This behavior GS-GOGAT cycle (Coschigano et al., 1998; Keys, 2006). The could be due to an inhibition of the NR enzyme, causing a conversion from glycine (Gly) to Ser in the photorespiratory deficiency in nitrate reduction. Because NR is located in the cycle results in the production of ammonia at a rate far ex- cytosol, the overexpression of Trx m might exert indirect con- ceeding that of primary N assimilation, by up to 10-fold (Keys trol over its activity. It is well known that a decrease in the avail- et al., 1978). Meanwhile, GS2 controls the reassimilation of the ability of reductant in the cytosol may limit NR activity (Kaiser released ammonia in chloroplasts (Nunes-Nesi et al., 2010), a et al., 2000, 2002; Hashida et al., 2018). Interestingly, a decrease process known as the photorespiratory N cycle. Interestingly, in the NADPH/NADP+ ratio and the size of the NADP pool the pool of Ser was higher in the o/exTrxm genotype than was observed in the o/exTrxm genotype (Supplementary Fig. in the Wt, while the pool of Gly remained unaltered (Fig. 3 S6), indicating a reduced flux of reducing power to the cytosol, Supplementary Table S2), indicating a higher rate of Gly to which may explain the large accumulation of nitrate that oc- Ser conversion that correlates with a significant increase in curred in o/exTrxm leaves. However, other factors occurring the ammonium concentration in o/exTrxm leaves (Fig. 6D). in the o/exTrxm genotype may also account for nitrate accu- Although three different pathways of Ser biosynthesis have mulation via a down-regulation of NR activity. Among them, been described in plants (the glycolate, phosphorylated, and sugar depletion (Kaiser and Huber, 2001) and decreases in net glycerate pathways), the glycolate pathway, which is associated photosynthesis (Quick et al., 1991) have been reported to re- with photorespiration, is considered to be the most important sult in a massive reduction in NR protein levels. Our results source of Ser, at least in photosynthetic tissues (Ros et al., 2014; collectively suggest that the overexpression of Trx m in chloro- Igamberdiev and Kleczkowski, 2018). Therefore, the higher plasts results in the increased reassimilation of photorespired amount of Ser observed in o/exTrxm plants was very likely ammonium while primary nitrate assimilation decreases. coming from an alteration of the glycolate pathway. Given the low glycerate levels observed in o/exTrxm plants (Table 1), the Putative role of Trx m in phenylpropanoid metabolism glycerate pathway could hardly contribute to increase the Ser pool. The phosphorylated pathway is more relevant at night Central metabolism is an important source of precursors for and under conditions in which the photorespiratory pathway the synthesis of secondary metabolites. There are several lines does not function (Benstein et al., 2013; Toujani et al., 2013). of evidence that the range and levels of secondary metabolites However, a putative activation of this pathway in o/exTrxm are regulated in response to the C and N status in tobacco plants cannot be ruled out given that the enzyme that cata- (Stitt et al., 2002; Fritz et al., 2006). Phenylpropanoids represent lyzes the first reaction of this route, 3-PGA dehydrogenase, was a diverse group of secondary metabolites, with roles in plant previously identified as a Trx target by proteomic approaches structure, defense, and signaling (Dixon and Paiva, 1995). They (Balmer et al., 2003; Alkhalfioui et al., 2007). Therefore, the are synthesized via the shikimate pathway in the plastid, with enhanced photorespiratory capacity in the transplastomic aromatic amino acids as precursors. In the o/exTrxm geno- genotype may be linked to the activation of the GS-GOGAT type there was a dramatic decrease in the concentrations of cycle. The reassimilation of ammonia into chloroplasts is ac- two shikimate pathway-related compounds, namely quinic and curately regulated by the transport of dicarboxylates across the caffeoylquinic acids (Table 1), the latter being one of the pre- chloroplast envelope via the 2-oxoglutarate/malate transporter dominant soluble phenylpropanoids in the Solanaceae (Vogt, (OMT or DiT1) and the general dicarboxylate transporter 2010). This result is in accordance with Fritz et al. (2006), who (DCT or DiT2) (Taniguchi et al., 2002; Renné et al., 2003). demonstrated a stimulation of phenylpropanoid metabolism DiT1 and DiT2 constitute a double translocator system that in response to low nitrate. Hence, the higher accumulation functions to coordinate the photorespiration and GS-GOGAT of nitrate (Fig. 6D) in the o/exTrxm genotype could account pathways (Weber and Flügge, 2002; Kinoshita et al., 2011). for the decrease in quinic and caffeoylquinic acids. Moreover, We demonstrated by qPCR that the o/exTrxm genotype phenylpropanoids are synthesized from Phe, and we also found showed an increase in the expression of DiT2 relative to the a specific decrease in the concentration of this amino acid Wt (Supplementary Fig. S5), thus reinforcing our proposal of in the o/exTrxm genotype (Fig. 3, Supplementary Table S2). an up-regulated photorespiratory N cycle in these plants. Alternatively, the decreased phenylpropanoid content in the o/ According to Nunes-Nesi et al. (2010), the occurrence of exTrxm genotype could be related to a decrease in the activity photorespiratory N recycling under photorespiratory con- of the phosphoenolpyruvate (PEP) translocator (Henkes et al., ditions may result in a lower net primary rate of inorganic 2001). Interestingly, an increase in the activity of PEPC, which N assimilation in the leaf, pointing to a stimulated use of the catalyzes the conversion of PEP and HCO3– to oxaloacetate photorespired ammonium. In agreement with this hypothesis, (OAA) and inorganic phosphate, occurred in these plants (Fig. Impact of thioredoxin m on N metabolism in chloroplasts | 4961 4). OAA is the precursor of most of the amino acids that are Skłodowska-Curie grant agreement no. 753301, the ‘Ramon y Cajal’ increased in the o/exTrxm genotype. Hence, our results sug- contract RYC2019-027244-I/AEI/10.13039/501100011033, and the gest that in the o/exTrxm genotype PEP preferentially serves European Social Fund. as PEPC substrate instead of being exported through the PEP translocator to the chloroplast, thus negatively affecting the phenylpropanoid content. In agreement with this, Arabidopsis Author contributions PEPC mutants showed a large reduction in the flux from PEP MA and IF designed the research and wrote the article; MA, LL, IFS, CB, to OAA and, thereby, suppression of the GS-GOGAT cycle and AFS, and IA performed the experiments; MA, IFS, JV, YG, ARF, IA, and subsequent ammonium assimilation (Shi et al., 2015), a pheno- IF analyzed the data; all authors read the article, provided comments, and type completely opposite to that of the o/exTrxm genotype. approved the final version. Here, we show that when Trx m is overexpressed in tobacco chloroplasts the observed decreases in the rate of photo- synthesis and carbohydrate concentrations is not accom- Data availability panied by an inhibition of N metabolites; on the contrary, All data supporting the findings of this study are available within the there is an increase in amino acids (with the exception of paper and within its supplementary data published online. Phe) and protein content. These results differ from those obtained in plants with a decreased rate of photosynthesis, where the levels of carbohydrates decrease along with those References of the N-derived compounds (Henkes et al., 2001; Matt Alkhalfioui F, Renard M, Vensel WH, Wong J, Tanaka CK, Hurkman WJ, et al., 2002). Our results, combined with those of published Buchanan BB, Montrichard F. 2007. Thioredoxin-linked proteins are re- studies, point towards a specific influence of Trx m inside duced during germination of Medicago truncatula seeds. Plant Physiology the chloroplast, regulating the so-called photorespiratory N 144, 1559–1579. cycle and the activity and stability of GS2, thereby unbalan- Ancín M, Fernández-San Millán A, Larraya L, Morales F, Veramendi J, Aranjuelo I, Farran I. 2019. Overexpression of thioredoxin m in tobacco cing C and N partitioning in plants. chloroplasts inhibits the protein kinase STN7 and alters photosynthetic per- formance. Journal of Experimental Botany 70, 1005–1016. Aranjuelo I, Tcherkez G, Jauregui I, Gilard F, Ancín M, Fernández- San Millán A, Larraya L, Veramendi J, Farran I. 2015. Alteration by Supplementary data thioredoxin f over-expression of primary carbon metabolism and its re- sponse to elevated CO2 in tobacco (Nicotiana tabacum L.). Environmental The following supplementary data are available at JXB online. and Experimental Botany 118, 40–48. Fig. S1. Enzyme activities in o/exTrxm tobacco plants ex- Balmer Y, Koller A, del Val G, Manieri W, Schürmann P, Buchanan BB. pressed on a protein basis. 2003. Proteomics gives insight into the regulatory function of chloroplast Fig. S2. RT–qPCR analysis of gln2, pgk, and gapB expression. thioredoxins. Proceedings of the National Academy of Sciences 100, 370–375. Fig. S3. Alignment of amino acid sequences of GS2. Bénard C, Gibon Y. 2016. Measurement of enzyme activities and opti- Fig. S4. Redox status of 2-Cys Prx in o/exTrxm plants. mization of continuous and discontinuous assays. Current Protocols in Fig. S5. RT–qPCR analysis of DiT1 and DiT2 expression. Plant Biology 1, 247–262. Fig. S6. Effect of Trx m overexpression on the pyridine nu- Benstein RM, Ludewig K, Wulfert S, Wittek S, Gigolashvili T, cleotide content. Frerigmann H, Gierth M, Flügge UI, Krueger S. 2013. Arabidopsis phosphoglycerate dehydrogenase1 of the phosphoserine pathway is es- Table S1. Parameters used for peak annotation. sential for development and required for ammonium assimilation and trypto- Table S2. Soluble protein and amino acid contents in Wt and phan biosynthesis. The Plant Cell 25, 5011–5029. o/exTrxm plants. Betti M, Arcondéguy T, Márquez AJ. 2006. Molecular analysis of two Table S3. Changes in the levels of the 44 annotated mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes. Planta 224, 1068–1079. metabolites. 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