Saijaliisa Kangasjärvi

Anna Lepistö

Kati Hännikäinen

Mirva Piippo

Eeva-Maria Luomala

Eva-Mari Aro

Eevi Rintamäki

Prof. Eevi Rintamäki

Diverse roles for chloroplast stromal and thylakoid-bound ascorbate peroxidases in plant stress

Keywords: Chloroplast ascorbate peroxidase, peroxiredoxin, photo-oxidative stress, acclimation, signaling, Arabidopsis thaliana

Photosynthetic light reactions comprise a significant source of hydrogen peroxide (H 2 O 2 ) in illuminated leaves. Ascorbate peroxidases (APXs) reduce H 2 O 2 to water, and play an important role in the antioxidant system of plants. We addressed the significance of chloroplast APXs in stress tolerance and signaling in Arabidopsis thaliana. To this end, T-DNA insertion mutants tapx, sapx and tapx sapx, lacking the thylakoid-bound APX (tAPX), stromal APX (sAPX) or both, respectively, were characterized. Photo-oxidative stress during germination lead to bleaching of chloroplasts in sapx single mutant and particularly in the tapx sapx double mutant plants, whereas the greening process of wild type and tapx plants was only partially impaired. Mature leaves of tapx sapx double mutants were also susceptible to short-term photo-oxidative stress induced by high light or methyl viologen treatments. After a twoweek acclimation period under high light or under low temperature, none of the mutants exhibited enhanced stress symptoms. Immunoblot analysis revealed that high-lightstress-acclimated tapx sapx double mutants compensated the absence of tAPX and sAPX by increasing the level of 2-cys peroxiredoxin. Furthermore, the absence of tAPX and sAPX induced alterations in the transcriptomic profile of tapx sapx double mutant plants already under quite optimal growth conditions. We conclude that sAPX is particularly important for photoprotection during the early greening process. In mature leaves, tAPX and sAPX are functionally redundant, and crucial upon sudden onset of oxidative stress. Moreover, chloroplast APXs contribute to chloroplast retrograde signaling pathways upon slight fluctuations in the accumulation of H 2 O 2 in chloroplasts.

INTRODUCTION

Generation of reactive oxygen species (ROS) is characteristic of a number of metabolic reactions that take place in different compartments of plant cells. Under favorable conditions, plants generally maintain ROS at low levels. Under adverse environmental conditions, however, imbalances in metabolic processes may lead to increased accumulation of ROS, forming a potential threat of oxidative damage to cellular components. On the other hand, transient increases in ROS levels also have a vital role in stress-signaling and thereby in the survival of plants under adverse environmental conditions [START_REF] Dat | Dual action of the active oxygen species during plant stress responses[END_REF]. In plants, ROS levels are controlled via a versatile antioxidant network.

Components of the antioxidant system reside in various cellular locations, and include a range of enzymatic scavengers, such as superoxide dismutases (SODs), ascorbate peroxidases (APXs), peroxiredoxins (PRXs), glutaredoxins, glutathione peroxidases and catalases. In addition, several nonenzymatic antioxidants like ascorbate, glutathione, tocopherols, carotenoids and phenolic compounds also contribute to the cellular redox balance [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF].

The water-water cycle of chloroplasts comprises a significant source of ROS in lightexposed green tissues [START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF]. Photoreduction of molecular oxygen via Photosystem I (PSI) leads to formation of superoxide (O 2 putative lumenal APX are each encoded by a single nuclear gene in Arabidopsis [START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF][START_REF] Chew | Molecular definition of the ascorbate-glutathione cycle in arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants[END_REF][START_REF] Kieselbach | A peroxidase homologue and novel plastocyanin located by proteomics to the arabidopsis chloroplast thylakoid lumen[END_REF][START_REF] Yoshimura | Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants[END_REF]. Outside chloroplasts, 25 kDa APX1 and APX2 have been found in the cytoplasm [START_REF] Santos | Cytosolic ascorbate peroxidase from arabidopsis thaliana L. is encoded by a small multigene family[END_REF], and a 31 kDa APX3 is located in peroxisomes and in oilseed glyoxisomes [START_REF] Narendra | The arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for arabidopsis growth and development[END_REF][15][16][START_REF] Jespersen | From sequence analysis of three novel ascorbate peroxidases from arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase[END_REF].

Members of the second group of H 2 O 2 detoxifying enzymes, peroxiredoxins, are [START_REF] Jespersen | From sequence analysis of three novel ascorbate peroxidases from arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase[END_REF][START_REF] Broin | The plastidic 2cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage[END_REF][START_REF] Danna | Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity[END_REF][START_REF] Yabuta | Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress[END_REF][21][START_REF] Miyake | Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses[END_REF] kDa enzymes that possess N-terminal cystein residue(s) responsible for peroxidase activity. In Arabidopsis, four PRXs are targeted to chloroplasts: 2-cystein peroxiredoxins A and B, peroxiredoxin Q and peroxiredoxin II E [START_REF] Dietz | The function of peroxiredoxins in plant organelle redox metabolism[END_REF]. Re-reduction of chloroplastic PRXs occurs via the action of various thioredoxins and thioredoxin-like proteins [START_REF] Dietz | The function of peroxiredoxins in plant organelle redox metabolism[END_REF][START_REF] Broin | The plastidic 2cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage[END_REF]. Ascorbate peroxidases have long been considered as key enzymes in the detoxification of H 2 O 2 in chloroplasts [START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF]. However, the functional specificities of tAPX and sAPX in various plant species and stress conditions, and particularly their functional overlaps with other cellular antioxidant agents remain poorly understood. In a wheat mutant line, 40% reduction in tAPX activity resulted in lowered photosynthetic carbon assimilation as well as reduced growth rate and seed production [START_REF] Danna | Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity[END_REF]. Moreover, unsuccessful attempts to create antisense tapx lines from tobacco suggested that suppression of tAPX in tobacco may be lethal [START_REF] Yabuta | Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress[END_REF]. On the other hand, transgenic tobacco and Arabidopsis plants over-expressing tAPX showed enhanced tolerance to methyl viologen-induced photo-oxidative stress [START_REF] Yabuta | Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress[END_REF]21]. Inconsistent with their role in photo-oxidative stress tolerance, chloroplastic APXs are known to become inactivated by H 2 O 2 if ascorbic acid is present at low levels [START_REF] Shigeoka | Regulation and function of ascorbate peroxidase isoenzymes[END_REF][START_REF] Miyake | Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses[END_REF]. Indeed, tAPX was recently connected with signaling events conferring resistance to heat stress in Arabidopsis [23].

In this study, we have characterized Arabidopsis thaliana T-DNA insertion mutants deficient in the thylakoid-bound and stromal forms of APX. Special emphasis was paid on (i) the significance of tAPX and sAPX in oxidative stress tolerance under various developmental stages and on (ii) signaling effects that the absence of tAPX and sAPX provokes in the mutant plants. We demonstrate that sAPX is particularly important for photoprotection during the early greening process. In mature leaves, tAPX and sAPX appear to be functionally redundant, and exhibit a key role in the maintenance of chloroplast functionality upon sudden onset of oxidative stress. Moreover, chloroplast

EXPERIMENTAL Material and control growth conditions

Homozygous T-DNA insertion mutants for chloroplast thylakoid-bound ascorbate peroxidase (tapx; At1g77490; SALK_027804) or stromal ascorbate peroxidase (sapx; At4g08390; SALK_083737) were identified from the SALK institute' s collection by PCR analysis of genomic DNA according to the institute' s protocols (http://signal.salk.edu/cgi-bin/tdnaexpress) [START_REF] Alonso | Genome-wide insertional mutagenesis of arabidopsis thaliana[END_REF]. The tapx and sapx lines were backcrossed to wild-type Columbia, selfed and re-identified to remove additional T-DNA inserts from the genome. A tapx sapx double mutant was created by crossing the tapx and sapx single mutants, and identified from the F2 generation by PCR analysis of genomic DNA using the same set of primers that were used to screen for the single mutants.

Arabidopsis thaliana ecotype Columbia wild type and the mutant plants were grown under control conditions of 130 µmol photons m -2 s -1 /20ºC with 60% humidity at an 8hour light period. We chose short day conditions to avoid general upregulation of antioxidative capacity, which was recently shown to be typical of plants grown under long day conditions [START_REF] Becker | Influence of the photoperiod on redox regulation and stress responses in arabidopsis thaliana L. (heynh.) plants under long-and short-day conditions[END_REF].

Germination tests

For germination tests, seeds were sown on filter paper on Petri dishes containing halfstrength Murashige and Skoog basal salt mixture (MS) (Sigma-Aldrich; http://www.sigmaaldrich.com ), and kept at 4ºC in darkness for two days. Subsequently, the sensitivity of germination to photo-oxidative stress was explored by supplementing

Stress treatments

Sensitivity of expanded, four-week-old rosettes to methyl viologen-induced photooxidative stress was explored by gently spraying the plants with 50 µM methyl viologen at the end of the dark period. After 30 min incubation under 50 µmol photons m -2 s - 1 /20ºC, plants were illuminated under 130 or 1000 µmol photons m -2 s -1 /20ºC for 6 or 24 hours. Control samples were sprayed with water and illuminated under normal growth conditions (130 µmol photons m -2 s -1 /20ºC). After the treatment, rosettes were excised and incubated in 30 ml of ion-exchanged water at 4ºC in darkness over night. The extent of cell death and membrane disruption was measured as ion leakage to the solution with a conductivity meter (Radiometer, Copenhagen, Denmark). Finally, the results were normalized to the value of ion leakage obtained after boiling the samples for five minutes.

For short term light treatments, four-week-old plants, grown under 130 µmol photons m -2 s -1 /20ºC, were shifted at the end of the dark period to light intensities of 130, 300, 450, 600, 1000 and 1300 µmol photons m -2 s -1 at 20ºC for two or six hours. Contribution of PSII repair cycle to the light sensitivity was assessed by incubating the plants under 300 µmol photons m -2 s -1 f or two hours on Petri di shes in the presence of 1 mM lincomycin.

For long term stress treatments, plants were first grown for two weeks under the control growth conditions (130 µmol photons m -2 s -1 /20ºC, 8-hour light period), and thereafter shifted for two weeks to moderately high light intensity (600 µmol photons m -2 s - 1 /20ºC), to high light stress (1300 µmol photons m -2 s -1 /28ºC), to low temperature (130 µmol photons m -2 s -1 /10ºC) or maintained at control growth conditions. For biochemical analysis, samples were collected from four-week-old plants, six hours after the lights were turned on.

Measurement of chlorophyll content, chlorophyll fluorescence and gas exchange

To determine the leaf chlorophyll content, two leaf discs, 3 mm in diameter, were incubated in 1 ml of dimethylformamide over night at 4ºC in darkness, and the chlorophyll content was determined spectrophotometrically according to (Inskeepand Bloom, 1985).

The photoinhibition state of PSII was monitored as a ratio of variable to maximal fluorescence, Fv/Fmax (Fv is a difference between maximal, Fmax, and initial fluorescence, Fo), measured from intact leaves with a Hansatech PEA fluorometer (King's Lynn, UK) after a 30-min dark incubation.

Gas exchange of intact Arabidopsis plants was measured with CIRAS-1 Combined infrared gas analysis system (PP Systems, Hitchin, UK) equipped with an Arabidopsis pot chamber (PP Systems, Hitchin, UK; http://www.ppsystems.com/). The response of net photosynthesis (A n ) to the reference CO 2 was measured under photosynthetically active photon flux density (PPDF) that was saturating for net photosynthesis (500 µmol m -2 s -1 /20ºC). The parameters for maximal carboxylation rate of Rubisco (Vcmax, µmol CO 2 m -2 s -1 ), maximal electron transport rate (Jmax, µmol m -2 s -1 ) and rate of mitochondrial respiration in light (Rd, µmol m -2 s -1 ) were obtained by modeling the response of net CO 2 assimilation to increasing extracellular CO 2 concentration according to [START_REF] Farquhar | A biochemical model of photosynthetic CO 2 assimilation in leaves of C 3 species[END_REF].

Isolation of thylakoid membranes, soluble extract and total leaf extract

For isolation of thylakoids and soluble leaf extract, leaves were harvested and immediately frozen in liquid nitrogen. After homogenization in ice cold shock buffer (25 mM Hepes-KOH, pH 7,4, 10 mM MgCl 2 , 10 mM NaF) and filtration through Miracloth, the suspension was centrifuged at 6000 g, for 5 min at 4°C. The thylakoid pellet was resuspended in isolation buffer, and the supernatant was further centrifuged at 12 000 g, for 15 min at 4°C to collect the soluble extract. For isolation of total leaf extract, six 12,56 mm 2 leaf discs from three individual rosettes were punched and immediately frozen in liquid nitrogen. The leaf discs were completely and carefully homogenized in 150 µl of ice cold isolation buffer (330 mM sucrose, 25 mM Hepes-KOH, pH 7,4, 10 mM MgCl 2 , 10 mM NaF) in a dark cold room. All samples were frozen in liquid nitrogen and stored at 80°C. The chlorophyll content of isolated thylakoids was determined according to [27], and the protein content of the soluble and total leaf extracts was determined with the Biorad assay kit.

SDS-PAGE and Western blotting

Thylakoid polypeptides corresponding to 1 µg chlorophyll and the soluble and total leaf extracts corresponding to 10 µg of protein were solubilized and separated by SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF], using 15% acrylamide and 6 M urea in the separation gel, and subsequently electroblotted to a PVDF membrane (Millipore, http://www.millipore.com). After blocking with 5 % BSA (fatty acid free, Sigma-Aldrich), the polypeptides were immunodetected with protein-specific antibodies using a PHOTOTOPE TM -Star Detection Kit (New England Biolabs, Beverly, USA; http://www.neb.com/). Thylakoid protein phosphorylation in isolated thylakoids was explored by using a phosphothreonine-spesific antibody (New England Biolabs, Beverly, USA; http://www.neb.com/) [START_REF] Rintamäki | Cooperative regulation of light-harvesting complex II phosphorylation via the Biochemical Journal Immediate Publication[END_REF]. An Arabidopsis ascorbate peroxidase-specific antibody was raised against a highly conserved region among tAPX, sAPX and the cytoplasmic APXs, corresponding to amino acids 204-215 of tAPX (EEGRLPDAGPPS-COOH).

An additional cystein was added to the C-terminus of the peptide to facilitate conjugation to bovine serum albumin. The peptide synthesis, conjugation to BSA and antibody production was performed at Innovagen (http://www.innovagen.se/) according to the commercial standard package offered for custom peptide antibody production.

Other protein-specific antibodies were purchased from Research Genetics (D1; http://www.resgen.com/about/index.php3), Agrisera (Lhcb1, Lhca2;

http://www.agrisera.se/), or were kindly provided by Professor H. Scheller (psaD) and Professor K.-J. Dietz (2-cysteine peroxiredoxin, peroxiredoxin 2E and peroxiredoxin Q).

In vivo detection of H 2 O 2

Accumulation of H 2 O 2 in the leaves was detected by using diaminobenzidine (DAB, Sigma-Aldrich; http://www.sigmaaldrich.com) as a substrate [START_REF] Thordal-Christensen | Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction[END_REF]. Briefly, rosettes were excised at the end of the light period, and incubated on Petri dishes supplied with a 0,1 mg/ml solution of DAB (pH 3.8) over night in darkness. At the onset of the light period, the dishes were transferred to growth light (130 µmol photons m -2 s -1 /20ºC) for two hours, or incubated under high light (1000 µmol photons m -2 s -1 /20ºC) for 30 minutes.

Thereafter the rosettes were incubated in 96% (v/v) ethanol until chlorophyll bleached, and finally photographed under a Zeiss Lumar V12 stereomicroscope (http://www.zeiss.com).

Microarray analysis

Global changes in gene expression were explored with spotted Arabidopsis 24k oligonucleotide arrays (MWG Biotech; http://www.mwg-biotech.com; ArrayExpress database accession number A-ATMX-2; http://www.ebi.ac.uk/arrayexpress). Twenty three-week-old rosettes of wild type and tapx sapx double mutant plants were collected from normal growth conditions (130 µmol photons m -2 s -1 /20ºC ) two hours after the onset of the light period. For analysis of high-light-induced modulations in gene expression, the plants were illuminated under 1000 µmol photons m -2 s -1 /20ºC for 30 minutes. Total RNA was isolated with Trizol as described previously [START_REF] Piippo | Chloroplast-mediated regulation of nuclear genes in arabidopsis thaliana in the absence of light stress[END_REF].

Subsequently, DNA was removed with Qiagen RNeasy Mini Kit, and cDNA synthesis was performed in the presence of 0,2 mM aminoallyl-dUTP using anchored poly-dT The aminoallyl-labeled cDNA was purified with QIAquick PCR purification kit (Qiagen), and stained with CY TM Post-labeling Reactive Dye Pack (Amersham). cDNA corresponding to 15 pg of each dye was hybridized to the arrays in 50 % formamide, 5 x SSC, 0,1% SDS, 0,1 mg/ml herring sperm, 5 x Denhart' s at 42ºC over night.

The arrays were scanned with Agilent scanner (http://agilent.com) and the spot intensities were quantified with ScanArray Express Microarray Analysis system 2.0 (Perkin-Elmer Life Sciences, http://las.perkinelmer.com/). The data from three biological replicates was analyzed with GeneSpring 7.2. Genes with Student' s t-test P value below 0,05 were chosen for further analysis. Gene annotation was derived from Arabidopsis Information resource (http://www.arabidopsis.org) and was based on TAIR7. Imaging of chlorophyll fluorescence by confocal microscopy revealed no structural differences in the cotyledons of wild type, tapx, sapx and tapx sapx double mutant plants when germinated on control plates containing half strength Murashige and Skoog (MS) medium (Fig. 2a). In the presence of methyl viologen, cotyledons of the wild type and tapx plants also showed accumulation of chloroplasts, which, however, appeared to be smaller than those observed in the control plants (Fig. 2a). On the contrary, hardly any chlorophyll fluorescence could be detected when cotyledons of the sapx single mutants or the tapx sapx double mutants were imaged with the same microscope settings that were used to examine the wild type and tapx plants (data not shown). By enhancing the sensitivity of detection by increasing the detector gain, however, accumulation of a drastically reduced number of chloroplasts in the cotyledons of sapx and the tapx sapx double mutant could be documented (Figs 2a andb). The severity of chloroplast photo-oxidation in the sapx single mutant and the tapx sapx double mutant plants varied to some extent. Typically, the methyl viologen-induced oxidative stress had lead to almost complete bleaching of mesophyll cell chloroplasts in the sapx single mutant and the tapx sapx double mutant plants (Fig. 2a). In some of the cotyledons, however, chlorophyll autofluorescence could still be observed from the guard cell chloroplasts of the abaxial side of the leaves, as demonstrated in Figure 2a. even under growth light conditions, and this effect became drastically pronounced under high light intensity (Fig. 2c).

RESULTS

Identification

The high light sensitivity of four-week-old tapx, sapx and tapx sapx double mutant plants was explored by exposing the mutant and wild type plants to increasing light intensities (130, 300, 450, 600, 1000 and 1300 µmol photons m -2 s -1 /20ºC) for six hours.

A gradual PSII photoinhibition of similar extent, measured as Fv/Fmax after a 30 minute dark incubation, was recorded for the wild type and the single mutants tapx and sapx with increasing irradiance (Fig. 2d). In the tapx sapx double mutant, however, a significant decrease in the Fv/Fmax ratio was observed under 1000 µmol photons m -2 s -1 (Fig. 2d). However, when the plants were illuminated under 300 µmol photons m -2 s -1 in the presence of lincomycin, which prevents the repair of PSII by inhibiting translation in chloroplasts, the wild type, tapx, sapx and tapx sapx double mutant plants were all equally sensitive to photoinhibition (Fig. 2d). Thus, the vulnerability of the tapx sapx double mutant plants to photoinhibition under 1000 µmol photons m -2 s -1 in the absence of lincomycin presumably resulted from disturbances in the repair process of PSII.

Long-term acclimation of tapx, sapx and tapx sapx d o u b l e m u t a n t p l a n t s t o environmental stress

To study the capability of tapx, sapx and tapx sapx lines for long-term acclimation to stressful conditions, plants were grown for two weeks under the control growth conditions of 130 µmol photons m -2 s -1 /20ºC, and thereafter shifted for two weeks to moderately high light (600 µmol photons m -2 s -1 /20ºC), to high light stress (1300 µmol photons m -2 s -1 /28ºC) or to low temperature (130 µmol photons m -2 s -1 /10ºC). Control plants were maintained at initial growth conditions. After the acclimation period, neither the tapx and sapx single mutants nor the tapx sapx double mutants exhibited any enhanced stress symptoms or any kind of visual phenotype under any of the stress conditions studied (Fig. 3a, Supplemental table 1.). Indeed, the mutant and wild type plants showed similar morphological adaptations (Fig. 3a), and similar modulations in the content of chlorophyll (Supplemental table 1). Also the photochemical efficiency of PSII, expressed as Fv/Fmax, was after two The similar long-term stress tolerance of WT, tapx, sapx and the tapx sapx double mutant plants (Fig. 3a, Supplemental table 1) raised a question whether the acclimation to contrasting environmental conditions had also induced cross-tolerance against methyl viologen-induced abrupt oxidative stress. Thus, the differentially stress-acclimated plants were sprayed at the end of the dark period with 50 µM methyl viologen, and the extent of cell death was measured after 24-hour treatment under standard growth conditions (130 µmol photons m -2 s -1 /20ºC, 8/16 h light/dark period) (Fig. 3b). Clearly, all the lines grown under normal growth conditions wilted during the treatment (Figs. 3b andc). In contrast, acclimation to high light or to low temperature induced tolerance against subsequent methyl viologen treatment in the wild type and the tapx and sapx single mutant plants (Fig. 3b ). In sh arp con trast to wi l d ty pe an d th e sin gl e m utan ts, th e dif f eren ti ally stressacclimated tapx sapx double mutant plants acquired only partial tolerance against methyl viologen-induced photo-oxidative stress, and showed cell death, bleaching of chlorophyll and wilting of leaves after the 24-hour illumination period (Figs. 3b andc).

Steady-state levels of APXs and chloroplast PRXs upon long-term acclimation to stress

Besides ascorbate peroxidases, chloroplast peroxiredoxins have been associated with the water-water cycle, and thus with the protection of chloroplasts against oxidative damage [START_REF] Dietz | The function of peroxiredoxins in plant organelle redox metabolism[END_REF]. It is therefore conceivable that different environmental stresses promote differential adjustments in these antioxidant enzymes in plants. Moreover, increases in the levels of tAPX and/or sAPX would explain why the differentially stress-acclimated wild type and the tapx and sapx single mutant plants, but not the tapx sapx double mutant plants, acquired tolerance against methyl viologen-induced oxidative damage (Fig. 3b). Thus, we and4b).

Acclimation to increasing light intensities induced an increase in the steady-state level of the cytoplasmic APX in all the lines (Figs 4a andb). Notably, the high-light-stressacclimated tapx sapx double mutant plants also showed an increased level of 2-cys peroxiredoxin as compared to the wild type plants (Fig. 4b). No distinct light-intensitydependent modulations were observed for tAPX or sAPX (Figs 4a andb). On the contrary, in low-temperature-acclimated wild type and tapx plants, a strict up-regulation of sAPX occurred. Under these conditions, the amounts of peroxiredoxin IIE and also of 2-cys peroxiredoxin rather decreased (Fig. 4a). Low-temperature-acclimated tapx and sapx single mutants, however, maintained somewhat higher levels of these enzymes as compared to wild type plants (Fig. 4a). The 31 kDa polypeptide (depicted in Fig. 1) showed no distinct modulations under any of the conditions studied (data not shown). In conclusion, the absence of tAPX and/or sAPX induced no drastic compensatory increases in the steadystate levels of other ascorbate peroxidase isoforms. Moreover, only under high light stress, the absence of both tAPX and sAPX induced apparent compensatory increase in the steady-state level of 2-cys peroxiredoxin in the tapx sapx double mutant plants (Figs. 4a andb).

Photosynthetic adjustments in tapx sapx double mutant plants

The wild type and tapx sapx double mutant plants showed similar accumulation of the light-harvesting antenna I and II polypeptides Lhca1 and Lchb2 as well as the psaD and D1 proteins, representatives of the PSI and PSII core complexes, respectively, under normal growth conditions (Fig. 4c). Likewise, acclimation of the wild type and tapx sapx double mutant plants to high light intensity induced similar decreases in the levels of Lhca1, Lchb2 and psaD (Fig. 4c). The D1 protein, on the other hand, showed no consistent changes after two weeks light acclimation in either of the lines (Fig. 4c). Low temperature induced no significant modulations in any of the polypeptides in either the wild type or the tapx sapx double mutant plants (Fig. 4c). Moreover, the contents of Rubisco large subunit remained quite invariable under all conditions explored (Fig. 4c). Consistently, the response of net CO 2 assimilation to increasing atmospheric CO 2 concentrations in wild type and tapx sapx double mutants grown under control growth conditions (130 µmol photons m -2 s -1 /20ºC) did not drastically differ from each other (data not shown). Modeling of photosynthetic parameters revealed quite similar values for maximal carboxylation rate of Rubisco (Vcmax) and for maximal photosynthetic electron transport rate (Jmax) in the wild type and tapx sapx double mutant plants (Supplemental Table 2). Interestingly, however, the calculated parameters indicated reduced rate of mitochondrial respiration (Rd) in tapx sapx double mutant plants as compared to wild type plants (Supplemental Table 2).

Redox regulation of LHCII protein phosphorylation in tapx sapx double mutant plants T h e r e d o x c o n d i t i o n s o f c h l o r o p l a s t s a r e k n o w n t o m o d u l a t e s i g n a l i n g e v e n t s t h a t

dynamically regulate photosynthetic processes [START_REF] Nott | PLASTID-TO-NUCLEUS RETROGRADE SIGNALING[END_REF]. In the thylakoid membrane, the reversible phosphorylation of the PSII light-harvesting antenna polypeptides Lhcb1 and Lchb2 on stroma-exposed threonine residues is particularly sensitive to modulations in the thiol redox state of chloroplast stroma [START_REF] Rintamäki | Cooperative regulation of light-harvesting complex II phosphorylation via the Biochemical Journal Immediate Publication[END_REF]. To explore whether tAPX and sAPX contribute to chloroplast redox signaling, we studied the light-dependent adjustments in the level of LHCII protein phosphorylation in wild type and the tapx sapx double mutant plants. Under normal growth light conditions (130 mol photons m -2 s -1 /20ºC), the LHCII showed no drastic differences in the phosphorylation level between wild type and the tapx sapx double mutant plants (Fig. 4d). When the plants were shifted to higher light intensity (300 mol photons m -2 s -1 /20ºC) for two hours, LHCII protein phosphorylation became downregulated in the wild type plants. However, the LHCII of tapx sapx double mutant plants remained slightly more phosphorylated as compared wild type plants (Fig. 4d). Under these experimental conditions, no distinct differences were observed in the phosphorylation of the PSII core proteins D1, D2 and CP43 between wild type and the tapx sapx double mutant plants (Fig. 4d). ). Exposure of the rosettes to a 30-minute high-light illumination period under 1000 µmol photons m -2 s -1 /20ºC enhanced the accumulation of H 2 O 2 to some extent in the wild type plants (Supplemental Fig. 1). In the leaves of tapx sapx double mutant plants, the accumulation of H 2 O 2 was slightly more pronounced as compared to the wild type plants (Supplemental Fig. 1).

When the gene expression was analyzed under control growth conditions, only one gene, At1g26170 encoding an importin beta-2 subunit family protein, showed statistically significant (t-test p-value < 0,05) up-regulation in the tapx sapx double mutant as compared to wild type plants. On the contrary, distinctly lower transcript levels for 67 genes were observed in the tapx sapx double mutant as compared to wild type plants (Supplemental Fig. 1, Supplemental Table 3). Twelve of these genes, including TAPX and SAPX, were found to encode for chloroplast proteins (Supplemental Table 3). Furthermore, distinct down-regulation of six transcription factors that control gene expression in the nucleus occurred in the tapx sapx double mutant plants as compared to wild type plants (Supplemental table 3). Among these transcriptional regulators, At1g04400, which encodes the blue light photoreceptor cryptochrome 2 (CRY2), was down-regulated in the tapx sapx double mutant plants (SRO2). RCD1 and the SRO family members have crucial functions in the regulation of stress-responsive genes in Arabidopsis [START_REF] Ahlfors | Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses[END_REF][START_REF] Borsani | Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in arabidopsis[END_REF]. Notably, despite of the absence of both tAPX and sAPX, no compensatory up-regulation of genes related to the antioxidant system was observed. The high-light treatment further strengthened the down-regulation of several genes in tapx sapx double mutant plants as compared to wild type plants (Supplemental Fig. 1, Supplemental Table 3). Interestingly, the down-regulated genes included a dynamin superfamily GTPase, At1g03160, which has been assigned crucial functions in the biogenesis of chloroplasts [37] (Supplemental Table 3, Sheet 2). white and green sectors in leaves [START_REF] Yu | Variegation mutants and mechanisms of chloroplast biogenesis[END_REF]. In accordance with the crucial importance for tAPX and sAPX in the maintenance of optimal chloroplast development (Fig. 2a), the high-light treated tapx sapx double mutant plants showed down-regulation of a recently identified dynamin superfamily GTPase, which is required for proper biogenesis of chloroplasts (Supplemental Table 3.; [37]). Moreover, both sAPX and tAPX have been shown to accumulate in high amounts in pea etioplasts in darkness, and then to decrease during the progress of the greening process upon illumination [START_REF] Kanervo | Expression of protein complexes and individual proteins upon transition of etioplasts to chloroplasts in pea (pisum sativum)[END_REF]. Likewise, the genes encoding for chloroplast PRXs and APXs are highly expressed during the first days after germination in Arabidopsis [41].

Another intriguing phenomenon was that the chloroplasts located in the guard cells or along the emerging veins in the cotyledons of the sapx single mutants and the tapx sapx double mutants were less sensitive to oxidative stress than the mesophyll cell chloroplasts (Figs. 2a andb). Similarly, high-light illumination of a tapx sapx vtc2 triple mutant deficient in chloroplast ascorbate peroxidases and also of ascorbate resulted in bleaching of interveinal leaf tissues [START_REF] Giacomelli | Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low[END_REF]. Indeed, there is increasing evidence indicating that the different cell types possess specialized roles in the regulation of leaf development in response to environmental cues [START_REF] Yu | Variegation mutants and mechanisms of chloroplast biogenesis[END_REF][START_REF] Mullineaux | Spatial dependence for hydrogen peroxide-directed signaling in light-stressed plants[END_REF]. The bundle sheath cell chloroplasts are the primary site for accumulation of hydrogen peroxide during highlight-illumination of leaves [START_REF] Fryer | Control of ascorbate peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of arabidopsis leaves[END_REF](see also Supplemental Fig. 1), and several genes related to plant antioxidant network seem to be preferentially expressed in the veinal regions in Arabidopsis leaves. These gene products include the cytoplasmic ascorbate peroxidase 2 [START_REF] Fryer | Control of ascorbate peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of arabidopsis leaves[END_REF], a microRNA (miR398) that targets Cu/Zn superoxide dismutase [START_REF] Sunkar | Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance[END_REF] and a chloroplastic glutaredoxin [START_REF] Cheng | AtGRXcp, an arabidopsis chloroplastic glutaredoxin, is critical for protection against protein oxidative damage[END_REF]. It is conceivable that cell-type-specific expression of antioxidant genes also contributed to the differential oxidative stress tolerance of various cell types in the tapx sapx double mutant and wild type plants (Figs. 2a and2b; [START_REF] Giacomelli | Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low[END_REF]). were suddenly exposed to high light intensity (Fig. 2d). An abrupt increase in excitation energy lead to an enhanced photoinhibition of PSII in tapx sapx double mutant plants, which could be recorded as a pronounced decline in the photochemical efficiency of PSII (Fig. 2d). This corroborates with results obtained for transgenic tobacco plants over-expressing tAPX, which were capable of maintaining photosynthetic activity under a combination of short-term high light and low temperature stress [START_REF] Yabuta | Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress[END_REF]. These data indicate that chloroplastic APX activity is crucial for photoprotection, yet the mechanism of protection is not fully understood.

On one hand, the water-water cycle may attenuate over-excitation of the photosynthetic electron transfer chain by utilizing molecular oxygen as an alternative electron acceptor, and thereby diminishing ROS-induced photodamage to PSII [START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF]. On the other hand, detoxification of H 2 O 2 is linked to the maintenance of Calvin cycle [START_REF] Yabuta | Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress[END_REF] and translational activity [START_REF] Nishiyama | A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II[END_REF] in chloroplasts. Indeed, in cyanobacterial cells, the accumulation of H 2 O 2 led to enhanced photoinhibition due to impairment of the PSII repair cycle, while the rate of photodamage to PSII was not affected [START_REF] Nishiyama | Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery[END_REF]. This was also the case for the tapx sapx double mutant plants, which were as sensitive as the wild type plants to light-induced inactivation of PSII when the repair of damaged PSII complexes during illumination was inhibited by the chloroplast translation inhibitor lincomycin (Fig. 2d). This observation strongly suggests that the enhanced susceptibility of the tapx sapx double mutant to photoinhibition resulted from translational perturbations thereby hampering the repair cycle of PSII.

The tapx sapx double mutant plants also showed slightly altered regulation of LHCII protein phosphorylation upon a shift of plants to higher light intensity (Fig. 4d). In wild type plants, LHCII protein phosphorylation becomes strongly down-regulated under [29]). Such inhibition can, however, be restored by hydrogen peroxide, which serves as an oxidant reactivates the LHCII kinase [START_REF] Martinsuo | Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes[END_REF]. Thus, it is likely that minor accumulation of H 2 O 2 in the chloroplasts of the tapx sapx double mutant plants upon a shift to higher light intensity maintained the LHCII kinase at more active state as compared to the wild type plants (Fig. 4d; [START_REF] Rintamäki | Cooperative regulation of light-harvesting complex II phosphorylation via the Biochemical Journal Immediate Publication[END_REF]).

It is noticeable that both the chloroplast translation and the activity of the LHCII kinase have been connected to retrograde signaling, which initiates in the plastids to regulate gene expression in the nucleus [START_REF] Nott | PLASTID-TO-NUCLEUS RETROGRADE SIGNALING[END_REF]. It was therefore of interest to explore whether the minor differences in the tapx sapx double mutant as compared to wild type plants in the level of H 2 O 2 are reflected in the regulation of nuclear genes (Supplemental Fig. 1).

Apparently due to only a low level of H 2 O 2 production, no preferential up-regulation of genes generally regarded as hallmarks for oxidative stress responses [49,[START_REF] Ma | Integration of arabidopsis thaliana stressrelated transcript profiles, promoter structures, and cell-specific expression[END_REF] was observed for the double mutant despite of the absence of both tAPX and sAPX in chloroplasts (Supplemental Table 3.). Rather, as deduced from the altered regulation of LHCII protein phosphorylation (Fig. 4d), the moderate accumulation of H 2 O 2 in the tapx sapx double mutant plants modulates the thiol-redox-state of chloroplasts, which in turn is likely to modulate the retrograde signaling pathways from chloroplasts to the nucleus.

Functional redundancy between thylakoid-bound and stromal APXs

Among four-week-old plants, predominantly only the tapx sapx double mutant plants showed both the acceleration of high-light-induced decline in PSII activity (Fig. 2d) and the enhanced methyl viologen-induced cell death (Fig. 2c), whereas the single mutants tapx and sapx showed less severe stress symptoms. Thus, it is clear that tAPX and sAPX are functionally redundant with respect to photo-oxidative stress in mature Arabidopsis leaves. Besides scavenging by the two types of ascorbate peroxidases, especially if ascorbic acid is present at suboptimal levels [START_REF] Mano | Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: Its relevance to monodehydroascorbate radical detected with in vivo ESR[END_REF]52].

Plants deficient in chloroplast APXs acquire compensatory mechanisms to tolerate long-term stress

Considering the distinct susceptibility of the tapx sapx double mutant plants to shortterm photo-oxidative stress, it was intriguing that no stress symptoms appeared under any of the long-term stress conditions studied (Fig. 3a). Recently, Giacomelli et al. [START_REF] Giacomelli | Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low[END_REF] showed that this is not due to compensatory accumulation of the low-molecular-weight ROS scavengers ascorbate, glutathione or alpha-tocopherol.

Our study revealed that the deficiency of tAPX and sAPX in the high-light-stressacclimated tapx sapx double mutant plants resulted in accumulation of chloroplast 2-cys peroxiredoxin (Fig. 4b), which exhibits over-lapping activities in ROS scavenging with the chloroplast APXs [START_REF] Dietz | The function of peroxiredoxins in plant organelle redox metabolism[END_REF]. Conversely, transgenic Arabidopsis plants with reduced amounts of 2-cys PRX were shown to up-regulate genes encoding for chloroplast APXs [START_REF] Baier | Antisense suppression of 2-cysteine peroxiredoxin in arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism[END_REF]. Furthermore, a distinctly enhanced accumulation of cytoplasmic APX occurred in all high-light acclimated plants, the wild type, tapx, sapx and tapx sapx double mutant plants (Fig. 4). These results suggest that upon prolonged high light exposure of plants, The expression of genes encoding for different chloroplast PRXs has been shown to respond specifically to various environmental conditions and developmental stages, presumably due to complex regulation via overlapping signaling pathways [START_REF] Baier | Antisense suppression of 2-cysteine peroxiredoxin in arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism[END_REF][START_REF] Horling | Divergent light-, ascorbate-, and oxidative stressdependent regulation of expression of the peroxiredoxin gene family in arabidopsis[END_REF].

These regulatory pathways seem to involve highly specific chloroplast retrograde signaling systems, as the accumulation of 2-cys peroxiredoxin became specifically enhanced in high-light-stress-acclimated tapx sapx double mutant plants (Fig. 4b). The retrograde signaling mechanisms require functional chloroplasts with intact metabolic pathways [START_REF] Koussevitzky | Signals from chloroplasts converge to regulate nuclear gene expression[END_REF], and are capable of sensing even slight alterations in the chloroplast redox state to induce specific responses and scavenging systems according to the prevailing environmental challenges. Altogether, stress tolerance in plants is acquired

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T APXs are required for accurate fine-tuning of chloroplast signaling pathways upon slight fluctuations in the accumulation of H 2 O 2 in chloroplasts. On the contrary, in the course of long-term acclimation to various stress conditions, the chloroplast APXs can be compensated by other components of the chloroplast antioxidative system.

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T half-strength MS medium with 1,5 µM methyl viologen (Sigma-Aldrich) and following greening at 130 µmol photons m -2 s -1 /20ºC at 8-hour light period for seven days.

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T Laser scanning confocal microscopy Confocal microscope images of seven-day-old seedlings were obtained with an inverted confocal laser scanning microscope (Zeiss LSM510 META; http://www.zeiss.com) using a 20×/0.50 water objective. Chlorophyll fluorescence was excited at 488 nm with an argon diode laser, and detected with a 650-710 nm passing emission filter. Maximal projections of the sequential confocal images were created with the Zeiss LSM Image Browser Version 3,5,0,376 available through http://www.zeiss.com.

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T primer (Oligomer, Finland) and the reverse transcriptase SuperScript III (Invitrogen).

  Photo-oxidative stress in germinating tapx, sapx and tapx sapx double mutant plants

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T

For

  photo-oxidative stress than the mesophyll cell chloroplasts (Figs2a and b).

  Biochemical Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T weeks stress treatment almost the same as the control value of the wild type plants (Supplemental table 1.).

  examined the levels of chloroplastic and cytoplasmic APXs and chloroplast PRXs in the differentially stress-acclimated plants by Western-blotting. Again, plants were grown for two weeks under the control growth conditions of 130 µmol photons m -2 s -1 /20ºC, and thereafter shifted for two weeks to moderately high light intensity (600 µmol photons m -2 s - Biochemical Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T 1 /20ºC), to high light stress (1300 µmol photons m -2 s -1 /28ºC) or to low temperature (130 µmol photons m -2 s -1 /10ºC). Then comparisons in the levels of H 2 O 2 -detoxifying enzymes between the wild type, the tapx and sapx single mutants and the tapx sapx double mutant plants were made. These experiments also confirmed the absence of tAPX and/or sAPX in the respective mutants under all the experimental conditions explored (Figs. 4a

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T

BiochemicalFig. 1

 1 Fig.1). Exposure of the rosettes to a 30-minute high-light illumination period under

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T (Supplemental table 3). CRY2 was recently shown to function as a positive regulator of flowering in the vascular tissue of Arabidopsis leaves [33]. Recently, tapx single mutant plants were reported to induce flowering a few days earlier than wild type plants [23]. Under our control growth conditions, both tapx and sapx single mutant plants also start to flower one or two days earlier than the wild type plants, whereas the tapx sapx double mutant plants show floral induction simultaneously with the wild type plants (data not shown). The short 30-minute high-light illumination period induced further adjustments in the pattern of gene expression in the tapx sapx plants as compared to wild type plants (Supplemental Fig. 1, Supplemental

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION Chloroplast ascorbate peroxidases have been assigned a distinct role in scavenging reactive oxygen species produced by photosynthetic electron transfer reactions in chloroplasts[START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF][START_REF] Danna | Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity[END_REF][START_REF] Yabuta | Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress[END_REF][START_REF] Lee | Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses[END_REF]. In the water-water cycle, electron transfer from PSI to molecular oxygen leads to generation of superoxide on the stromal side of the thylakoid membrane. Superoxide is highly reactive, and becomes rapidly dismutated to hydrogen peroxide (H 2 O 2 ) by a thylakoid-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD)[START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons[END_REF][START_REF] Foyer | Hydrogen peroxide-and glutathione-associated mechanisms of acclimatory stress tolerance and signalling[END_REF]. H 2 O 2 in turn becomes enzymatically reduced to water by two distinct groups of water-water cycle enzymes, ascorbate peroxidases (APXs) and peroxiredoxins (PRXs), both of which comprise several isoforms[START_REF] Shigeoka | Regulation and function of ascorbate peroxidase isoenzymes[END_REF]. Here we attempted to dissect the roles of the thylakoid-bound (tAPX) and the stromal (sAPX) ascorbate peroxidases in the antioxidative network of Arabidopsis thaliana as influenced by the different developmental stage and by the exposure of plants to stress conditions.Stromal APX has a key role in oxidative stress tolerance during early greening phasesArabidopsis seedlings lacking the stromal APX (sAPX) were sensitive to mild photooxidative stress, and produced pale green seedlings when germinated in the presence of methyl viologen in low concentration (Fig2a). The single mutants lacking the thylakoid-bound APX (tAPX), on the contrary, developed like wild type plants (Fig.2a). Nevertheless, the tapx sapx double mutant exhibited even more severe failure in the greening process than that observed for the sapx single mutant (Fig2a), demonstrating the contribution of both tAPX and sAPX to the antioxidant defence in young seedlings. Thus, chloroplast APXs, and particularly the stromal APX, are of primary importance for photoprotection during the initial greening process. Light absorption by the newly accumulating chlorophylls in developing chloroplasts poses a threat of photo-oxidative damage, especially if the metabolic balance between the absorption and utilization of light energy has not yet been attained. Such photo-oxidative disturbances during chloroplast development have been suggested also to cause the formation of white plastids in the class of variegated mutants, which are characterized by alteration of Biochemical Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T Chloroplast APXs are crucial for photoprotection and signaling The tapx and sapx single mutants and the tapx sapx double mutant plants exhibited no visible stress symptoms after long-term growth under high light (Fig 3; [42]) or under l o w t e m p e r a t u r e ( F i g . 3 ) . H o w e v e r , t h e i m p o r t a n c e o f c h l o r o p l a s t A P X s i n t h e maintenance of photosynthetic activity in mature leaves became evident when plants

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T high light intensities upon inhibition of the LHCII kinase by a thiol reductant (Fig. 4d;

H 2 O

 2 2 can be detoxified in chloroplasts also by peroxiredoxins, glutaredoxins, and glutathione peroxidases as well as by the low-molecular-weight antioxidants ascorbate and glutathione, both of which are present in millimolar concentrations in chloroplasts[START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. Indeed, the stress-acclimation and cross-tolerance of plants involve multiple H 2 O 2 Biochemical Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T detoxification systems that efficiently prevent oxidative damage by controlling the level of H 2 O 2 in chloroplasts. Accordingly, the differentially stress-acclimated wild type plants as well as the tapx and sapx single mutants had acquired tolerance against sudden attack by methyl viologen-induced accumulation of ROS (Figs 3b and c) (see below), whereas the stress-acclimated tapx sapx double mutant plants exhibited only partial tolerance. This indicates that the capacity of the double mutant to efficiently scavenge the ROS induced by a sudden MV-treatment was exceeded, contrary to that of the wild type plants and single mutants (Fig 3c

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T both the chloroplastic peroxiredoxins and the cytoplasmic APXs have a crucial role in the removal of H 2 O 2 that originates from photosynthetic processes [54].

  via a complex network of antioxidant activities, signaling components and cellular regulators that ultimately determine the metabolic status of the cells [23, 54]. In this e n t i r e t y , t h e c h l o r o p l a s t A P X s s e e m t o b e i n d i s p e n s a b l e u p o n t h e o n s e t o f environmental stresses, whereas various compensatory systems may take over during the long-term acclimation to contrasting environmental cues. Biochemical Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 .

 1 Figure 1. Characteristics of tapx and sapx single mutant and tapx sapx double mutant lines. (a) Molecular structures and T-DNA insertion sites of TAPX (At1g77490; SALK_027804) and SAPX (At4g08390; SALK_083737) genes. T-DNA insertion sites are indicated with triangles, and black and gray boxes represent exons and untranslated regions, respectively. (b) Immunoblots with Arabidopsis anti-APX antibody. The left panel demonstrates the linear range of the Arabidopsis APX antibody. The middle panel depicts the APX content of isolated thylakoids and the soluble leaf extract. The right-hand panel confirms the absence of tAPX, sAPX or both in total leaf extracts isolated from tapx, sapx and the tapx sapx double mutant. DM, tapx sapx double mutant; WT, wild type; tAPX, thylakoid bound APX; sAPX, stromal APX; cAPX, cytoplasmic APX.

Figure 2 .

 2 Figure 2. Sensitivity of wild type, tapx, sapx and tapx sapx double mutant plants to short-term photo-oxidative stress. (a) Susceptibility of seedlings to photo-oxidative stress during germination. Seedlings were germinated in the presence of 1,5 µM methyl viologen under 130 µmol photons m - 2 s -1 /20ºC. The confocal images are projections of chlorophyll fluorescence from 30 µm stacks of sequential images starting from the abaxial side of the cotyledons. The scale bars correspond to 100 µm. (b) Confocal images of chlorophyll fluorescence demonstrating cell-type-specific effects of photo-oxidative stress on chloroplast development in germinating tapx sapx double mutant plants. Seedlings were germinated in the presence of 1,5 µM methyl viologen under 130 µmol photons m -2 s -1 /20ºC. The confocal images are projections of 50 µm stacks of sequential images starting from the abaxial side of the cotyledons. The scale bars correspond to 100 µm. (c) Methyl viologen -induced photo-oxidative stress in four-week old plants. Plants were sprayed with 50 µM methyl viologen and illuminated under 130 or 1000 µmol photons m -2 s -1 /20ºC for six hours prior to measurement of ion leakage from the leaves. Control samples were sprayed with water and illuminated under normal growth

Figure 3 .

 3 Figure 3. Stress tolerance of wild type, tapx, sapx and tapx sapx double mutant plants upon long-term acclimation to adverse environmental conditions. (a) Phenotypes after two-week acclimation period under high light, high light stress or low temperature. Plants were grown for two weeks under normal growth conditions, and thereafter shifted for another two weeks to various light and temperature conditions as indicated in the figure. (b) Methyl viologen-induced photo-oxidative stress in differentially stress-acclimated plants. Plants were sprayed with 50 µM methyl viologen and illuminated under 130 µmol photons m -2 s -1 /20ºC for 24 hours prior to measurement of ion leakage from the leaves. The values are means ± SD of four independent experiments. ***, P<0,005 in Student' s T-test. (c) Photograph depicting the tolerance of high-light-acclimated wild type plants and sensitivity of tapx sapx double mutant plants to methyl viologen-induced cell death.

Figure 4 .Biochemical

 4 Figure 4. Modulations in the steady-state levels of H 2 O 2 -detoxifying enzymes, photosynthetic proteins and LHCII protein phosphorylation under contrasting environmental cues. (a) Immunoblots depicting the levels of H 2 O 2 -detoxifying enzymes in the tapx and sapx single mutants and wild type plants. (b) Immunoblots depicting the levels of H 2 O 2 -detoxifying enzymes in the tapx sapx double mutants (DM) and wild type plants.

  Biochemical Journal Immediate Publication. Published on 28 Feb 2008 as manuscript BJ20080030 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080030 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 3

 3

; Sheet 2). The up-regulated genes included At5g55910, a chloroplastic member of the thus far poorly characterized Arabidopsis AGC family protein kinases (Supplemental Table

3

; sheet 2). Members of the AGC family protein kinases mediate signals from key secondary messengers, including cAMP, cGMP and calcium, and act as crucial regulators of basic cellular metabolism

[34]

. Another intriguing observation in the tapx sapx double mutant plants was a highlight-induced up-regulation of At1g23550, which encodes for SIMILAR TO RCD1 2

  ). It is conceivable that the thylakoid-bound and stromal APXs operate in concert during the initial detoxification of H 2 O 2 , and are capable of poising the H 2 O 2 level unless the production of H 2 O 2 suddenly exceeds the threshold level (Fig. 2). This interpretation is consistent with the observation that chloroplastic APXs become rapidly inactivated by high concentrations of H 2 O 2 ,

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

We thank Minna Lintala, Mika Keränen and Colin Ruprecht for excellent technical assistance, and Dr. Paula Mulo for critically reading the manuscript. Jouko Sandholm at the Cell Imaging Core of the Turku Center for Biotechnology of the University of Turku and Åbo Academi University is acknowledged for excellent technical support in laser scanning confocal microscopy. The Salk Institute Genomic Analysis Laboratory, funded by the National Science Foundation, is acknowledged for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. This work was financially supported by the Academy of Finland (Projects number 107039 and 204521).