Linking Chloroplast Antioxidant Defense to Carbohydrate Availability: The Transcript Abundance of Stromal Ascorbate Peroxidase Is Sugar-Controlled via Ascorbate Biosynthesis

Molecular Plant  •  Volume 7  •  Number 1  •  Pages 58–70  •  January 2014

RESEARCH ARTICLE

Linking Chloroplast Antioxidant Defense to Carbohydrate Availability: The Transcript Abundance of Stromal Ascorbate Peroxidase Is Sugar-Controlled via Ascorbate Biosynthesis a Former address: Plant Physiology and Biochemistry, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany b Plant Physiology, Free University of Berlin, Dahlem Center of Plant Sciences, Königin-Luise-Str. 12–16, 14195 Berlin, Germany

ABSTRACT  All genes encoding chloroplast antioxidant enzymes are nuclear-encoded and posttranscriptionally targeted to chloroplasts. The transcript levels of most of them decreased upon sucrose feeding like the transcript levels of many genes encoding components of the photosynthetic electron transport chain. However, the transcript abundance of stromal ascorbate peroxidase (s-APX; At4g08390) increased. Due to mild sugar application conditions, the plants kept the phosphorylation status of the ADP+ATP pool and the redox states of the NADPH+NADP+ and the ascorbate pools under control, which excludes them as signals in s-APX regulation. Correlation with ascorbate pool size regulation and comparison of transcript abundance regulation in the starch-biosynthetic mutant adg1, the ascorbate biosynthesis mutant vtc1, and the abscisic acid (ABA) biosynthetic mutant aba2 showed a link between sugar induction of s-APX and ascorbate biosynthesis. Key words:  ascorbate; antioxidant; carbohydrate; chloroplast; gene expression; stromal ascorbate peroxidase.

Introduction Sugar availability regulates the expression of many genes for chloroplast proteins (Rook and Bevan, 2003; Rolland et al., 2006). While low sugar concentrations support seedling growth, excess carbohydrates antagonize greening and seedling development (Koch, 1999). Carbohydrate surplus inhibits the Calvin-Cycle (Macdonald and Buchanan, 1992) and promotes generation of reactive oxygen species (ROS) via feedback inhibition of photosynthetic electron transport (Rook et al., 2006a). Antioxidant enzymes and low-molecular-weight antioxidants, such as ascorbate (Asc) and glutathione (GSH), form a ROS-protective network (Asada, 1999; Dat et  al., 2001; Dietz et al., 2002; Chang et al., 2009). Stromal and thylakoidbound ascorbate peroxidase (s-APX (At4g08390) and t-APX (At1g77490)) detoxify H2O2 on the expense of ascorbate (Asada, 1999). The co-substrate is regenerated by monodehydroascorbate and dehydroascorbate reductases (MDHAR and DHAR). In parallel, glutathione peroxidases (GPX) and peroxiredoxins (PRX) reduce peroxides via ascorbate-independent thiol-mediated pathways (Dietz et al., 2002). These enzymes are nuclear-encoded and posttranslationally targeted to the organelles by N-terminal transit peptides (Pitsch et al., 2010). Most proteins, like the four peroxiredoxins and t-APX, for

example, are exclusively targeted to chloroplasts. s-APX and MDHAR (At1g63940) can be alternatively targeted to chloroplasts and (the intermembrane space of) mitochondria (Chew et al., 2003), yet they display strong preference towards chloroplasts. To acclimate the chloroplast antioxidant protection upon (photo-)oxidative stress, the genes for chloroplast antioxidant enzymes respond to organellar signals. Organelle-to-nucleus signaling has been best studied for photosynthesis associated genes, like cab (encoding chlorophyll-a/b-binding proteins/light-harvesting complex proteins) and rbc-S (encoding the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). They are suppressed by carbohydrates (Arenas-Huertero et  al., 2000), while apl3 (encoding a large subunit of ADP-glucose pyrophosphorylase; At4g39210) is induced by sugars (Rook et al., 2006b).

1 To whom correspondence should be addressed. E-mail margarete.baier@ fu-berlin.de, fax +49-30-838-51688, tel. +49-(0)30-838-53183

© The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst154, Advance Access publication 7 November 2013 Received 25 June 2013; accepted 27 October 2013

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Isabelle Heibera, Wenguo Caib, and Margarete Baiera,1

Heiber et al.  •  s-APX Transcript Abundance Is Regulated by Ascorbate   

RESULTS Optimization of the Experimental Set-Up In nature, the mucilage of the outer seed coat provides the first carbohydrate source upon germination. In experiments, most of the mucilage is washed off during seed sterilization. Here, Arabidopsis was screened for the optimal background sucrose (Suc) concentration prior to gene-expression analyses. In continuous moderate light (50  μmol quanta m–2  s–1), the seedling biomass increased on Suc-supplemented MS-media up to 0.8% (w/v) Suc (data not shown). With Suc concentrations higher than 1.6% (w/v), anthocyanins accumulated in hypocotyls and cotyledons indicating excess carbohydrates (Solfanelli et  al., 2006). Suc concentrations higher than 2.5% (w/v) resulted in growth inhibition. Based on these

observations, 1% (w/v) Suc was chosen as optimal sugar concentration and 0.5% (w/v) Suc was used to establish moderate sugar starvation. Sorbitol (Sor) was used as an osmotic control. Compared to Suc, it is taken up and metabolized much less efficiently (Shabala and Lew, 2002; Gibson, 2005). Chemically, the osmolarities of Suc and Sor are similar. In planta, hydrolysis of Suc into glucose and fructose increases the osmolarity and the heterotrophic metabolism decreases it. The Sor concentration best suited as an osmotic control for Suc treatments was determined empirically based on biomass comparison. At 10 d, the fresh weights of seedlings grown on 1% (w/v) Suc supplemented with 0.8–1.3% (w/v) Sor were in the range of seedlings grown on 2% (w/v) Suc (data not shown). Finally, 1% Sor was chosen as control for 2% Suc application.

Long-Term Carbohydrate Effect in Arabidopsis Seedlings Long-Term Carbohydrate Effect on the Transcript Abundance of Nuclear-Encoded Chloroplast Proteins Long-term effects of Suc and Sor on the expression of genes encoding chloroplast antioxidant enzymes were studied in ACTIN-2-standardized cDNA samples by RT–PCR (Figure 1). As secondary control, the transcript level of ubiquitin-11 (UBQ11; At4g05050) was quantified. Relative to ACTIN-2 transcript levels, the UBQ11 transcript levels were not significantly changed by any of the treatments (Figure 1). APL3 (encoding a large subunit of ADP-glucose-pyrophosphorylase) served as a control for induction by Suc (Rook et  al., 2001). RBC-S (encoding the small subunit of Ribulose-1,6-bisphosphatcarboylase/oxygenase; At5g38430) and STP1 (encoding a high-affinity monosaccharide/proton symporter; At1g11260) were included to monitor suppression by surplus amounts of carbohydrates (Figure 1; Koch, 1999; Sherson et al., 2000). This study focuses on regulation of chloroplast antioxidant enzymes. On 2% (w/v) Suc, the transcript levels of the four chloroplast peroxiredoxins, 2CPA (At3g11630), 2CPB (At5g06290), Prx-Q (At3g26060), and Prx-IIE (At3g52960), of CuZn-superoxide dismutase 2 (CSD2; At2g28190), MDHAR (At1g63940), t-APX (At1g77490), the Rieske protein PET-C (At4g03280), the photosystem-II antenna protein LHCB2.2 (At2g05070), and of the cytochrome b6f proteins PET-M (At2g26500) were decreased compared to 1% (w/v) Suc and the osmotic control containing 1% (w/v) Sor demonstrating a broad inactivation of genes encoding chloroplast proteins. On 3% (w/v) Suc, the transcript levels were below those observed on 1% (w/v) and 2% (w/v) Suc. On 1% (w/v) Suc supplemented with 2% (w/v) Sor, they were barely detectable (Figure 1). PET-E1 (At1g76100) and PET-E2 (At1g20340) levels, encoding plastocyanins, were unchanged irrespective of the treatment. The s-APX mRNA level was increased on 2% (w/v) Suc, but not on 1% (w/v) Suc supplemented with 1% (w/v) Sor demonstrating Suc-dependent induction.

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Screens for sugar signaling mutants demonstrated a strong, but diverse, crosstalk of ABA, sugar, and ROS/redox signaling (Rook et  al., 2001; Gonzalez-Guzman et  al., 2002; Staneloni et al., 2008). So far, little is known about the transcriptional regulation of genes encoding chloroplast antioxidant enzymes. Array studies and RT–PCR demonstrate that transcript abundances often increase only by mild stress, but are decreased upon severe stress due to general inactivation of chloroplast function (summarized in Baier et al. (2010)). Regulation of the 2-Cys peroxiredoxin-A gene (At3g11630) has been investigated in detail (Baier et al., 2004b): cis-acting motifs located within 200 bp upstream of the transcription initiation site mediate developmental responses. Photosynthetic redox signals and ABA-signals integrate antagonistically at a more distally located promoter region, designated as redoxbox. There, the APETALA-2-type transcription factor RAP2.4a induces 2cpa transcription upon moderate oxidative stress (Shaikhali et al., 2008). Upon severe stress, RAP2.4a oligomerizes. As a consequence, 2cpa transcription activity decreases (Shaikhali et al., 2008). Transcriptional regulation of other genes for chloroplast antioxidant enzymes has not been studied so far. Mutants impaired in redox regulation of the 2cpa promoter (Heiber et  al., 2007) and knockout lines of the 2cpa redox-regulating transcription factor RAP2.4a (Shaikhali et al., 2008) show links between s-APX, t-APX, and 2CPA in redox regulation. To investigate the impact of the photosynthetically controlled parameters, carbohydrate availability, light, and ABA on the expression of nuclear genes for chloroplast antioxidant enzymes, here the transcript abundances were analyzed in Arabidopsis following light and sugar treatments. The responses were compared to those in the ascorbate-biosynthetic mutant vtc1 (Conklin et  al., 1997), the starch-biosynthetic mutant adg1 (Lin et al., 1988), and the ABA-biosynthetic mutant aba2 (Léon-Kloostersiel et al., 1996) after short- and long-term Suc treatment. In this comparison, a specific regulation is shown for s-APX. It is concluded that s-APX regulation is indirectly sugar-responsive via ascorbate biosynthesis.

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Chlorophyll Levels after Long-Term Treatment After 10-day growth on MS medium supplemented either with 1–3% (w/v) Suc or 1% (w/v) Suc + 1% or 2% (w/v) Sor, the chlorophyll (chl) contents were very similar in all samples (Figure 2) reflecting acclimation and the mildness of the treatments.

Sugar Metabolite Levels and Energetization and Carbohydrate Redox Status after Long-Term Sugar Treatment

Ascorbate Levels and the Ascorbate Redox State after Long-Term Sugar Treatment Total ascorbate and reduced ascorbate contents were measured to determine the ascorbate pool size and to calculate the ascorbate redox state. The ascorbate level was 3.7-fold increased on 2% (w/v) Suc, 3.9-fold on 3% (w/v) Suc, and 1.6-fold on 1% (w/v) Suc + 1% (w/v) Sor and 1% (w/v) Suc + 2% (w/v) Sor compared to plant material grown on 1% (w/v) Suc (Figure  2), demonstrating a carbohydrate flux into the ascorbate pool. The ascorbate redox state was not affected (Figure 2).

Anthocyanin Levels after Long-Term Treatment In response to excess carbohydrate availability, Arabidopsis accumulated anthocyanins (Solfanelli et  al., 2006). The anthocyanin contents were 2.73-, 4.35-, 1.32-, and 1.83-fold increased after 10-day growth on 2% and 3% (w/v) Suc and 1% (w/v) Suc supplemented with 1% or 2% (w/v) Sor, respectively (Figure 2).

Effect of Short-Term Sucrose Application on Arabidopsis thaliana To analyze short-term responses, the seedlings grown on 0.5% (w/v) Suc were either floated on liquid MS medium containing 0.5 or 1.5% (w/v) Suc for 24 h. For a set of 22 genes, the transcript

Effect of Ascorbate, Starch, and ABA-Biosynthesis on the Suc Effect The use of mutants enables short- and long-term studies with modified strains. The mutant vtc1 carries a mutation in GDPD-mannose pyrophosphorylase which catalyzes a final step in the main ascorbate biosynthesis pathway (Conklin et al., 1999). Adg1 has a point mutation in the small subunit of ADP-glucose pyrophosphorylase and is limited in chloroplast starch biosynthesis, while aba2 is restricted in ABA-biosynthesis (Rook et al., 2001; Cheng et al., 2002). Here, the mutant in short- and longterm acclimation response was compared to that of wildtype (wt) plants.

Long-Term Effect of Suc on Transcript Abundance in the Mutants vtc1, adg1, and aba2 After 10-day growth on 2% (w/v) Suc, the transcript levels of most of the analyzed genes were decreased in vtc1, adg1, and aba2 (Figure  4, top). Exceptions were s-apx and apl3, whose transcript levels were increased. The PET-E1, PET-E2, and PET-C transcript levels were barely altered. Consistently with the hypothesis that ABA signaling supports the sugar effect on the apl3 promoter (Rook et al., 2001), the transcript levels of APL3 were most induced in aba2. s-APX regulation resembled APL3 induction. s-APX and APL3 transcripts were similarly accumulated in adg1 as they were in wt, indicating that starch biosynthesis capacities have a minor effect on the induction. Using the vtc1 mutant distinguished s-APX and APL3 regulation: s-APX transcripts accumulated less in vtc1 than in wt. In contrast, the APL3 transcript levels increased in a wt-like manner. This demonstrates a specific sucrose-linked impact of ascorbate biosynthesis on s-APX regulation. APL3 transcript levels were (consistently with previous reports by Rook et  al. (2001)) induced in wt in response to sorbitol and decreased when ABA signaling was affected. Similarly, the transcript levels of most other tested genes were slightly increased in adg1, decreased in aba2, and was hardly affected in vtc1. In contrast, s-APX levels were decreased in all four tested genotypes in response to sorbitol application (Figure 4, bottom). The effect was strongest in aba2 and vtc1,

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To study the effect of sugar feeding on the metabolic signature, DHAP (dehydroxyacetone phosphate), FruBP (fructose6-bisphosphate), 3PGA (3-phosphoglycerate), ATP (adenosine triphosphate), ADP (adenosine diphosphate), ascorbate, and anthocyanin levels were analyzed as indicators for the cellular carbohydrate, redox, and energy status. Most metabolite levels were only slightly changed between the treatments, demonstrating the mildness of the treatments (Figure 2). The ATP and ADP contents increased in response to Suc application. However, the ATP/ADP ratio, like the 3PGA/DHAP ratio, which reflects the carbohydrate redox status, and the redox status of the NADPH pool (NADPH+NADP+), and, consequently, the [ ATP ] [NADPH ] assimilatory force, (Dietz and Heber, 1989), [ ADP ][Pi ] NADP + ] were hardly affected, demonstrating that the metabolic control capacity was not exhausted in the seedlings (Figure 2).

abundance regulation factors were calculated from the transcript levels quantified for ACTIN-2 standardized cDNA samples. The transcript levels of all genes encoding chloroplast antioxidant enzymes, except again s-APX, and, of all tested genes, encoding proteins of the thylakoid membrane (LHCA5– PET-E2) and of RBC-S were decreased in response to Suc application (Figure 3). The transcripts of the sugar-inducible apl3 gene accumulated and that of the sugar-suppressible stp1 were less abundant. The transcript levels of the ROS-marker genes bap1 (BONZAI associated protein I; At3g61190) and fer1 (Ferritin-1 precursor; At5g01600) (op den Camp et  al., 2003) were hardly affected, demonstrating the mildness of the conditions.

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demonstrating the impact of ABA and ascorbate biosynthesis on the osmotic regulation of s-APX.

Short-Term Effect of Sucrose on Transcript Abundance in Arabidopsis in the Mutants vtc1, adg1, and aba2 For comparison of short-term responses, vtc1, adg1, and aba2 seedlings were grown for 9 d on 0.5% (w/v) Suc and

afterwards floated for 24 h either on 1.5% (w/v) Suc in MS medium or on 0.5% (w/v) Suc in MS medium. The transcript abundances relative to wt (Figure  3) were calculated from RT–PCR data of three independent plant sets. For most genes encoding chloroplast proteins, the transcript levels were decreased the most in vtc1, demonstrating a wide positive impact of ascorbate biosynthesis in regulation of chloroplast function via nuclear gene expression (Figure  5). The s-APX,

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Figure 1.  Transcript Level Regulation in Response to Sucrose and Sorbitol. Transcript abundance levels of genes encoding chloroplast peroxiredoxins (2CPA, 2CPB, Prx-Q, Prx-IIE), chloroplast CuZn-superoxide dismutase 2 (CSD2), monodehydroascorbate reductase (MDHAR), and stromal and thylakoid-bound ascorbate peroxidase (s-APX and t-APX) in comparison to genes encoding proteins of the photosynthetic membrane (PET-E1, PET-E2, PET-M, PET-C, and LHCB2.2), carbohydrate-induced APL3 and RBC-S, and carbohydrate-repressed STP1 and constitutively expressed ACTIN-2 and UBQ11 in Arabidopsis seedlings grown for 10 d on MS plates supplemented with 1%, 2%, or 3% sucrose or 1% sucrose plus 1% or 2% sorbitol. RT–PCR analysis was performed in three biological replicates with at least two technical replicates. For better comparison, here, band intensities are shown for a characteristic data set after separation of the PCR products on ethidium bromide gels and the means ± standard deviation after qPCR are presented.

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Figure 2.  Metabolic Indicators in 10-Day-Old Seedling Grown for 10 d on MS plates Supplemented with 1%, 2%, or 3% Suc or 1% Suc plus 1% or 2% Sor. The ascorbate content, ascorbate redox state, the concentration of dihydroxyacetone phosphate (DHAP) fructose-bisphosphate (FruBP) and 3-phophoglycerate (3PGA), the 3PGA/DHAP ratio, the ATP and ADP content, the ATP/ADP ratio, the redox state of the NADP+NADPH pool, the assimilarity force, the chlorophyll content, and the anthocyanin content were analyzed in 6–10 biological replicates.

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MDHAR, and APL3 transcript levels were higher in vtc1 than in wt, indicating that ascorbate synthesis impacts one gene regulation. While the MDHAR transcript level was decreased less in vtc1 than in wt in response to higher sugar levels, the APL3 and s-APX levels were increased more (Figures 3 and 5). The relative effect was stronger for APL3 than for s-APX. Similarly to long-term sucrose feeding, limitations in starch biosynthesis (adg1) increased the APL3 levels more strongly and the s-APX levels less. The aba2-mutation had a promoting effect on s-APX regulation and a highly variable impact on APL3 transcript abundance. In contrast, for the other genes, the mutations did not affect gene regulation significantly or had a preferentially negative effect on transcript levels.

Ascorbate Levels in Arabidopsis wt and in the Mutants vtc1, adg1, and aba2 after Long- and Short-Term Treatment with Suc In all mutants, the ascorbate level was increased after 10-day growth on 2% (w/v) Suc, but not on 1% (w/v) Suc + 1% (w/v) Sor (Figure  6, top). The effect was similar in wt, adg1, and aba2, but weaker in vtc1. The redox state of the ascorbate pool was barely affected (Figure 6, top). After 9 d growth on 0.5% (w/v) Suc, the ascorbate levels were more consistent than after 10 d growth on 1% (w/v) Suc in wt, aba2, and adg1. Twenty-four hours after application of additional 1% (w/v) Suc, the ascorbate levels were slightly increased in wt, aba2, and adg1, but not in the ascorbate-biosynthetic

mutant vtc1 (Figure  6, bottom). The redox state of ascorbate was slightly lower on 0.5% (w/v) Suc than on 1% Suc (Figure 6, bottom). Application of additional 1% (w/v) Suc increased the redox state slightly in wt and significantly in aba2 and adg1, but not in vtc1 (Figure  6, bottom), demonstrating that overcoming sugar starvation promoted ascorbate redox control and indicating more subtle, but specific, signaling effects.

Light Regulation of Transcript Abundance and Ascorbate Availability To finally test the effect of photosynthetic activity in the context of carbohydrate regulation, plants were grown for 10 d on Suc or Sor media and were placed for 4 h under 50 μmol quanta m–2 s–1 and 600 μmol quanta m–2 s–1 or left under the growth conditions of 100 μmol quanta m–2 s–1. For transcripts, except PET-E1, PET-E2, and s-APX, an inhibitory effect of 2% Suc was observed compared to 1% Suc and 1% Suc + 1% Sor at all light intensities. While the transcript levels of the two pet-E genes did not respond, s-APX levels were increased in response to elevated Suc under all light regimes. For 2CPA, 2CPB, Prx-Q, and RBC-S, the gradual response was strongest at lower light intensities, and for t-APX, MDHAR, PRX-C, and STP1 at highest light intensity.

GeneMANIA and PlaNet Analysis of Transcript Abundance Co-Regulation Transcript abundance co-regulation analysis can provide insight into the gene regulation context. GeneMANIA (Zuberi et  al., 2013) and PlaNet (Mutwil et  al., 2011) use publicly

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Figure 3.  Relative Transcript Amount Regulation in Response to Elevated Sucrose Levels. The relative transcript amount of genes encoding chloroplast antioxidant enzymes (2CPA–MDHAR), extra-plastidic antioxidant enzymes (CAT– APX2), proteins of the photosynthetic membrane (LHCA5–PET–E2), marker genes for the carbohydrate status (APL3–GDH2), and reactive oxygen levels (BAP1 and FER1) in 10-day-old seedlings after a 24-hour increase in the sucrose availability to 1% (w/v) to 2% (w/v) (n = 3). The asterisk shows statistical significance (α < 0.05; t-test).

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available Arabidopsis array data to look for transcripts coregulated with the transcripts of interest. For t-APX transcript abundance regulation similarity with the regulation of various genes encoding chloroplast thylakoid proteins, such as PSB-P (At1g76450) and PSA-E (At2g20260), proteins involved in chlorophyll biosynthetic (CHLI2; magnesium chelatase subunit I) and electron transfer (the chloroplast NAD(P)oxidoreductase At1g04420, ferredoxin C1 (At4g14890) and a rubredoxin was shown by GeneMANIA (Supplemental Figure  1B). The more selective PlaNet filters show links to At3g63140 (encoding a chloroplast riboprotein), At3g24930 (encoding a thylakoid-lumen protein), and At4g39970 (encoding a chloroplast hydrolase), and indirectly also to various photosynthesis-related genes (Supplemental Figure 1D). In contrast, s-APX regulation is most similar to the transcript abundance regulation of cytochrome-C1 and C2, the

heat shock factor 4, and ferritin-1 in the GeneMANIA analysis (Supplemental Figure  1A). Especially the co-regulation with the cytochrome-c genes suggests predominantly mitochondria-related control. The more selective PlaNet analysis revealed strongest co-regulation with the chloroplast-targeted tryptophan α-subunit (At3g54640; Zhao and Last, 1995; Supplemental Figure 1C). The HRR levels (highest reciprocal ranks) of s-APX or t-APX with other transcripts were only low (≤30) indicating distinct (sets of) signaling pathways.

Discussion Excess availability of carbohydrates inactivates photosynthesis and induces carbohydrate storage (Koch, 1999). Consistently, most genes encoding proteins of the thylakoid membrane,

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Figure 4.  Log-term Effect of Sucrose and Sorbitol on Transcript Levels. The long-term effect of increased sucrose availability (top) and sorbitol-induced osmotic stress (bottom) on the transcript levels of antioxidant enzymes (left) and carbohydrate-regulated genes in Arabidopsis mutants with defects in ascorbate biosynthesis (vtc1), starch biosynthesis (adg1), and abscisic acid biosynthesis (aba2) after 10-day growth on 1 or 2% (w/v) sucrose and 1% (w/v) sucrose plus 1% (w/v) sorbitol, which contains the same amount of metabolizable sucrose as the 1% (w/v) sucrose sample and is almost identical in its long-term osmotic effect as 2% (w/v) sucrose. Means and standard deviations from three biological replicates with at least two technical replicates are depicted. Significant differences from wt regulation (α = 0.05; t-test) are labeled with an asterisk.

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such as pet-M, pet-C, and lhcb2.2, were inactivated and genes encoding starch storage enzymes, like apl3, were activated (Figure 1). Carbohydrate-dependent metabolism was shifted and ascorbate accumulated. In addition to being a carbohydrate sink, it is a major low-molecular antioxidant and an essential co-factor of s-APX. Its good availability avoids enzyme inactivation (Miyake and Asada, 1996). In parallel to inhibition of photosynthesis, the transcript levels of most genes for chloroplast antioxidant enzymes decreased under excess carbohydrate conditions (Figures 1, 3, and 5). Strongest of all, the transcript levels of the genes encoding thylakoid-bound APX and thylakoid-associated 2CPA and 2CPB decreased. For 2cpa, it was shown (Baier et al., 2004b) that a promoter element located in close vicinity to the transcription initiation site correlates transcription activity with chloroplast development. A  more upstream-located redox-box responds to the acceptor availability at photosystem I  and adjusts the gene activity in green tissues to the chloroplast redox status via the transcription factor RAP2.4a (Shaikhali et  al., 2008). In Rap2.4a-KO lines, t-APX, s-APX, and 2CPB were, like 2CPA, decreased in expression activity. Furthermore, analysis of transcript abundance regulation in the rimb-mutants which are defective in 2cpa-activating mechanisms (Heiber et al., 2007) demonstrated that expression of

the four genes for chloroplast peroxidases is under the control of the same signaling pathway which induces 2cpa upon redox imbalances, although the transcript levels are distinctly regulated during development and in response to other stimuli (Pena-Ahumada et al., 2006; Juszczak et al., 2012). Here, the transcript levels of the ROS indicator genes bap1 and fer1 (op den Camp et al., 2003) were not elevated upon Suc treatment under any condition tested (Figures 1, 3, and 5), demonstrating that ROS-signaling pathways were unaffected in the seedlings grown on 2% Suc. Upon mild sucrose application, s-APX showed a distinct regulation pattern compared to the other genes encoding chloroplast antioxidant enzymes and the transcript levels increased upon sugar feeding (Figures 1, 3, and 5). Sucrose induction of s-APX took place at growth light intensity, upon exposure to low light and high light, and upon short- and long-term sucrose feeding (Figures 1, 3, 5, and 7), demonstrating a solid connection between sucrose application and gene induction. Compared to induction of the sucrose-inducible APL3, s-APX was less responsive to sucrose in the ascorbate-biosynthetic vtc1-mutant (Figure 5). Consistently with previous analysis of s-APX regulation in response to high CO2-application (Wormuth et  al., 2006), short-term sucrose application increased the s-APX transcript amount stronger in vtc1 and

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Figure 5.  The Effect of Short-Term Sucrose Feeding on the Transcript Levels (top) and the Relative Transcript Abundance Regulation (Bottom) in vtc1, adg1, and aba2 as Compared to Wild-Type Plants. Means and standard deviations from three biological replicates with at least two technical replicates are depicted. The asterisk show statistical significance relative to wt (α = 0.05; t-test).

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less in adg1 than in wt (Figure  5). This demonstrates that a specific ascorbate biosynthesis-related process dominates the sucrose consumption effect, which ascorbate and starch biosynthesis have in common. In the case of 2cpa, ascorbate suppresses transcription via a signaling pathway depending on photosynthetic electron transport activity (Shaikali and Baier, 2010). It has been concluded that ascorbate/monodehydro- and dehydroascorbate cycling in response to ROS quenching and NAD(P)H consumption regulates 2cpa transcription. In contrast to ascorbate application experiments, the redox status of the ascorbate pool was stable upon long-term sucrose feeding (Figure 2), but shifted to a higher reduction status upon short-term sucrose application (Figure  6). The massive biosynthesis of ascorbate consumes electrons from the respiratory chain, suggesting that, in addition to the ascorbate redox status, mitochondrial redox signals impact on the signal transduction process. Inhibition of the respiratory electron transport at complex III by antimycin-A results in increased s-APX transcript amounts (Chew et al., 2003). Thus, if respiratory electron transport is involved in the excess-sugar increase of s-APX, the signal would correlate with a decrease in electron transport activity in the electron transport chain

downstream of complex III. This is consistent with increased electron consumption between complexes III and IV due to ascorbate biosynthesis and supports the conclusion on a link between the ascorbate biosynthesis rate and s-APX regulation. Due to the mild conditions, the chlorophyll content, the chl-a/chl-b-ratio, and the ATP/ADP ratio were not changed in plants grown on 2% Suc compared to 1% Suc (Figure 2), and can more or less be excluded as putative signals controlling s-APX levels. Ascorbate availability or ascorbate synthesis control the transcript levels of many genes (Veljovic-Jovanovic et  al., 2001). Partly, the ascorbate response has been described to be linked to ABA signaling. However, Suc-induced s-APX promotion was stronger in aba2-mutants and lower in vtc1-mutants than in wt (Figure 4) excluding also a prominent role of ABA in sucrose regulation of s-APX. Compared to strong down-regulation of transcripts for chloroplastic antioxidant enzymes and most proteins of the photosynthetic membrane, the increase of s-APX upon short- and long-term Suc application demonstrated specific regulation (Figure  2). The long-term effect was less pronounced in vtc1 (Figure  4) and stronger in response to short-term Suc feeding in adg1 (Figure  5), indicating that

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Figure 6.  Mutant Effects on the Ascorbate Availability and the Redox State of Ascorbate. Ascorbate levels and the redox state of the ascorbate pools in Arabidopsis wild-type, aba2, adg1, and vtc1 plants grown for 10 d on 1% or 2% (w/v) sucrose or 1% (w/v) sucrose plus 1% (w/v) sorbitol (top) and in plants treated for 24 h with 1% extra sucrose (bottom, black bars) compared to plants kept at the basal sucrose level (bottom, white bars) (n = 5–6). The asterisk show statistical significance of difference compared to the wt response (α = 0.05; t-test).

Heiber et al.  •  s-APX Transcript Abundance Is Regulated by Ascorbate   

Conclusion Within the group of genes encoding antioxidant enzymes, s-apx showed the most specific gene-expression pattern throughout all experiments (Figures 1, 3, 4, and 5). The transcript levels were higher in response to Suc feeding, which otherwise inhibits genes encoding chloroplast photosynthesis-related proteins (Koch, 1999; Gibson, 2000). The Suc effect was maintained between 50 and 600  μmol quanta m–2  s–1 illumination. If affected, it negatively correlated with the osmotic effect of sugar feeding (Figure 7). Previously, we studied the regulation of the chloroplast antioxidant system during the first days of seedling development (Pena-Ahumada et  al., 2006). While PRX were quickly activated upon germination, it took until the third day after root emergence to fully induce the ascorbate peroxidase system. Slow activation of s-apx (and mdhar) expression correlated with low ascorbate availability. Here, vice versa, higher s-APX transcript levels correlated with high ascorbate levels on high Suc media. It is concluded that high carbohydrate availability promotes the stromal ascorbate-dependent

water–water-cycle (Asada, 1999) by supporting ascorbate accumulation and stimulating s-APX (and to a lesser extent MDHAR) levels, while the expression of genes encoding thylakoids associated antioxidant enzymes, such as t-APX (Asada, 1999) or 2CP (König et al., 2003) are inactivated (Figures 1, 3, and 7). In summary, the specific regulation of s-APX could stabilize the chloroplast antioxidant poise, when photosynthesis is inactivated and chloroplasts shift towards carbohydrate storage.

METHODS Plant Growth Seeds of the Arabidopsis thaliana Col-0 and mutants generated in the Col-0 background were surface-sterilized and stratified as described previously (Baier et  al., 2004a). The plants were grown in continuous light on MS medium (Duchefa, The Netherlands) adjusted to pH 5.8 with KOH, buffered with 5 mM MES, and supplemented with 2.5 g L–1 phytagel (Roth, Germany) and sugars (as indicated in the ‘Results’ section) at 24°C and 100 μmol quanta m–2 s–1 light, respectively. For illumination experiments, 10-day-old seedlings were transferred for 4 h to 50  μmol quanta m–2  s–1 or high light of 600  μmol quanta m–2  s–1. For short-term Sucfeeding experiments, the seedlings were grown for 8 d on MS plates containing 0.5% Suc in a day/night cycle (10 h light/14 h dark). One hour after onset of light, the seedlings were treated with 0.5–1.5% Suc in MS-media for 24 h. The plant material was shock-frozen in liquid nitrogen during the harvest.

Metabolite Analyses Ascorbate and chlorophyll contents were quantified as described by Baier et  al. (2000), anthocyanins according to Mancinelli et  al. (1975), and chlorophylls according to Porra et al. (1989). ATP and ADP contents were determined luminometrically according to Kaiser and Urbach (1977), the assimilatory force and the reduction state of NADPH as described by Dietz and Heber (1989), and 2-PGA, DHAP, and fructose-6-phosphate according to Dietz and Heber (1984).

RNA Isolation and RT–PCR Analysis For each treatment, plant material from at least four MS plates with at least 40 individual plants were pooled and RNA was extracted as described by Baier and Dietz (1999) or by using the Roboklon RNA extraction Kit (Roboklon, Berlin, Germany) according to the manufacturer’s recommendations. cDNAsynthesis, standardization on ACTIN-2 transcript amount, RT– PCR, detection, and quantification were performed with at least three biological replicates as described by Baier et  al. (2000) or with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, USA). For qPCR, 108–140 bp

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s-APX regeneration is under control of carbohydrate-driven ascorbate biosynthesis. Ascorbate biosynthesis takes place in the cytosol and in mitochondria, and consumes cytosolic hexose-phosphates (Smirnoff et al., 2001). In its final step, it is linked to respiratory electron transport activity between complexes III and IV (Bartoli et al., 2000). GeneMANIA analysis for transcripts co-regulated with s-APX showed that s-APX transcript abundance is most similarly regulated to cytochrome-c1 (CYT-C1) and cytochrome-c2 (CYT-C2) expression, indicating a mitochondrial driving force in s-APX regulation. Within the first 1000 bp upstream of the transcription initiation site, no elements described for the organ- and celltype-specific regulation of the cyt-C-promoters, such as site II elements (TGGGCC/T) and telo-boxes (AAACCCTAA) (Welchen and Gonzalez, 2005), were found (data not shown), indicating that the co-regulation depends on unknown regulatory mechanism. s-APX has been described to be dually targeted to chloroplasts and mitochondria (Chew et al., 2003). The full-length protein shows a much higher preference for chloroplasts than for mitochondria (TargetP: probability for chloroplast targeting 86.4%; for mitochondria targeting 8.2%). In the truncated s-APX used by Chew et  al. (2003), which lacks the first 25 amino acids, the relative probability for mitochondria targeting (as compared to chloroplast targeting) increases as indicated by the TargetP-values (TargetP probability: 36.2% for mitochondria and 94.8% for chloroplasts) and import studies (Chew et  al., 2003). Therefore, mitochondrial or mitochondria-related signals may control the composition of the chloroplast antioxidant system. The weaker response of s-APX to sucrose in vtc1 (Figure 5) demonstrates that ascorbate biosynthesis impairs on s-APX regulation.

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DNA fragments were amplified from actin-standardized cDNA samples in the presence of SYBR Green I  using the 2 QuantiTect SYBR Green PCR Master Mix (Qiagen, Germany) or the Robokolon SYBR Green Master Mix (Roboklon, Berlin, Germany) in the DNA Engine Opticon System (PTC-200 DNA Engine Cycler plus CFD-3200 Opticon detector; MJResearch Waltham, USA) or the Biorad CFX96 System (Biorad, Munich, Germany). Prior to the quantification, the reaction condition was optimized and the melting kinetics (10 s per °C) were determined for each primer pair. Background fluorescence was subtracted. The transcript abundance was determined from the threshold (CT(T) relative to the actin threshold (CN(T)) according to Manthey et  al. (2004). Each experiment

was performed in at least three biological replicates and with two to three technical replicates.

In Silico Transcript Abundance Correlation Analysis Co-expression of genes was analyzed in Arabidopsis thaliana microarray databases by using GeneMANIA (Zuberi et  al., 2013) and PlaNet (Mutwil et  al., 2011) with default setting.

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

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Figure 7.  Light Intensity Effect. The ascorbate content, ascorbate redox state, and the relative transcript amounts of genes encoding chloroplast antioxidant enzymes and reference genes for oxidative stress, carbohydrate availability, and photosynthetic membrane support in response to increased sucrose availability (sugar effect) and increased osmotic stress (osmotic effect) in 10-day-old seedlings grown at 100 μmol quanta m–2 s–1 after four additional hours of illumination at 50, 100, and 600 μmol quanta m–2 s–1. The asterisk shows statistical significance of regulation (α = 0.05; t-test).

Heiber et al.  •  s-APX Transcript Abundance Is Regulated by Ascorbate   

FUNDING We acknowledge funding by the German Research Foundation, the Chinese Scholarship Council, and the Freie Universität Berlin.

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Chew, O., Whelan, J., and Millar, A.H. (2003). Molecular definition of the ascorbate–glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J. Biol. Chem. 278, 46869–46877.

We thank Dr Christiane Hedtmann, Dr Rainer Bode, and Jörn van Buer for critical reading of the manuscript. No conflict of interest declared.

Conklin, P.L., Pallanca, J.E., Last, R.L., and Smirnoff, N. (1997). L-ascorbic acid metabolism in the ascorbate-deficient Arabidopsis mutant vtc1. Plant Physiol. 115, 1277–1285.

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