Plant, Cell and Environment (2015) 38, 2 6 41–2651

doi: 10.1111/pce.12575

Original Article

Subunits B′γ and B′ζ of protein phosphatase 2A regulate photo-oxidative stress responses and growth in Arabidopsis thaliana Grzegorz Konert1, Moona Rahikainen1, Andrea Trotta1, Guido Durian1, Jarkko Salojärvi2, Sergey Khorobrykh1, Esa Tyystjärvi1 & Saijaliisa Kangasjärvi1 1

Molecular Plant Biology, Department of Biochemistry, University of Turku, FI-20014 Turku, Finland and 2Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland

ABSTRACT Plants survive periods of unfavourable conditions with the help of sensory mechanisms that respond to reactive oxygen species (ROS) as signalling molecules in different cellular compartments. We have previously demonstrated that protein phosphatase 2A (PP2A) impacts on organellar crosstalk and associated pathogenesis responses in Arabidopsis thaliana. This was evidenced by drastically enhanced pathogenesis responses and cell death in cat2 pp2a-b′γ double mutants, deficient in the main peroxisomal antioxidant enzyme CATALASE 2 and PP2A regulatory subunit B′γ (PP2A-B′γ). In the present paper, we explored the impacts of PP2A-B′γ and a highly similar regulatory subunit PP2A-B′ζ in growth regulation and light stress tolerance in Arabidopsis. PP2A-B′γ and PP2A-B′ζ display high promoter activities in rapidly growing tissues and are required for optimal growth under favourable conditions. Upon acclimation to a combination of high light, elevated temperature and reduced availability of water, however, pp2a-b′γζ double mutants grow similarly to the wild type and show enhanced tolerance against photo-oxidative stress. We conclude that by controlling ROS homeostasis and signalling, PP2A-B′γ and PP2A-B′ζ may direct acclimation strategies upon environmental perturbations, hence acting as important determinants of defence responses and light acclimation in plants. Key-words: growth regulation; light acclimation; PP2A; reactive oxygen species (ROS); signalling.

INTRODUCTION Plants cope with alternating stress factors by integrating environmental signals to optimize growth and acclimation under unfavourable conditions. In a number of stress responses, vital signalling functions have been attributed to transient formation of reactive oxygen species (ROS) in intracellular and extracellular compartments of plant cells (Bechtold et al. 2008; Sierla et al. 2013). Hence, delicate responses to ROS as signalling molecules have presumably provided significant evolutionary competitiveness to plants. Under high light stress, which in natural growth habitats is Correspondence: S. Kangasjärvi. e-mail: [email protected] © 2015 John Wiley & Sons Ltd

commonly intensified by elevated temperature and limited availability of water, the photosynthetic machinery forms a primary site of ROS production (Hideg et al. 1998; Karpinski et al. 2012). Strong sunlight may pose a threat of photoinhibition and oxidative damage to the photosystems (Suorsa et al. 2012; Tyystjärvi 2013). On the other hand, numerous studies have highlighted important functional relationships between ROS signalling, reprogramming of nuclear gene expression and attainment of stress resistance in photosynthetic organisms (Karpinski et al. 1999, 2003, 2012; Pogson et al. 2008; Kangasjärvi et al. 2014). Plants have evolved overlapping protective systems that control the formation of ROS by sensing and responding to excess irradiance levels. In the light harvesting (LHCII) antenna of photosystem II (PSII), excess excitation energy can be dissipated as heat via non-photochemical quenching (NPQ) that is triggered by low pH of the thylakoid lumen and assisted by the xanthophyll pigments zeaxanthin and antheraxanthin which are enzymatically converted from violaxanthin (Niyogi et al. 2005; Müller et al. 2001; Li et al. 2004; Jahns & Holzwarth 2012). In addition, plants avoid over-accumulation of ROS by coordinating the levels of antioxidative pigments, such as carotenoids and tocopherols (Mittler et al. 2004). Among antioxidant enzymes, ASCORBATE PEROXIDASE 2 (APX2) is transcriptionally responsive to reduction of the photosynthetic electron transfer chain, abscisic acid (ABA) and metabolic signals, and has become a model for studies on photo-oxidative stress responses in plants (Karpinski et al. 1999; Fryer et al. 2003; Rossel et al. 2007; Galvez-Valdivieso et al. 2009; Estavillo et al. 2011). Besides metabolic reprogramming, long-term acclimation to high light induces developmental responses, such as compact morphology, thickening of the cuticular layer and increased abundance of trichomes (Anderson & Aro 1994). On the other hand, mechanisms promoting growth and preventing unnecessary stress reactions under favourable conditions are also beneficial for the plant and form a major contributor to plant productivity. Protein phosphatase 2A (PP2A) is a key signalling component that modulates stress signalling and regulates cell division and developmental programmes in eukaryotic organisms (Ahn et al. 2011; Di Rubbo et al. 2011; Tang et al. 2011; Heidari et al. 2013; Uhrig et al. 2013). PP2A is predominantly trimeric 2641

2642 G. Konert et al. and consists of a catalytic subunit C, a scaffold subunit A and a highly variable regulatory subunit B, which in Arabidopsis thaliana (hereafter Arabidopsis) are encoded by 5, 3 and 17 subunits, respectively. How the distinct PP2A regulatory B subunits cooperate to modulate the stress resistance and growth of plants has remained poorly understood. We have shown that a cytosolic PP2A regulatory subunit B′γ controls day length-dependent responses to intracellular oxidative stress (Trotta et al. 2011; Li et al. 2014). Analysis of cat2 pp2a-b′γ double mutants, deficient in PP2A-B′γ and the main peroxisomal antioxidant enzyme CATALASE 2, demonstrated that PP2A-B′γ is required for the control of salicylic acid signalling and cell death upon formation of photorespiratory ROS signals (Li et al. 2014). Analysis of pp2a-b′γ single mutants, in turn, revealed that the constitutive immune responses are conditional and become evident under low humidity and moderate light intensity (Trotta et al. 2011; Li et al. 2014; Rasool et al. 2014). This finding is in contrast to the behaviour of many lesion mimic mutants that show enhanced cell death when organellar ROS production becomes triggered by high light (Lorrain et al. 2003). In this paper, we have explored the impacts of PP2A-B′γ (GAMMA) and a highly similar regulatory subunit PP2AB′ζ (ZETA) in growth regulation and light stress tolerance in Arabidopsis. We show that promoter activities of PP2A-B′γ and PP2A-B′ζ overlap in rapidly growing areas of developing tissues. Double mutants deficient in PP2A-B′γ and PP2AB′ζ show growth reduction under normal growth conditions. Under abiotic stress, however, the growth penalty levels off and pp2a-b′γζ mutants grow similarly to wild type. Transcript profiling of plants acclimated to high light and elevated temperature indicates that PP2A-B′γ and PP2A-B′ζ affect mitotic cell cycle and photo-oxidative stress responses. The latter was evidenced by increased transcript abundance for APX2, HEAT SHOCK FACTOR A3 (HSFA3) and a cluster of co-regulated heat shock proteins involved in plant responses to abiotic stress. Associated with this, pp2a-b′γζ double mutants acquire enhanced tolerance against longterm abiotic stress induced by a combination of high light, elevated temperature and reduced availability of water. We conclude that the subunit composition of PP2A is a key determinant of ROS signalling responses, and is therefore essential in growth regulation and stress signalling in plants.

MATERIALS AND METHODS Plant material Homozygote knock-down pp2a-b′γ (SALK_039172 for At4g15415), knockout pp2a-b′ζ1-1 and pp2a-b′ζ1-2 (SALK_ 107944C and SALK_150586 for At3g21650, respectively), a pp2a-b′γ pp2a-b′γζ1-1 double mutant (constructed by crossing the SALK_039172 and SALK_107944C single mutants) and a pp2a-b′γ line complemented by 35S-driven expression of the PP2A-B′γ gene were generated and genotyped before (Trotta et al. 2011; Rasool et al. 2014). Unless otherwise stated, 4-week-old wild-type A. thaliana ecotype Columbia and mutant plants grown under 8 h/16 h day/night photoperiod at 50% relative humidity were used for the experi-

ments. For growth under favourable conditions, plants were grown under 130 μmol photons m−2s−1/23 °C. For analysis of long-term stress responses, plants were first grown under favourable conditions for 2 weeks and thereafter shifted to high light and elevated temperature (800 μmol photons m−2s−1/28 °C) for 2 weeks. For well-watered growth conditions, the soil was kept moist by daily irrigation. Analysis under water-limited conditions was performed by limiting the irrigation to once per week. Analysis of rosette size was conducted by using ImageJ (http://fiji.sc/). Water loss from detached rosettes was measured according to Leung et al. (1997).

Analysis of gene expression Rosettes of wild-type pp2a-b′γ, pp2a-b′ζ1-1 and pp2a-b′γζ (Agilent Technologies, Santa Clara, USA) double mutant plants acclimated to 800 μmol photons m−2s−1 at 28 °C were collected 4 h after the onset of light period. RNA from four biological replicates was isolated using Agilent Plant RNA isolation mini kit and 200 ng of total RNA was amplified and Cy-3 labelled using Agilent one-color Low Input Quick Amp Labeling kit (Product No. 5190-2331) and processed with the RNA Spike in kit (Product No. 5188-5282). RNA/cRNA quality control was performed using Agilent 2100 bioanalyzer RNA 6000 Nano kit (Product No. 5067-1511). Up to 1.65 μg Cy-3 labelled samples were hybridized to Agilent Arabidopsis (V4) Gene Expression Microarrays, 4x44K (Design ID 021169) according to the manufacturer’s instructions, and finally scanned with Agilent Technologies Scanner G2565CA with a profile AgilentHD _GX_1Color. Numeric data were produced with Agilent Feature Extraction program, version 10.7.3. Gene expression data were analysed using the R statistical software version 3.1.1 (http://www.r-project.org). Firstly, the average of processed signal of probes for each gene was computed.Then, a linear model with genotype, treatment and their interaction as fixed effects was estimated. The P-values from the linear model were then subjected to false discovery rate correction using the qvalues package (Storey 2002). Genes with absolute log2 fold change >1 and FDR-corrected P-values < 0.05 were considered significant. Model comparisons and their P-values were estimated using the multcomp package in R (Hothorn et al. 2008). Gene ontology (GO) annotations were derived from TAIR (http:// www.arabidopsis.org), and used as gene sets in Gene Set Analysis, carried out in R using GSA package (Efron & Tibshirani 2007). Gene sets with FDR-corrected P-values < 0.05 were considered significantly enriched.

β-glucuronidase (GUS) staining Wild-type Arabidopsis plants were transformed with a promoter:uidA fusion construct containing a 2811 bp region of genomic DNA upstream of the translational start codon of the PP2A-B′ζ coding sequence in pGREENII0029 (Rozhon et al. 2010). After sequencing of the resulting constructs, wildtype plants were transformed by floral dipping. The transgenic line expressing proPP2A-B′γ:uidA was available from our previous study (Trotta et al. 2011). GUS staining was performed according to Weigel & Glazebrook (2006).

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

PP2A B′γ and B′ζ mediate photo-oxidative stress responses and growth

Chlorophyll fluorescence measurements The photoinhibition state of PSII in intact leaves was recorded as the ratio of variable to maximal fluorescence [Fv/Fm, where Fv is the difference between maximal fluorescence (Fm) and initial fluorescence (Fo)], measured with a Hansatech PEA fluorometer (Hansatech Instruments, UK) after a 30 min dark incubation. Before NPQ measurements, plants were incubated in darkness for 2 h. A fully expanded leaf of each individual plant was detached in semi-darkness and immediately analysed with PAM-101 fluorometer (Heinz Walz GmbH, Effeltrich, Germany). Actinic light (PPFD 260 or 1500 μmol m−2s−1, as indicated) was switched on after measurement of F0, and saturating flashes (1 s, PPFD 4000 m-2s-1) were fired after 1 and 140 s of actinic illumination to measure Fm and Fm′, respectively. NPQ was calculated as (Fm − Fm′)/Fm′.

Measurement of leaf pigments and α-tocopherol Pigments (chlorophylls a and b, neoxanthin, violaxanthin, lutein and beta-carotene) and α-tocopherol were analysed as described in Lehtimäki et al. (2011). Pigments were extracted from 5-mm-diameter leaf discs with 300 μL of pure methanol pre-cooled to −80 °C. The extracts were centrifuged twice at 18 600 g for 15 min and filtrated with 0.2 μm syringe filters. Photosynthetic pigments were thereafter separated by highperformance liquid chromatography (HPLC) according to Gilmore & Yamamoto (1991) with a reverse phase C18 column (LiChroCART 125-4; Hewlett Packard, Germany), series 1100 HPLC device with diode array and fluorescence detector (Agilent Technologies, Palo Alto, CA, USA). Buffer A consisted of acetonitrile–methanol–Tris–HCl buffer 0.1 m, pH 8.0 (72:8:3, v/v) and buffer B consisted of methanol–hexane (4:1, v/v). A constant flow rate of 0.5 L min−1 was used and the temperature of the column was maintained at about 25° C. The program started with an isocratic run with buffer A for 4 min followed by a linear gradient for 15 min from 0% buffer B to 100% buffer B. The isocratic run of buffer B lasted for 26 min. The column was re-equilibrated between samples for a minimum of 10 min with buffer A. Pigments were detected by absorbance at 440 nm, and α-tocopherol by fluorescence (lex = 295 nm, lem = 340 nm). Standards were supplied by DHI Lab Products (Denmark).

Statistical analyses The numerical data were subjected to statistical analysis using Student’s t-test, with statistical significance at the level of P < 0.05.

RESULTS Deficiencies in PP2A-B′γ and/or PP2A-B′ζ promote conditional light intensity and humidity-dependent phenotypes in Arabidopsis Knock-down pp2a-b′γ and pp2a-b′γζ double mutants show 21–40% reduction in the level of PP2A-B′γ mRNA as compared with wild-type plants (Trotta et al. 2011; Rasool et al. 2014), while PP2A-B′ζ transcripts in knockout pp2a-b′ζ1-1

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and pp2a-b′ζ1-2 single mutants or the pp2a-b′γζ double mutants could not be observed (Rasool et al. 2014). The pp2a-b′γ mutant displays a highly conditional phenotype with constitutive immune responses and age-dependent yellowing when grown under moderate light intensity and 50% relative humidity in a short day photoperiod (Trotta et al. 2011; Li et al. 2014). Here we first assessed how abrupt changes in humidity affect the phenotypic characteristics of wild type, pp2a-b′γ, pp2a-b′ζ1-1, pp2a-b′ζ1-2 and pp2a-b′γζ double mutants and the pp2a-b′γ line complemented by 35S-driven expression of the PP2A-B′γ gene. To minimize possible developmental effects caused by high humidity, the plants were first grown under 130 μmol photons m−2s−1 at 50% humidity for 2 weeks, and shifted to 85% humidity for 1 week. Thereafter, their visual appearance was followed upon lowering the relative humidity back to 50%. Within 3 d after the shift back to 50% relative humidity, pp2a-b′γ leaves showed symptoms of bleaching at the peripheral parts of the leaf blade, while wild type, pp2a-b′ζ1-1, pp2a-b′ζ1-2 and the pp2a-b′γζ double mutants as well as the pp2a-b′γ line complemented by 35S-driven expression of the PP2A-B′γ gene remained visually unaltered (Fig. 1a).Thus, the low humidityinduced bleaching of pp2a-b′γ leaves required the presence of PP2A-B′ζ, suggesting that the two regulatory subunits of PP2A are functionally connected with each other. The control plants grown at 50% relative humidity for 24 d were all visually healthy and did not develop any symptoms of premature yellowing at this time point (Fig. 1a). Next we applied different growth conditions to further examine the functional impacts of PP2A-B′γ and PP2A-B′ζ under 50% relative humidity. No drastic differences in water loss from excised rosettes of wild type, pp2a-b′γ, pp2a-b′ζ1-1 and pp2a-b′γζ double mutants were observed (Fig. 1b), suggesting that the yellowing pp2a-b′γ phenotype was unlikely to arise from imbalanced stomatal function. To assess growth regulation in pp2a mutants, plants were first grown under 130 μmol photons m−2s−1 for 4 weeks. These normal growth conditions revealed additive growth reduction in pp2a-b′γζ double mutant plants as compared with wild type, pp2a-b′γ, pp2a-b′ζ1-1 and pp2a-b′ζ1-2 single mutants (Fig. 1c). To study growth under abiotic stress conditions, plants were first grown under 130 μmol photons m−2s−1 for 2 weeks, and thereafter shifted to high light and elevated temperature (800 μmol photons m−2s−1/28 °C) for 2 weeks. To further harden the stress conditions, plants were first grown under growth light with normal irrigation for 2 weeks, and thereafter either kept in growth light or shifted to high light and elevated temperature with limited watering during the following 2 weeks. Under all these abiotic stress conditions, the wild type, pp2a-b′γ and pp2a-b′ζ produced significantly smaller rosettes than in control conditions and the differences to pp2a-b′γζ double mutant levelled off (Fig. 1c).

Gene expression profiling of high light-acclimated plants To gain insight into abiotic stress-induced signalling in the pp2a mutant backgrounds, we utilized Agilent Arabidopsis

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

2644 G. Konert et al. (a)

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Figure 1. Impacts of relative humidity, light intensity and irrigation on Arabidopsis thaliana wild type and pp2a mutants. (a) Representative photograph depicting the impact of humidity on wild type, the pp2a mutants and a pp2a-b′γ line complemented by 35S-driven expression of the PP2A-B′γ gene. The plants were first grown under 130 μmol photons m−2s−1 at 50% humidity for 2 weeks, and subsequently shifted to 85% humidity for 1 week. Thereafter, their visual appearance was recorded upon lowering the relative humidity back to 50%. The control plants were grown at 50% relative humidity for 24 d. (b) Water loss from excised rosettes of wild type and the pp2a mutants grown at 50% relative humidity. The loss of water was followed under growth light (130 μmol photons m−2s−1) by weighing excised rosettes at regular intervals and is expressed as a percentage of the value at time point 0. Data are means ± SD, n = 8. (c) Box plot representation of leaf area of wild type and the pp2a mutants after acclimation to different growth conditions. Plants were grown under growth light (GL control; 130 μmol photons m−2s−1/23 °C, daily irrigation) for 4 weeks, or shifted at 2 weeks of age to high light and elevated temperature (HL control, 800 μmol photons m−2s−1/28 °C, daily irrigation) for additional 2 weeks. Analysis under water-limited conditions was performed by limiting the irrigation to once per week (GL drought; 130 μmol photons m−2s−1/23 °C, limited irrigation and HL drought; 800 μmol photons m−2s−1/28 °C, limited irrigation). Analysis of rosette size was conducted by using ImageJ, n = 8. The bottom of the box indicates the 25th percentile, the line within the box marks the median (50th percentile), and the top of the box indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles; asterisk, t-test P < 0.05. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

PP2A B′γ and B′ζ mediate photo-oxidative stress responses and growth Table 1. Abundance of selected transcripts in pp2a-b′γ and pp2a-b′ζ1-1 single mutants and pp2a-b′γζ double mutant relative to wild-type plants grown in high light conditions (800 μmol photons m−2s−1/28 °C) log2 FC AGI

Annotation

pp2a-b′γ

pp2a-b′ζ

pp2a-b′γζ

AT3G09640 AT5G03720 AT2G26150 AT1G32330 AT3G02990 AT3G46230 AT1G53540 AT5G59720 AT4G10250 AT4G27670 AT1G27730 AT5G59820 AT4G34410 AT5G04490 AT3G22370 AT3G22360 AT3G27620 AT1G32350 AT5G64210 AT5G10140 AT5G04240

APX2 HSFA3 HSFA2 HSFA1D HSFA1E HSP17.4 HSP17.6C-C1 HSP18.2 HSP22 HSP21 ZAT10 ZAT12 RRTF1 VTE5 AOX1a AOX1b AOX1c AOX1d AOX2 FLC ELF6

1.965 1.20 0.03 0.14 0.06 1.50 1.14 0.46 1.59 2.50 1.099 2.112 2.403 1.883 0.881 0.779 0.968 3.468 0.858 7.196 1.903

2.679 0.41 0.73 0.27 0.09 1.22 1.62 0.92 1.90 3.74 1.458 1.094 0.661 1.130 1.110 1.121 1.265 1.157 0.896 0.670 1.130

4.247 1.51 -0.72 0.32 0.03 1.73 2.05 1.80 3.24 5.68 1.129 1.649 3.992 2.287 0.974 1.010 1.179 2.392 0.853 8.342 2.214

Values are the means of four independent biological replicates. Statistically significant values with P < 0.05 are indicated in bold.

gene expression microarrays for analysis of pp2a-b′γ and pp2a-b′ζ1-1 single mutants and the pp2a-b′γζ double mutant acclimated to high light and elevated temperature (800 μmol photons m−2s−1/28 °C). In accordance with our previous analysis with custom-made spotted arrays (Trotta et al. 2011), the pp2a-b′γ single mutant acclimated to high light and elevated temperature did not show drastically elevated mRNA levels for salicylic acid-related defence genes compared with high light-acclimated wild-type plants (Supporting Information Table S1). A number of genes encoding F-box proteins and miscellaneous signalling proteins in turn were up-regulated in pp2a-b′γ (Supporting Information Table S1). High light-acclimated pp2a-b′ζ1-1 mutants did not show drastic transcriptional adjustments when compared with wild-type plants (Supporting Information Table S1), although slightly reduced transcript abundance of the key flowering repressor FLOWERING LOCUS C (FLC) in line with the early flowering phenotype (Rasool et al. 2014) was observed (Table 1). The late flowering pp2a-b′γ mutant (Heidari et al. 2013), in contrast, showed increased abundance of FLC mRNA (Table 1). The pp2a-b′γζ double mutant in turn displayed differential mRNA abundance for 162 genes that were not differentially expressed in pp2a-b′γ or pp2a-b′ζ1-1 single mutants (Supporting Information Table S1). The pp2a-b′γζ double mutant displayed elevated transcript levels for APX2, HSFA3 and the heat shock proteins HSP18.2, HSP21 and HSP22, indicating enhanced responses

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elicited by the combination of high light and elevated temperature (Table 1). Querying publicly available datasets at ATTED-II (http://atted.jp) indicated that these stressinducible genes form a transcriptionally co-regulated cluster (Supporting Information Fig. S1). Furthermore, analysis of GO categories enriched in the mutant transcript profiles revealed increased activity of processes related to mitotic cell cycle regulation, response to ionizing radiation and DNA modification in pp2a-b′γζ double mutant compared with wild-type plants (Fig. 2). Differential expression of components related to cell expansion, such as expansions, xyloglucan endotransglucosylase/hydrolases or cell wall peroxidases (Cosgrove 2005), was not observed in any of the pp2a mutants studied (Supporting Information Table S1). Taken together, these transcriptomic adjustments suggested that PP2A-B′γ and PP2A-B′ζ modulate developmental processes and photo-oxidative stress responses in plants.

Promoters of PP2A-B′γ and PP2A-B′ζ are active in developing tissues Next we took advantage of promoter-driven GUS activity to analyse the spatiotemporal patterns of PP2A-B′γ and PP2AB′ζ gene expression in Arabidopsis plants. Comparative analysis of developing seedlings demonstrated that promoters of both PP2A-B′γ and PP2A-B′ζ were active in the root tips of germinating 2-day-old seedlings, cotyledons and roots of 6-day-old plants, and newly emerging leaves and lateral roots of developing seedlings and floral stalks and flowers of 6-week-old plants (Fig. 3). In 4-week-old rosettes grown under moderate light, promoters of both PP2A-B′γ and PP2A-B′ζ were active in the centre of the rosettes and displayed partially overlapping profiles in expanding leaves. During leaf maturation, the activity of the PP2A-B′ζ promoter shifted towards leaf periphery and, in contrast to the promoter of PP2A-B′γ, was barely detectable in mature fully expanded leaves. Similar patterns of GUS staining were also observed after a 2 d shift to high light (800 μmol photons m−2s−1/28 °C; Fig. 4). After a 4 week growth period in high light and elevated temperature (800 μmol photons m−2s−1, 28 °C), the activities of PP2A-B′γ and PP2A-B′ζ were also strong in newly emerging rosette leaves (Fig. 4). These results suggested that PP2A-B′γ and PP2A-B′ζ may have overlapping functions in promoting growth in developing Arabidopsis plants.

pp2a-b′γζ double mutant acquires enhanced tolerance to long-term light stress Previously, we reported that wild type and all the pp2a-b′γ mutants showed gradual PSII photoinhibition of similar extent, measured as Fv/Fm after a 30 min dark incubation, when plants were first grown under normal growth light for 4 weeks and then suddenly shifted to high light for 4 h (Trotta et al. 2011; Rasool et al. 2014). Here, to evaluate the ability of pp2a mutants to cope with long-term light-induced oxidative stress, we first measured NPQ and the contents of antioxidative pigments embedded in LHCII. As shown in

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

2646 G. Konert et al. Interphase of mitotic cell cycle GO :0051329 Response to ionizing radiation GO :0010212 Regulation of mitotic cell cycle GO :0007346 Proteasome accessory complex GO :0022624 Proteasome regulatory particle GO :0005838 Regulation of cell cycle process GO :0010564 DNA modification GO :0006304 Cell cycle process GO :0022402 DNA alkylation GO :0006305 DNA methylation GO :0006306 M phase GO :0000279 Nuclear division GO :0000280 Stomatal complex morphogenesis GO :0010103 Cell cycle phase GO :0022403 M phase of mitotic cell cycle GO :0000087 Mitotic cell cycle GO :0000278 Structural constituent of cytoskeleton GO :0005200 Protein phosphatase type 2A complex GO :0000159 Protein phosphatase type 2A regulator activity GO :0008601 Phosphatase regulator activity GO :0019208 Protein phosphatase regulator activity GO :0019888 Sesquiterpenoid biosynthetic process GO :0016106 Sesquiterpene biosynthetic process GO :0051762 Abscisic acid metabolic process GO :0009687 Apocarotenoid metabolic process GO :0043288 Sesquiterpenoid metabolic process GO :0006714 Sesquiterpene metabolic process GO :0051761 Protein serine/threonine phosphatase complex GO :0008287 Starch biosynthetic process GO :0019252 Dioxygenase activity GO :0051213 Terpene metabolic process GO :0042214 Oxidoreductase activity GO :0016701 Regulation of secondary metabolic process GO :0043455 Oxidoreductase activity GO :0016706 Lactose metabolic process GO :0005988 Lactose catabolic process GO :0005990 Starch metabolic process GO :0005982 Cytoskeletal part GO :0044430

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–2.0

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Figure 2. Comparison of gene ontology (GO) categories enriched in the transcript profile of pp2a-b′γζ double mutant compared with wild type. GSA, gene set analysis.

Table 2, wild type, pp2a-′γ, pp2a-b′ζ and pp2a-b′γζ double mutants did not show differences in the extent of NPQ when assessed upon growth under normal growth conditions or upon a 2 week acclimation period to high light and elevated temperature. Likewise, in plants grown under 130 μmol photons m−2s−1, the levels of xanthophyll cycle pigments, β-carotene and α-tocopherol, relative to chlorophyll a, did not significantly differ between wild type and any of the pp2a mutants (Table 3). After a 2 week acclimation period to high light and elevated temperature, however, the levels of β-carotene and antheraxanthin had significantly increased (P < 0.05) in high light-acclimated and pp2a-b′ζ1-1 and pp2ab′ζ1-2 single mutant and pp2a-b′γζ double mutant leaves, while the levels of violaxanthin, zeaxanthin or α-tocopherol did not significantly differ from wild type (Table 3).The pp2ab′γ mutant, in contrast, did not significantly differ from wild type (Table 3). Thus, with respect to the accumulation of protective pigments, pp2a-b′γζ double mutant resembled the pp2a-′ζ1-1 and pp2a-b′ζ1-2 single mutant plants. Finally, we compared the extent of photo-oxidative stress tolerance in wild type, pp2a-b′γ, pp2a-b′ζ1-1, pp2a-b′ζ1-2 and pp2a-b′γζ double mutants grown and acclimated to stressful conditions. Plants were first grown under moderate light for 2 weeks, and thereafter shifted to high light and elevated temperature (800 μmol photons m−2s−1/28 °C) for 2 weeks. These experimental conditions suggested a tendency where the Fv/Fm value, indicative of the extent of PSII photoinhibition, remained higher in pp2a-b′γ and pp2a-′γζ double mutants

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Figure 3. GUS analysis demonstrating promoter activities of PP2A-B′γ and PP2A-B′ζ in developing seedlings of Arabidopsis thaliana under normal growth light (130 μmol photons m−2s−1). The uppermost panel shows GUS activity in root tips of germinating 2-day-old seedlings and cotyledons and roots of 6-day-old plants. The middle panel demonstrates GUS staining in newly emerging leaves and lateral roots of developing seedlings. The lowest panel shows GUS staining in floral stalks and flowers of 6-week-old plants.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

PP2A B′γ and B′ζ mediate photo-oxidative stress responses and growth

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Hight light

Growth light

Natural habitats are characterized by periods of favourable and unfavourable conditions that commonly alternate during plant growth and development, and maximal agricultural crop yields are therefore rarely reached due to losses caused by biotic and abiotic stress agents. To survive in the hazardous environment, plants have evolved sophisticated mechanisms that sense the type and severity of the stress and trigger defence reactions accordingly, but the signalling networks determining whether stress-exposed tissues activate cell death or invest on acclimation remain poorly understood (Mullineaux & Baker 2010). It is clear, however, that negative regulators that prevent inappropriate stress reactions have vital functions in these interactions. Previous works demonstrated that the specific PP2A regulatory subunit B′γ is required to control intracellular oxidative stress responses and associated pathogenesis reactions in Arabidopsis (Li et al. 2014). Here we applied differentially severe light stress conditions to assess the roles of PP2A-B′γ and the highly similar PP2A-B′ζ in abiotic stress responses. We show that PP2A-B′γ and PP2A-B′ζ are expressed in rapidly growing tissues and required for optimal growth under favourable conditions (Figs 1, 3 & 4). Upon acclimation to light stress, however, deficiencies in PP2A-B′γ and PP2A-B′ζ lead to induction of photoprotective mechanisms and enhanced tolerance against abiotic stress (Figs 1c & 5, Tables 1 & 2). Thus, the subunit composition of PP2A is critical in determining the stress tolerance and growth in plants.

PP2A-B′γ and PP2A-′ζ are required for proper growth under optimal conditions

Figure 4. GUS analysis demonstrating promoter activities of PP2A-B′γ and PP2A-B′ζ in 4-week-old rosettes of Arabidopsis thaliana under growth light (130 μmol photons m−2s−1) and high light (800 μmol photons m−2s−1/28 °C). The high light shift depicts GUS analysis upon a 2 d shift from growth light to high light (800 μmol photons m−2s−1/28 °C).

than in wild type and pp2a-b′ζ single mutant plants (Fig. 5). To further harden the above described stress conditions, plants were additionally stressed by limited watering during the growth period. The combination of high light, elevated temperature and limited watering reinforced the higher Fv/Fm value for pp2a-b′γζ mutant plants (Fig. 5). Evidently, deficiencies in PP2A-B′γ and PP2A-B′ζ promote abiotic stress tolerance (Fig. 5) but do not cause additional growth penalties upon plant acclimation to harsh environmental challenges (Fig. 1).

NPQ GL NPQ HL

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Cessation of growth is one of the key physiological responses that allow plants to conserve nutrients and allocate energy for protective mechanisms. Recently, the concept of ‘stressinduced morphogenic response’ (SIMR) has emerged, stating that stress-exposed plants are generally smaller and display more compact morphology than unstressed plants (Potters et al. 2009). Cellular ROS homeostasis is one of the key mechanisms that regulate growth in plants (Potters et al. 2009), and the regulatory actions of ROS in SIMR have recently been exemplified by an intimate cross-talk between ROS and auxin signalling (Blomster et al. 2011; Brosché et al. 2014). However, even though pp2a-b′γζ double mutant plants display smaller and more compact rosettes than wild-type plants, we did not observe enrichment of growth hormonerelated signatures in the transcript profiles of pp2a-b′γ, pp2ab′ζ or pp2a-b′γζ double mutant plants (Figs 1 & 2, Supporting

WT Col

pp2a-b′γ

pp2a-b′ζ1-1

pp2a-b′ζ1-2

pp2a-b′γζ

0.74 ± 0.03 2.31 ± 0.09

0.69 ± 0.06 2.68 ± 0.30

0.56 ± 0.05 2.33 ± 0.26

0.68 ± 0.09 2.37 ± 0.15

0.93 ± 0.13 2.61 ± 0.22

NPQ was measured at 260 μmol photons m−2s−1 for growth light plants and 1500 μmol photons m−2s−1 for high light plants, equivalent to two times the light intensity of the respective growth conditions. Data are means ± SD, n = 3. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

Table 2. Non-photochemical quenching in wild type, pp2a-b′γ, pp2a-b′ζ1-1 and pp2a-b′ζ1-2 single mutants and pp2a-b′γζ double mutant grown under growth light (130 μmol photons m−2s−1/23 °C) or high light conditions (750 μmol photons m−2s−1/28 °C)

2648 G. Konert et al. Table 3. Pigment and α-tocopherol contents in wild type, pp2a-b′γ, pp2a-b′ζ1-1 and pp2a-b’ζ1-2 single mutants and pp2a-b′γζ double mutant grown under growth light (130 μmol photons m−2s−1/23 °C) or high light conditions (800 μmol photons m−2s−1/28 °C)

GL

HL

Neoxanthin Violaxanthin Antheraxanthin Lutein Zeaxanthin Chlorophyll b α-Tocopherol Pheophytin a β-carotene Neoxanthin Violaxanthin Antheraxanthin Lutein Zeaxanthin Chlorophyll b α-Tocopherol Pheophytin a β-carotene

Wild type

pp2a-b′γ

pp2a-b′ζ1-1

pp2a-b′ζ1-2

pp2a-b′γζ

0.03447 ± 3.68E-04 0.14578 ± 1.10E-02 0.00220 ± 9.62E-05 0.25266 ± 6.50E-03 0.00639 ± 3.28E-03 0.31401 ± 4.47E-03 0.00794 ± 4.15E-04 0.00851 ± 2.35E-04 0.05209 ± 7.30E-04 0.03598 ± 9.90E-04 0.17747 ± 2.03E-02 0.00453 ± 1.63E-03 0.26704 ± 1.31E-02 0.01015 ± 1.65E-03 0.31484 ± 3.11E-03 0.03633 ± 1.13E-02 0.00713 ± 8.17E-04 0.05196 ± 2.96E-04

0.03444 ± 7.92E-04 0.12962 ± 5.87E-03 0.00231 ± 4.99E-04 0.24784 ± 2.85E-03 0.00577 ± 1.19E-03 0.31455 ± 6.10E-03 0.00761 ± 4.48E-04 0.00790 ± 1.40E-04 0.05154 ± 1.03E-03 0.03621 ± 7.69E-04 0.18956 ± 2.62E-02 0.00682 ± 2.95E-03 0.28198 ± 1.41E-02 0.01332 ± 2.81E-03 0.30812 ± 2.82E-03 0.04463 ± 1.87E-02 0.00712 ± 7.26E-04 0.05258 ± 1.62E-03

0.03419 ± 6.54E-04 0.14179 ± 1.60E-02 0.00203 ± 2.96E-04 0.25024 ± 6.76E-03 0.00677 ± 6.93E-04 0.30879 ± 4.26E-03 0.00817 ± 4.44E-04 0.00764 ± 3.14E-04 0.05124 ± 9.56E-04 0.03866 ± 9.55E-04 0.18749 ± 2.36E-02 0.00770 ± 1.03E-03 0.28529 ± 9.21E-03 0.00832 ± 2.11E-03 0.30490 ± 6.30E-03 0.04456 ± 1.60E-02 0.00707 ± 5.28E-04 0.05479 ± 1.46E-03

0.03537 ± 6.22E-04 0.13751 ± 1.04E-02 0.00251 ± 2.73E-04 0.25435 ± 3.76E-03 0.00465 ± 6.47E-04 0.31893 ± 6.13E-03 0.00754 ± 6.04E-04 0.00779 ± 1.66E-04 0.05117 ± 6.49E-04 0.03825 ± 8.09E-04 0.16523 ± 1.67E-02 0.00894 ± 2.59E-03 0.28450 ± 1.31E-02 0.01089 ± 3.79E-03 0.30109 ± 6.26E-03 0.03060 ± 4.26E-03 0.00755 ± 5.60E-04 0.05568 ± 1.17E-03

0.03552 ± 2.96E-04 0.14085 ± 1.92E-02 0.00253 ± 2.20E-04 0.25286 ± 1.10E-02 0.00569 ± 9.61E-04 0.31454 ± 5.91E-03 0.00835 ± 9.81E-04 0.00789 ± 3.10E-04 0.05233 ± 8.96E-04 0.03942 ± 1.28E-03 0.15263 ± 1.16E-02 0.00890 ± 1.69E-03 0.27526 ± 1.01E-02 0.01080 ± 2.68E-03 0.29566 ± 1.65E-02 0.02767 ± 8.57E-03 0.00717 ± 6.96E-04 0.05475 ± 2.09E-03

Data are means ± SD, n = 5. Statistically significant differences with P < 0.05 between wild type and mutant lines are indicated in bold. Values are normalized to chlorophyll a and presented as pigment (ng)/chlorophyll a (ng).

Information Table S1; Trotta et al. 2011). Instead, the transcript profile of pp2a-b′γζ double mutants indicated enrichment of GO categories related to the regulation of the mitotic cell cycle (Fig. 2). Leaf development is a complex process coordinated through multiple genetic pathways, which regulate the proliferation, expansion and viability of cells according to inputs from internal and external cues (Skirycz et al. 2011; Owell & Lenhard 2012; Considine & Foyer 2014). The pp2a-b′γζ double mutant plants showed a statistically significant additive growth reduction when grown under favourable conditions (130 μmol photons m−2s−1, 23 °C), and the trend, although no longer statistically significant, was still observable under moderately unfavourable (2 weeks under 800 μmol photons m−2s−1/28 °C) conditions (Fig. 1c). Such growth defect was likely not a consequence of declined photosynthetic activity, since neither the light response curves nor CO2 response curves of photosynthetic carbon assimilation differed between wild type and the pp2a mutants grown under moderate or high light intensities (Rasool et al. 2014). Furthermore, when pp2a-b′γζ double mutants were grown under more severe abiotic stress, induced by a combination of high light, elevated temperature and reduced availability of water, the growth penalty completely levelled off (Fig. 1). Thus, PP2A-B′γ and PP2A-B′ζ are required to forward growth under optimal conditions but do not significantly modulate plant growth and development under harsh environmental stress.

PP2A-B′γ and PP2A-B′ζ impact the decision between ROS-induced cell death and acclimation PP2A-B′γ controls foliar ROS levels and cell death under moderate light intensities but does not significantly contrib-

ute to the redox status of the main antioxidants ascorbate and glutathione (Trotta et al. 2011; Li et al. 2014). By now, several works have indisputably demonstrated that cell death is under genetic control and is not a simple consequence of ROS-induced oxidative damage (Mullineaux & Baker 2010; Brosché et al. 2014; Li et al. 2014). The control of ROS signalling responses and the decision between cell death and acclimation become determined through opposing actions of pro-cell death and anti-cell death signalling mechanisms (reviewed by Mullineaux & Baker 2010). What determines the threshold, however, is not well understood. It is therefore of interest that intracellular oxidative stress in cat2 pp2a-b′γ double mutants triggers salicylic acid signalling and cell death, while the double mutant combination pp2a-b′γζ leads to enhanced activation of photo-oxidative stress responses and acclimation to light stress (Li et al. 2014; Table 3; Fig. 5). PP2A-B′γ and PP2A-B′ζ may therefore represent genetic components that impact the threshold between cell death and acclimation under various environmental cues. By utilizing pharmacological approaches and selected reaction monitoring (SRM) mass spectrometry, we demonstrated that PP2A-B′γ impacts on ROS homeostasis by negatively regulating the abundance of the alternative oxidases AOX1A and AOX1D in leaf mitochondria (Konert et al. 2015). Shuttling of redox-active intermediates between the organelles and the cytoplasm affects the probability of ROS accumulation in different cellular compartments, and even highly localized antioxidant activities may have far-reaching acclamatory effects in plants (Hanning & Heldt 1993; Igamberdiev & Gardeström 2003; Vandenabeele et al. 2004). Here we show that high light-acclimated pp2a-b′γζ double mutants contain elevated amounts of β-carotene and antheraxanthin (Table 3), which may quench excess excitation energy and mediate antioxidant functions in chloroplasts.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

PP2A B′γ and B′ζ mediate photo-oxidative stress responses and growth

GL control

HL control

GL drought

HL drought

0.9

*

Fv/Fm

0.8

0.7

0.6

0.5

Figure 5. Box plot representation of long-term light stress tolerance as depicted by photoinhibition state of PSII in wild type, pp2a mutants and a pp2a-b′γ line complemented by 35S-driven expression of the PP2A-B′γ gene (pp2a-b′γ 35S:PP2A-B′γ). Plants were grown under growth light (GL control, 130 μmol photons m−2s−1/23 °C, daily irrigation) for 4 weeks, or shifted at 2 weeks of age to high light and elevated temperature (HL control, 800 μmol photons m−2s−1/28 °C, daily irrigation) for additional 2 weeks. Analysis under water-limited conditions was performed by limiting the irrigation to once per week (GL drought, 130 μmol photons m−2s−1/23 °C, limited irrigation; and HL drought, 800 μmol photons m−2s−1/28 °C, limited irrigation). Photoinhibition of PSII in intact leaves was recorded as the ratio of variable to maximal fluorescence [Fv/Fm, where Fv is the difference between maximal fluorescence (Fm) and initial fluorescence (Fo)], measured with a Hansatech PEA fluorometer after a 30 min dark incubation. n = 24. In water-limiting conditions, Fv/Fmax was measured 4 d after the last watering. The bottom of the box indicates the 25th percentile, a line within the box marks the median (50th percentile), and the top of the box indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. Dots are outliers; asterisk, t-test P < 0.05.

High light-acclimated pp2a-b′γζ double mutants also display increased transcript abundance for APX2, HSFA3 and a subset of co-regulated heat shock proteins, including HSP18.2, HSP21 and HSP22, which are transcriptional targets of HSFs (Table 1). APX2 is specifically expressed in young developing areas as well as in bundle sheath cells of Arabidopsis rosettes, where it becomes induced in response to a multitude of signals, including those arising from high light, heat and drought (Karpinski et al. 1999; Rossel et al. 2007). Associated with this, APX2 gene expression correlates with ABA signalling from vascular parenchyma, reduction of

2649

the plastoquinone pool in the photosynthetic electron transfer chain, H2O2 generated in chloroplasts or by plasma membrane NADPH oxidases and metabolic signals including accumulation of phosphoadenosine 5′-phosphate (PAP) (Karpinski et al. 1999; Rossel et al. 2002, 2007; Fryer et al. 2003; Ball et al. 2004; Estavillo et al. 2011). Despite increasing interest towards HSFs and their potential in increased cross-tolerance and crop yield, their specific roles, genetic redundancy and the genetic components behind their regulation remain to be uncovered (Nover et al. 2001; Scharf et al. 2012). Wu et al. (2012) resolved a transcriptional cascade involving consecutive actions of JUNGBRUNNEN1 (JUB1; ANAC042), the dehydrationresponsive element binding protein 2A (DREB2A) and HSFA3, which were discussed in terms of negative regulation of oxidative stress and leaf ageing. In contrast to HSFA3, neither JUB1 nor DREB2A was significantly up-regulated in pp2a-b′γζ (Table 1; Supporting Information Table S1). A cluster of genes encoding HSPs, including HSP18.2 and HSP21 that are transcriptionally co-expressed with HSFA3, was instead observed in the transcript profile of pp2a-b′γζ double mutants (Table 1; Supporting Information Fig. S1). Thus, rather than controlling unspecific broad range responses to environmental stress, PP2A-B′γ and PP2A-B′ζ appear to specifically modulate a subset of stress-inducible regulatory components in plants. Overexpression of HSFA2 and HSFA3 has been implicated in APX2 regulation even in the absence of stress (Nishizawa et al. 2006; Ogawa et al. 2007; Yoshida et al. 2008). Recently, Jung et al. (2013) suggested that the transcriptional regulation of APX2 involves functional hierarchy among HSFA1d, HSFA2 and HSFA3. Exposure of single and higher order mutants for hsfa1d, hsfa2 and hsfa3 to abrupt light stress, heat (40 °C) or the photosynthetic electron transfer inhibitor DBMIB revealed that HSFA3 modulates APX2 expression under high light and elevated temperature, but does not mediate signals arising from DBMIB-induced reduction of the plastoquinone pool (Jung et al. 2013). A good candidate for mediating the HSFA3-dependent regulation of APX2 is H2O2, which is generated in both heat- and light-induced stresses (Volkov et al. 2006). This is well in line with the identification of APX2 and the co-expressed heat shock protein genes HSP18.2 and HSP21 as thermomemory expressed genes (Shahnejat-Bushehri et al. 2012). Cross-communication is characteristic to cellular signalling in plants and often crosses the boundaries between biotic and abiotic stress responses. By controlling intracellular ROS homeostasis and signalling, PP2A-B′γ and PP2A-B′ζ may direct the choice between cell death, growth and acclimation upon environmental perturbations. Altogether, our findings highlight the PP2A subunit composition as a key determinant of defence responses and light acclimation in plants.

ACKNOWLEDGMENTS This work was supported by the Academy of Finland projects 263772, 218157, 259888, 130595 and 271832, the Finnish Graduate Program in Plant Biology and the University of

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

2650 G. Konert et al. Turku Graduate School. We thank Jouni Kujala and the Finnish Microarray Center for excellent technical assistance. The Salk Institute Genomic Analysis Laboratory is acknowledged for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. The authors have no conflict of interest to declare.

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Received 7 October 2014; received in revised form 18 May 2015; accepted for publication 19 May 2015

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Co-expression network depicting heat shockrelated genes with elevated transcript levels in pp2a-b′γζ double mutant compared with wild type. APX2, ASCORBATE PEROXIDASE 2; HSFA3, HEAT SHOCK FACTOR A3; HSP21, HEAT SHOCK PROTEIN21; At4g10250 is also annotated as HEAT SHOCK PROTEIN22; At5g59720, HEAT SHOCK PROTEIN 18.2. The network was drawn with the Networkdrawer tool in Atted II (http://atted.jp/ top_draw.shtml#NetworkDrawer). Table S1. Adjustments in gene expression in pp2a mutant plants after a 2 week acclimation period under 800 μmol photons m−2s−1 at 28 °C.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 2641–2651

Subunits B'γ and B'ζ of protein phosphatase 2A regulate photo-oxidative stress responses and growth in Arabidopsis thaliana.

Plants survive periods of unfavourable conditions with the help of sensory mechanisms that respond to reactive oxygen species (ROS) as signalling mole...
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