Published online: August 24, 2017

Article

Redox regulation of plant stem cell fate Jian Zeng1, Zhicheng Dong2, Haijun Wu1, Zhaoxia Tian1 & Zhong Zhao1,*

Abstract Despite the importance of stem cells in plant and animal development, the common mechanisms of stem cell maintenance in both systems have remained elusive. Recently, the importance of hydrogen peroxide (H2O2) signaling in priming stem cell differentiation has been extensively studied in animals. Here, we show that different forms of reactive oxygen species (ROS) have antagonistic roles in plant stem cell regulation, which were established by distinct spatiotemporal patterns of ROS-metabolizing enzymes. The superoxide anion (O
 2 ) is markedly enriched in stem cells to activate WUSCHEL and maintain stemness, whereas H2O2 is more abundant in the differentiating peripheral zone to promote stem cell differentiation. Moreover, H2O2 negatively regulates O
 2 biosynthesis in stem cells, and increasing H2O2 levels or scavenging O
 2 leads to the termination of stem cells. Our results provide a mechanistic framework for ROS-mediated control of plant stem cell fate and demonstrate that the balance between O
 2 and H2O2 is key to stem cell maintenance and differentiation. Keywords plant stem cell; reactive oxygen species; superoxide anion; superoxide dismutase; WUSCHEL Subject Categories Plant Biology; Stem Cells DOI 10.15252/embj.201695955 | Received 25 October 2016 | Revised 12 July 2017 | Accepted 28 July 2017

Introduction The growth and development of multicellular organisms depend on the maintenance and constant differentiation of stem cells. In plants, all of the aboveground organs except cotyledons originate from the shoot stem cells that reside in the central zone (CZ) of the shoot apical meristem (SAM), while the differentiated transient amplifying cells in the peripheral zone (PZ) immediately surround the central zone (Weigel & Jurgens, 2002; Stahl & Simon, 2005; Aichinger et al, 2012). Multiple signaling molecules and transcription factors are known to tightly control stem cell fate in Arabidopsis thaliana. The homeodomain transcription factor WUSCHEL (WUS), which is expressed in niche cells in the organizing center (OC), moves to the CZ to maintain plant stem cell fate (Yadav et al, 2011; Daum et al, 2014). The downregulation or loss of WUS functions causes a reduction or termination of plant stem cells (Mayer et al, 1998; Yadav

et al, 2011; Daum et al, 2014). However, stem cells express the secreted peptide CLAVATA3 (CLV3), which serves as a genetic marker for plant stem cells, to negatively regulate the expression of WUS. This negative feedback loop between WUS and CLV3 is required to define plant stem cell fate and is tightly integrated with more widespread signaling molecules (Mayer et al, 1998; Fletcher et al, 1999; Brand et al, 2000; Schoof et al, 2000). In all living organisms, reactive oxygen species (ROS), including the superoxide anion (O
 2 ), hydrogen peroxide (H2O2), and the hydroxyl radical (˙OH), are the natural by-products of aerobic metabolism. O
 2 , which is the precursor for most other ROS, can be catalyzed into H2O2 by superoxide dismutases (SODs) and further reduced to the hydroxyl radical or water by peroxidases. Reactive oxygen species are well-known stress signaling molecules in both plants and animals (Finkel & Holbrook, 2000; Swanson & Gilroy, 2010), and can be increased dramatically by environmental stress and disease. Over the past decade, as one of the ROS species, H2O2 has been discovered as a key signaling molecule in animal stem cell regulation. Although the functional significance of H2O2-mediated priming of differentiation in various animal stem cell populations has been well proved (Owusu-Ansah & Banerjee, 2009; Kanda et al, 2011; Le Belle et al, 2011; Tormos et al, 2011; Malinska et al, 2012; Hamanaka et al, 2013; Ludin et al, 2014; Mantel et al, 2015), several recent studies have suggested that a low level of H2O2 signaling is required for the self-renewal of hematopoietic stem cells (Jang & Sharkis, 2007), mouse and human airway basal stem cells (Paul et al, 2014), and spermatogonial stem cells (Morimoto et al, 2013), as well as the pluripotency induction of mouse fibroblasts (Zhou et al, 2016). However, the biological significance of low H2O2 in stem cells and whether H2O2 exhibits the same functions in plant stem cell regulation remain unanswered. In plant, ROS has been seen as important signaling molecules that regulate plant development and growth (Tsukagoshi et al, 2010; Considine & Foyer, 2014; Schmidt & Schippers, 2015; Schippers et al, 2016). The UPBEAT1 (UPB1) transcription factor-mediated balance between H2O2 and O
 2 in the root influences the transition from cell proliferation to cell expansion and differentiation (Tsukagoshi et al, 2010). The mutation of an ATP-dependent mitochondrial protease, AtFTSH4, causes internal oxidative stress to accumulate in the SAM at a high temperature of 30°C and affects the morphology of the mitochondria and functions of the SAM (Dolzblasz et al, 2016). Until recently, most studies in stem cell biology have mainly focused on H2O2-mediated signaling pathways. However, whether the superoxide is involved in stem cell regulation,

1 School of Life Sciences, University of Science and Technology of China, Hefei, China 2 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China *Corresponding author. Tel: +86 551 63600640; Fax: +86 551 63600640; E-mail: [email protected]

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and the biological significance of establishing tissue-specific ROS balance in the cell fate specification, still needs to be determined.

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Results Superoxide accumulation is essential for stem cell maintenance To study the functions of ROS in plant stem cell fate regulation, we first examined the O
 2 distribution in the SAM using nitroblue tetrazolium (NBT), which is specific for O
 2 staining (Bielski et al, 1980). Amazingly, O
 2 showed the strongest signals in the CZ that harbored stem cells in both the reproductive (Fig 1A; Appendix Fig S1) and vegetative stages (Appendix Fig S2A–D). When stem cells were over-proliferated in the clv3 mutant, O
 2 was also increased accordingly in the enlarged stem cell region (Fig 1B; Appendix Fig S2E). We further confirmed the O
 2 distribution in the SAM using the fluorescent dye dihydroethidium (DHE) (Owusu-Ansah et al, 2008) in both the longitudinal and transverse sections (Fig 1E and I). Consistent with this finding, we observed a higher content of O
 2 in the clv3 mutants by quantification, indicating that O
 2 was highly accumulated in the plant stem cells (Appendix Fig S2F). To explore the biological functions of O
 2 accumulation in stem cell regulation, we reduced the O
 2 levels in the stem cells by treating plants with n-propyl gallate (PG) (Zhang & Kirkham, 1996) or N,N0 dimethylthiourea (DMTU) (Di Marco et al, 2008), two scavengers of free radicals, to eliminate O
 2 (Fig 1M; Appendix Fig S3). Seven days after germination (DAG) on MS media supplemented with different concentrations of PG or DMTU, the ability of the seedlings to generate the first pair of true leaves was drastically compromised (Fig 1C and D). The seedlings treated with lower concentrations of PG or DMTU showed reduced SAM sizes (Appendix Fig S2G–J). However, high concentration-treated plants did not generate any true leaves (Fig 1F–H), and shoot apical meristems and plant stem cell marker CLV3 gene expression were not observed (Fig 1J–L), suggesting that stem cells were terminated by removing O
 2 . NADH dehydrogenase of Mitochondrial Complex I in the respiratory chain and NADPH oxidase in the plasma membrane are two primary sources of O
 2 in living cells (Malinska et al, 2012; Hamanaka et al, 2013). To test the biological functions of endogenous O
 2 in plant stem cell regulation, we examined the ndufs4 and ndufv1 mutants of the NADH dehydrogenase subunits (Andreyev et al, 2005; Kuhn et al, 2015; Wu et al, 2015) and atrbohD/F mutants of the NADPH oxidase subunits (Torres et al, 2002). All the ndufs4, ndufv1, and atrbohD/F mutant plants showed quite similar defects that the generation of true leaves was delayed at the early seedling stage (Fig 2A–D). By in situ hybridization, we found that all three mutants of ndufs4, ndufv1, and atrbohD/F had less CLV3 expression (Fig 2E–H) and smaller SAMs (Fig 2V). After flowering, the ndufs4, ndufv1, and atrbohD/F mutants showed fewer floral buds than the wild-type plants, indicating functional defects in SAM regulations in the mutants (Fig 2I–L). To test whether the O
 2 contents in the stem cells was affected by the mutations in NADH dehydrogenase and NADPH oxidase, we examined them using the fluorescent dye DHE and observed remarkably low levels of O
 2 in the mutants (Fig 2M–P and U). In addition, more importantly, the CLV3-expressing regions (Fig 2Q–T) and SAM sizes were greatly reduced in all mutants (Fig 2V), suggesting that there were fewer stem cell populations in the mutant plants.

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Figure 1. Superoxide is accumulated in stem cells. A, B NBT staining of the WT and clv3 mutant shows that O
 2 is highly accumulated in the stem cells. Scale bars, 50 lm. NBT, nitroblue tetrazolium. C, D The percentages of plants with the first pair of true leaves after 7 days after germination (DAG) on media with different PG (C) or DMTU contents (D). More than 200 plants were counted for each treatment. Mean  SD. ***P < 0.001, Student’s t-test. PG, n-propyl gallate; DMTU, N,N0 dimethylthiourea; SD, standard deviation. E The wild-type inflorescence with dihydroethidium (DHE) staining shows that O
 2 is highly accumulated in stem cells using longitudinal sections. Scale bar, 50 lm. DHE, dihydroethidium. F–H Seven DAG of wild-type seedlings on mock medium (F), 0.5 mM PG medium (G), and 10 mM DMTU medium (H). Scale bars, 500 lm. I DHE staining of the wild-type inflorescence using transverse sections. Scale bar, 50 lm. J–L CLV3 expression patterns of the 7 DAG wild type on mock medium (J), 0.5 mM PG medium (K), and 10 mM DMTU medium (L). All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. M Diagram of ROS metabolism in plants. KI, potassium iodide; AT, amino-1,2,4-triazole.

To shed light on the mechanism underlying the ability of superoxide to regulate plant stem cell fate, we tested WUS expression in the superoxide-deficient mutants. We found that the transcripts of WUS were reduced in all three mutants of ndufs4, ndufv1, and atrbohD/F at the vegetative stage (Fig 3A–D and I). Consistent with this observation, WUS expression was also found to be downregulated at the reproductive stage (Fig 3E–H and J). To test the shortterm effects of O
 2 on stem cell regulation, we transiently reduced endogenous O
 2 levels using stem cell-specific ethanol-inducible promoter-derived artificial microRNA against NDUFS4 (CLV3::ALR, ALA::amiRNA-NDUFS4). After 12 h of 1% ethanol induction, the NDUFS4 transcript levels were significantly reduced in the transgenic plants (Fig 4D), along with the endogenous O
 2 levels (Fig 4A–C), while ethanol itself did not affect the O
 levels in the CLV3::ALR, 2

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Figure 2. Endogenous superoxide is essential for stem cell maintenance. A–D

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ALA::GUS plants (Appendix Fig S4). Together with the decrease in O
 2 in the stem cells, we observed a dramatic reduction of in both WUS and CLV3 expression in the transgenic plants (Fig 4E and F), suggesting that endogenous O
 2 accumulation is essential for stem cell maintenance. SODs are repressed in plant stem cells How did plants establish the high O
 2 content pattern in their stem cells? As a precursor of other ROS, O
 has been previously 2 suggested to be unstable and quickly catalyzed by SODs into H2O2, which is further reduced to water by peroxidase (Fig 1M). To explore the mechanism behind O
 2 accumulation in stem cells, we examined the expression patterns of all seven SODs in the Arabidopsis genome by in situ hybridization. Six SODs, including FSD2, CSD1, CSD3, MSD1, CSD2, and FSD1, were found to be strongly expressed in the differentiating PZ instead of the stem cells, which was complementary to the O
 2 distribution (Figs 5A–F and EV1). Only one SOD, FSD3, was not detected in the entire meristem. We also checked the expressions of all seven SODs in the published

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The top view of 7-day-old seedlings of the wild-type plant (A) and the ndufs4 (B), ndufv1 (C), and atrbohD/F (D) mutants. Scale bars, 500 lm. E–H CLV3 expression patterns of the 7-day-old wild-type plant (E) and the ndufs4 (F), ndufv1 (G), and atrbohD/F (H) mutants. All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. I–L Top view of inflorescence in the wild-type plant (I) and in the ndufs4 (J), ndufv1 (K), and atrbohD/F (L) superoxide-deficient mutants shows that there are fewer floral buds than the wild-type plant. Scale bars, 1 mm. M–P DHE staining of the wild-type (M), ndufs4 (N), ndufv1 (O), and atrbohD/F (P) inflorescences. Scale bars, 50 lm. Q–T CLV3 expression patterns in the wild-type plant (Q) and the ndufs4 (R), ndufv1(S), and atrbohD/F (T) mutants show reduced SAM sizes and CLV3 expression domains in the mutants at the reproductive stage. All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. U Quantification of DHE fluorescent intensity in (M–P) (WT, n = 10; ndufs4, n = 7; ndufv1 and atrbohD/F, n = 8;  SD). ***P < 0.001, Student’s t-test. V Quantification of the SAM size in the wild-type plant and the ndufs4, ndufv1, and atrbohD/F mutants in both the vegetative and reproductive stages. In the vegetative stage: WT and ndufv1, n = 12, ndufs4 and atrbohD/F, n = 11, in the reproductive stage: WT, n = 25, ndufv1, ndufs4, and atrbohD/F, n = 16. Upper and lower limits of box correspond to upper Q3 and lower Q1 quartiles, the horizontal line within the box represent the data medians, the single value (open square) plotted inside the box indicate the data means, error bars indicating minimum to maximum range, whiskers indicate data range within 1.5× of the interquartile range and outliers are not shown. ***P < 0.001, Student’s t-test.

expression data (Yadav et al, 2014). We found that most of SODs (five out of six) fit well with our in situ hybridization patterns (Appendix Fig S5). These data suggested that SODs were downregulated in plant stem cells. To address the biological functions of low SODs in the stem cells, we ectopically expressed one of the PZ-specific SODs, FSD2, into the stem cells under the CLV3 promoter. The resulting transgenic plants showed a wus-like phenotype and stopped growing after producing several leaves (Fig 5G and H). Likely, overexpression of FSD2 under the promoter of UBQ10 would also cause very similar phenotypes (Fig 5G and I). However, increasing the expression of FSD2 in the PZ and floral primordia in the MP::FSD2 transgenic plants could not cause the same phenotypes (Appendix Fig S6), suggesting that it is essential to repress the SODs expression in the stem cells. Consistent with the wus-like phenotypes, the typical SAM structure was not found in the CLV3::FSD2 and UBQ10::FSD2 transgenic plants, but rather a group of irregular and enlarged cells indicated that stem cells and meristematic cells were differentiated (Fig 5J–O). Moreover, we could not detect any expression of WUS (Fig 5J–L) and CLV3 (Fig 5M–O) in the transgenic plants, confirming that the stem cells were terminated. To further support this observation, we ectopically expressed three other SODs—CSD1, MSD1, CSD2—into the stem cells, and we obtained very similar results to those of the transgenic plants (Appendix Fig S7). Given that the SODs were repressed by miR398 (Sunkar et al, 2006, 2007), we found that miR398 was more strongly expressed in the stem cells (Appendix Fig S8). We came to the conclusion that downregulation of SODs was crucial for stem cell maintenance, and increasing the SOD levels to reduce the O
 2 content in stem cells terminated the plant stem cell fate process. While the presence of SODs in the PZ could on the one hand remove superoxide from the differentiating

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Figure 3. Endogenous superoxide positively regulates WUS expression. A–D WUS expression patterns in 7-day-old seedlings of the wild-type plant (A) and the ndufs4 (B), ndufv1 (C), and atrbohD/F (D) mutants. All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. E–H WUS expression patterns of the wild-type plant (E) and the ndufs4 (F), ndufv1 (G), and atrbohD/F (H) mutants at the reproductive stage. All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. I Quantitative measurements of the WUS and CLV3 expression levels in the wild-type plant and the ndufs4, ndufv1, and atrbohD/F mutants in the vegetative stages. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test. J Quantitative measurements of the WUS and CLV3 expression levels in the wild-type plant and the ndufs4, ndufv1, and atrbohD/F mutants in the reproductive stages. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, Student’s t-test.

cells, on the other hand it converts the O
 2 to H2O2, indicating such a possibility that H2O2 might be involved in stem cell differentiation. H2O2 promotes plant stem cell differentiation at the peripheral zone Previously, H2O2 was found to promote differentiation of various stem cell populations in mammals (Owusu-Ansah & Banerjee, 2009; Kanda et al, 2011; Le Belle et al, 2011; Tormos et al, 2011; Malinska et al, 2012; Hamanaka et al, 2013; Ludin et al, 2014; Mantel et al, 2015). Likewise, in Arabidopsis, H2O2 was observed to be more abundant in the PZ, which harbored differentiated amplifying cells, as determined by 3,30 -diaminobenzidine (DAB) staining (Fig 6A; Appendix Fig S9). Using the fluorescent dye 20 ,70 -dichlorofluorescein (DCF) (Owusu-Ansah et al, 2008), we further confirmed that there was less H2O2 in the stem cells in both the longitudinal and transverse sections (Fig 6B and C). To test whether H2O2 was involved in stem cell differentiation in plants, we reduced endogenous H2O2 by increasing the peroxidase levels in the peroxidase repressor mutant upb1-1. UPB1 was previously identified as a transcription factor that directly repressed a set

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Figure 4. A transient decrease in the superoxide level affects stem cell regulation. A, B DHE staining in the CLV3::ALR, ALA::amiRNA-NDUFS4 transgenic plants with (B) and without (A) ethanol induction. Scale bars, 50 lm. C Quantification of DHE fluorescent intensity in CLV3::ALR, ALA::amiRNANDUFS4 transgenic plants with or without ethanol induction (n = 7,  SD). **P < 0.01, Student’s t-test. D–F Quantitative measurement of NDUFS4 (D), WUS (E), and CLV3 (F) expression levels in CLV3::ALR, ALA::amiRNA-NDUFS4 and CLV3::ALR, ALA:: GUS plants with or without ethanol induction. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t-test.

of peroxidases in the root (Tsukagoshi et al, 2010). We found that UPB1 was also expressed in the SAM, and knocking out its function caused increased expression of several peroxidases and decreased the H2O2 levels in the SAM (Fig EV2A–D and F). The size of the SAM in the upb1-1 mutant was greatly reduced (Fig 6D, E and G), while the stem cell region that was marked by CLV3 expression was not noticeably changed (Fig 6D, E and H), indicating that differentiating cells in the PZ were dramatically decreased. Conversely, elevating endogenous H2O2 by overexpressing the peroxidase repressor UPB1 (Fig EV2C, E and F) caused the increase in the SAM size in the transgenic plants (Fig 6D, F and G). However, the CLV3 expression domain was not significantly affected in the transgenic plants (Fig 6D, F and H), suggesting that the number of differentiating cells in the PZ was dramatically increased. We further confirmed this observation by treating the plants with general H2O2 scavenger

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However, increasing the H2O2 levels in UPB1 overexpressing plants in turn activated CYCB1;1, CYCD4;1, and CYCA3;3 expression (Fig 6L). To further confirm this observation, we used a KI treatment to scavenge endogenous H2O2 (Tsukagoshi et al, 2010) and found that all three cell cycle regulatory genes were repressed (Fig 6M). Conversely, the expression of CYCB1;1, CYCD4;1, and CYCA3;3 was activated by the exogenous H2O2 treatment (Fig 6M). Together, these results demonstrate that similar to animals, H2O2 promotes stem cell differentiation in plants. H2O2 negatively regulates O
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Figure 5. Establishing ROS spatiotemporal patterns in stem cells. A–F

The spatiotemporal expression patterns of six SODs in the wild-type plants, including FSD2 (A), CSD1 (B), CSD3 (C), MSD1 (D), CSD2 (E), and FSD1 (F). Scale bars, 50 lm. G–I The phenotypes of the wild-type plant (G), CLV3::FSD2 (H, 6 out of 39 transgenic T1 plants showed the meristem termination phenotypes) and UBQ10::FSD2 (I, 7 out of 36 transgenic T1 plants showed the meristem termination phenotypes). Scale bars, 1 mm. J–L WUS expression patterns in the wild-type plant (J), CLV3::FSD2 (K), and UBQ10::FSD2 (L) transgenic plants. All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. M–O CLV3 expression patterns in the wild-type (M), CLV3::FSD2 (N), and UBQ10::FSD2 (O) transgenic plants. All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm.

potassium iodide (KI) (Tsukagoshi et al, 2010). Eleven days after treatment, the seedlings showed reduced H2O2 levels in the SAM (Appendix Fig S10A–C). Consistent with the H2O2-deficient mutant upb1-1, we observed reduced sizes of the SAMs after the KI treatment (Appendix Fig S10D–F), suggesting that the low level of H2O2 affected differentiation in the SAM. Previous studies have revealed that the differentiating cells in the PZ divided at a faster rate than the stem cells in the CZ (Laufs et al, 1998), and CYCB1;1 was found to be specifically expressed in the differentiating PZ, activating cell division (Reddy et al, 2004). In this study, we found that not only CYCB1;1 but also CYCD4;1 and CYCA3;3 were specifically expressed in the PZ (Fig 6I–K). In the H2O2-deficient mutant upb1-1, the mRNA levels of CYCB1;1, CYCD4;1, and CYCA3;3 were remarkably decreased (Fig 6L).

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Even though H2O2 was more abundant in the PZ, very low levels of H2O2 were found in the plant stem cells (Fig 6A–C). Interestingly, further reducing the H2O2 level by increasing the peroxidase activity in the upb1-1 mutant caused a greater accumulation of O
 2 (Figs 7A and B, and EV2G, H and J). Conversely, the O
 2 content was decreased in the UPB1 overexpression lines, which was due to the elevated H2O2 levels caused by peroxidase repressing (Figs 7A and C, and EV2G, I and J). This indicated that H2O2 negatively regulated
 O
 2 accumulation in the stem cells. Considering that O2 accumulation is crucial for stem cell maintenance, it is important for H2O2 levels to remain low in stem cells. Along these lines, we found that three peroxidases, AT5G64100, PRX33, and PRX52, were specifically expressed in the stem cells to limit H2O2 accumulation (Fig 7D–F; Appendix Fig S11). Consistent with an earlier observation, we found that these three stem cell-specific peroxidases were negatively regulated by UPB1 (Fig EV2B; Appendix Fig S12). To further investigate the mechanism by which H2O2 negatively regulated the O
 2 levels in stem cells, all three major sources of endogenous O
 2 in plant aerobic metabolism were examined under the KI or H2O2 treatments, including NADH dehydrogenase, NADPH oxidase, and the alternative oxidase. After the KI treatment to reduce endogenous H2O2, most subunits of the above metabolic enzymes were upregulated at the transcriptional level, including ATRBOHA, ATRBOHB, ATRBOHD, ATRBOHE, ATRBOHF, ATRBOHH, and ATRBOHI in NADPH oxidase, NDUFS4 in NADH dehydrogenase, and AOX1A, AOX1D, and AOX2 in the alternative oxidase (Fig 7G). Consistent with this observation, these genes were repressed by the H2O2 treatments (Fig 7G). In the H2O2-deficient mutant upb1-1, most of the NADPH oxidases, NADH dehydrogenases, and the alternative oxidases were upregulated (Fig 7H). However, in the H2O2accumulated UPB1 overexpression lines, the transcriptional levels of these metabolic enzymes were downregulated (Fig 7H). To functionally test whether increased H2O2 could negatively regulate stem cell fate, we treated plants with H2O2 and found that the emergence of the first pair of true leaves was drastically compromised (Fig 7I–K; Appendix Fig S13). These data strongly suggested that the high level of H2O2 could terminate stem cell fate by negatively regulating O
 2 homeostasis and that maintaining a low level of H2O2 was essential for stem cell maintenance in Arabidopsis thaliana. Self-maintenance of ROS balance in stem cells Reactive oxygen species accumulations in the SAM showed distinct spatial patterns, and stem cells maintained high levels of O
 2 and low H2O2, while differentiating cells showed opposite patterns. To investigate the mechanism of the ROS balance regulation in stem

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Figure 6. H2O2 promotes plant stem cell differentiation. A–C Wild-type inflorescences stained with DAB (A) and DCF (B, longitudinal section; C, transverse section) show that H2O2 is more abundant in the PZ. Scale bars, 50 lm. DAB, 3,30 -diaminobenzidine; DCF, 20 ,70 dichlorofluorescin diacetate. D–F CLV3 expression patterns of the wild-type (D), upb1-1-mutant (E), and 35S:UPB1 transgenic plants (F). All in situ hybridizations were performed in the same time and same conditions. Scale bars, 50 lm. G Quantification of the SAM size in the wild-type, upb1-1 mutant, and 35S:UPB1 transgenic plants. Upper and lower limits of box correspond to upper Q3 and lower Q1 quartiles, the horizontal line within the box represent the data medians, the single value (square) plotted inside the box indicate the data means, error bars indicating minimum to maximum range, whiskers indicate data range within 1.5× of the interquartile range and outliers are not shown. For each genotype, n = 12, ***P < 0.001, Student’s t-test. H Quantification of the CLV3 expression domains in the wild-type, upb1-1, and 35S::UPB1 plants. Boxplot representation is as described in panel (G). For each genotype, n = 12. I–K The expression patterns of CYCB1;1 (I), CYCD4;1 (J), and CYCA3;3 (K) in the wild-type SAM. Scale bars, 50 lm. L Quantitative measurement of the CYCB1;1, CYCD4;1, and CYCA3;3 expression levels in the wild-type, upb1-1, and 35S::UPB1 plants. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, Student’s t-test. M Quantitative measurement of the CYCB1;1, CYCD4;1, and CYCA3;3 expression levels under the 16-h H2O2 or KI treatments. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, Student’s t-test. ND, not detected.

over-accumulating. Therefore, the ROS balance of high O
 2 and low H2O2 was indispensable for plant stem cell maintenance and functions. ROS controls plant stem cell fate by antagonistically regulating WUS expression

cells, several key enzymes that are involved in ROS biosynthesis and metabolism were further analyzed. When we reduced the O
 2 content in the stem cells using PG treatments, we found that ATRBOHA, ATRBOHB, ATRBOHC, ATRBOHD, ATRBOHG, ATRBOHH, and ATRBOHI in NADPH oxidase, and AOX1B, AOX1C, and AOX1D in the alternative oxidase were downregulated (Fig EV3A), indicating that O
 2 could at least partially positively regulate the expression of its own biosynthesis enzymes. Consistent with the observation that SODs were absent from the stem cells, we found that all seven SODs were upregulated by the PG treatments (Fig EV3B). However, the stem cell-specific peroxidases PRX33, PRX52, and AT5G64100 were significantly reduced by the PG treatments (Fig EV3B). These data demonstrated that in the stem cells, O
 2 maintained its high levels by positively regulating NADPH oxidase and alternative oxidase expression and inhibiting SOD expression. However, O
 2 reduced H2O2 levels in the stem cell by activating peroxidase expression. We observed that stem cell-specific peroxidases could also be repressed by H2O2 treatments (Fig EV3C), indicating that H2O2 antagonized O
 2 repression to maintain its low level in stem cells. Given that H2O2 negatively regulated the O
 2 levels in stem cells by repressing its biosynthesis enzymes (Fig 7G and H), it is likely that the low level of H2O2 in stem cells was also necessary to prevent O
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To illuminate the mechanism of ROS in stem cell regulation at the molecular level, two scavengers of O
 2 —DMTU and PG—were used to treat plants for 16 h. Both the transcript and protein levels of the stem cell master regulator WUS were dramatically decreased (Fig 8A–D and J), which explained the phenotypes of stem cell elimination by removing O
 2 . Consistently, significant decreases in WUS expression (Fig 3) and SAM size (Fig 2V) were observed in ndufs4, ndufv1, and atrbohD/F mutants with less endogenous O
 2 . In contrast, WUS expression was dramatically upregulated by applying of a methyl viologen (MV) treatment to increase endogenous O
 levels (Fig 8A, E and J), while GFP 2 signals themselves were not affected by the increased or decreased O
 2 levels in plant cells (Appendix Fig S14). These data demonstrate that a high level of O
 2 is essential for WUS expression and plant stem cell maintenance. Because H2O2 negatively regulated O
 2 accumulation in stem cells, we increased H2O2 levels exogenously by applying H2O2 or endogenously using an amino-1,2,4-triazole (AT) treatment to inhibit catalase activity (Jiao et al, 2011). We found that both the transcript and protein levels of WUS were strongly repressed by increased H2O2 levels (Fig 8A, F–H and K). In contrast, WUS was upregulated when we decreased the H2O2 levels using the H2O2 scavenger KI treatments (Fig 8A, I and K), while GFP signals themselves were not affected by the changes of H2O2 levels in plant cells (Appendix Fig S14). In accordance with this finding, increasing the peroxidase levels to reduce endogenous H2O2 in the

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peroxidase repressor mutant upb1-1 or in the peroxidase overexpression Per57 (Tsukagoshi et al, 2010) could also increase WUS expression levels, whereas WUS was repressed in the UPB1 overexpression lines (Fig EV4). Taken together, these data lead to the conclusion that O
 2 is crucial for stem cell maintenance through activation of WUS, whereas H2O2 promotes stem cell differentiation by repressing WUS expression in transient amplifying cells (Fig 9).

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NADPH-oxidase- and NADH-dehydrogenase-derived superoxides are important for stem cell fate maintenance. Previously, O
 2 was also reported to be broadly accumulated in the root apical meristem (RAM) blocking the transition of cells from proliferation to differentiation (Tsukagoshi et al, 2010). These data suggest that superoxide, as a signaling molecule, is mainly accumulated in undifferentiated cells to inhibit differentiation in plants. ROS balance defines plant stem cell fate

Discussion Superoxide is a signaling molecule that blocks plant cell differentiation Stress-induced high levels of ROS have long been suggested to be harmful to cells. Even though ROS triggers stress response signaling, they are eventually eliminated by antioxidants and metabolic enzymes to prevent oxidative damage to cells. However, physiological levels of ROS have been widely proven to operate as intracellular signaling molecules in higher eukaryotes. Among the reactive oxygen species, H2O2 has been shown as a signaling molecule that primes animal stem cell differentiation (Owusu-Ansah & Banerjee, 2009; Kanda et al, 2011; Le Belle et al, 2011; Tormos et al, 2011; Malinska et al, 2012; Hamanaka et al, 2013; Ludin et al, 2014; Mantel et al, 2015). However, in this study, we found that O
 2 was specifically enriched in plant shoot stem cells (Fig 1A, E and I). The high levels of O
 were crucial for maintaining WUS expression, 2 which is the key regulator of plant stem cell maintenance (Figs 3 and 8). Elimination of O
 2 from stem cells could cause significant decreases in both the transcript and protein levels of WUS, and the termination of stem cell activity (Figs 1, 3, and 8). Therefore, O
 2 functions are essential for shoot stem cell maintenance. Although O
 2 has two major sources in plant cells, NADH dehydrogenase in the mitochondria and NADPH oxidase from the plasma membrane (Keller et al, 1998; Mittler et al, 2004; Belhaj et al, 2009; Malinska et al, 2012; Hamanaka et al, 2013; Hao et al, 2014), knocking out their subunits caused very similar phenotypes in stem cell maintenance (Fig 2), suggesting that both

Reactive oxygen species accumulation in the shoot apical meristem showed distinct spatiotemporal patterns and antagonistic roles. O
 2 mainly accumulated in the CZ to maintain the stem cells, whereas H2O2 accumulated in the PZ to promote cell differentiation. As two

A

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G

H Figure 7. H2O2 negatively regulates

O
 2

accumulation in stem cells.

A–C O
 2 distribution patterns in the SAMs of the wild-type (A), upb1-1-mutant (B), and 35S:UPB1 transgenic plants (C). Scale bars, 50 lm. D–F Peroxidase gene expression patterns in the wild-type SAM, including AT5g64100 (D), PRX33 (E), and PRX52 (F). Scale bars, 50 lm. G Quantitative measurement of the NADH dehydrogenase, NADPH oxidase, and alternative oxidase expression levels in the SAM under the 16-h H2O2 or KI treatments. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, Student’s t-test. H Quantitative measurement of the NADH dehydrogenase, NADPH oxidase, and alternative oxidase expression levels in the SAMs of the wild-type, upb1-1, and 35S::UPB1 plants. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, Student’s t-test. I, J Seven days after the germination of the wild-type seedlings in the mock (I) and H2O2-containing media (J). Scale bars, 500 lm. K The percentage of wild-type plants with the first pair of true leaves after 7 days of germination on media with different H2O2 concentrations. More than 200 plants were counted for each treatment. Mean  SD. ***P < 0.001, Student’s t-test.

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I

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Jian Zeng et al

Figure 8. ROS controls plant stem cell fate by antagonistically regulating WUS activity. WUS expression levels in 11-day-old wild-type seedlings under the shortterm (16-h) treatments with DMTU, PG, MV, H2O2, AT, and KI were measured by qRT–PCR. Mean  SD with three independent biological replicates. *P < 0.05, **P < 0.01, Student’s t-test. B–E DHE staining (top row) and the WUS protein (bottom row) in the 11day-old wild-type seedlings under the short-term (16-h) treatments with mock (B), DMTU (C), PG (D), and MV (E). The dotted yellow line represents the L1 layer of the meristem. Scale bars, 50 lm. F–I DCF staining (top row) and the WUS protein (bottom row) in 11-day-old wild-type seedlings under the short-term (16-h) treatments with mock (F), H2O2 (G), AT (H), and KI (I). The dotted yellow line represents the L1 layer of the meristem. Scale bars, 50 lm. J Quantification of the fluorescent intensity of DHE in (B–E) (mock and PG, n = 10,  SD; DMTU and MV, n = 7,  SD). *P < 0.05 and ***P < 0.001, Student’s t-test. K Quantification of the fluorescent intensity of DCF in (F–I) (mock and KI, n = 10,  SD; AT, n = 6,  SD; H2O2, n = 12,  SD). **P < 0.01 and ***P < 0.001, Student’s t-test.

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Figure 9. Redox regulation of plant stem cell fate.

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key enzymes in ROS metabolism, SOD and peroxidase are indispensable for establishing this distinct spatiotemporal pattern in the SAM. In stem cells, SOD expressions were repressed to maintain high levels of O
 2 , and peroxidases were activated to reduce the accumulation of H2O2. However, in the differentiating cells, six SODs were expressed in the PZ to generate high H2O2 levels and reduce O
 2 levels (Fig 5). Even though these six SODs are localized to different organelles in plant cell, CSD2, FSD2, FSD3 in the chloroplast, MSD1 in the mitochondria, CSD3 in the peroxisome, and FSD1 and CSD1 in the cytoplasm, they all seem to be recruited to the reduced O
 2 levels in the differentiating cells (Bowler et al, 1992, 1994; Streller & Wingsle, 1994; Bueno et al, 1995; Kliebenstein et al, 1998; Alscher et al, 2002; Myouga et al, 2008).

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High levels of O
 2 accumulate in the stem cells of the CZ (the red color) by repressing SODs to maintain WUS expression (the yellow color) and plant stem cell fate. Moreover, stem cell-specific peroxidases (PRXs) are expressed to ensure the optimal low levels of H2O2 in stem cells and fine-tune the O
 2 levels via negative feedback. In the PZ (the green color), H2O2 is generated by SODs to promote stem cell differentiation partially by repressing WUS activity. The blue arrows highlight the major functions of O
 2 in plant stem cell fate determination.

As H2O2 negatively regulated the O
 2 levels by repressing the expression of NADH dehydrogenase, NADPH oxidase, and the alternative oxidase (Fig 7G and H), a further reduction in the H2O2 levels by overexpressing peroxidase caused a sizeable accumulation of O
 2 (Figs 7B and EV2H), indicating that the low H2O2 content in the stem cells might still be functional toward fine-tuning the O
 2 level and preventing it from over-accumulating. The high O
 2 and low H2O2 balance might play a pivotal role in stem cell fate regulation. Reducing the ratio of O
 2 /H2O2 by the decreasing O
 2 or increasing H2O2 levels could eliminate stem cell activity and promote differentiation. Therefore, the spatial distribution of the O
 2 /H2O2 balance defined the boundary between the stem cells and non-stem cells (Fig 9) and served as a key switch for stem cell maintenance or differentiation. Interestingly, in the root, the UPB1-mediated O
 2 /H2O2 balance also defined the boundary between the meristematic cells in the RAM and the expanded cells in the elongation zone, suggesting that ROS balance is a key regulator in determining plant cell fate. However, how this O
 2 /H2O2

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 2 /H2O2 balance defines plant stem cell fate

balance cooperates with cytokinin and auxin to regulate plant cell fate remains an open question. Given the fact that ROS can be induced by environmental stress, it would be expected that the endogenous and exogenous stress signals might be integrated via influencing the balance of O
 2 /H2O2 to adjust stem cell behavior for the adaptive growth of the plant. Redox regulation of stem cells is conserved in both plants and animals Despite the large evolutionary distance between plants and animals, stem cells in both kingdoms share very similar concepts and functions, such as pluripotency, self-renewal, low cell division rate, and germline transmission. However, the specific transcription factors that are recruited from each system to specify the stem cells are quite different. The similarities in stem cell organization and metabolism that are achieved by these distinct transcription factors are not purely coincidental due to the convergent evolution of both kingdoms. A common feature in the redox regulation of H2O2 is its priming of stem cell differentiation. In animals, H2O2 has been shown to promote differentiation in different stem cell populations (OwusuAnsah & Banerjee, 2009; Kanda et al, 2011; Le Belle et al, 2011; Tormos et al, 2011; Malinska et al, 2012; Hamanaka et al, 2013; Ludin et al, 2014; Mantel et al, 2015). In plants, we found that H2O2 accumulated in differentiating cells in the PZ and promoted stem cell differentiation by repressing plant stem cell master regulator WUS and subsequent activation of cell division. These lines of evidence suggest that the mechanism of H2O2-regulated stem cell differentiation is highly conserved across the eukaryotic kingdom. Interestingly, in the multiple stem cell populations of animals, low levels of H2O2 signaling were also found to be required for stemness and pluripotency induction (Jang & Sharkis, 2007; Morimoto et al, 2013; Paul et al, 2014; Zhou et al, 2016). The high levels of H2O2 exhausted the hematopoietic stem cells of mammals (Mantel et al, 2015), while the increased superoxide levels in the stem cell niche or differentiated dshKD inner germarial sheath cells substantially impaired stem cell differentiation in Drosophila (Bailey et al, 2015; Wang et al, 2015), suggesting that maintaining H2O2 at low levels is also crucial for animal stem cell maintenance. In plants, we demonstrated that the high levels of O
 2 in the stem cells were essential for plant stem cell maintenance, while H2O2 negatively regulated its accumulation. The high O
 2 and low H2O2 patterns in the plant stem cells were established by repressing the SODs and activating the peroxidases. Likewise, during the induction of human fibroblasts iPSCs (induced pluripotent stem cells) and mouse neuronal stem cells, the c-Myc and Oct4 treatments repressed all three SODs in mammals and activated the expression of various peroxidases (Kim et al, 2009; Soldner et al, 2009) (Fig EV5A and B). The downregulated SODs, including SOD1, SOD2, and SOD3 in humans or mice, were highly conserved with the SODs that we found in the differentiating PZ of plants (Figs 5A–F and EV5C). These data strongly suggest that the functions of O
 2 in stem cell regulation are highly conserved across the plant and animal kingdoms and might also play key roles in animal stem cell maintenance. Although this seems to be a basic mechanism of stem cell regulation that is conserved in both kingdoms, how different regulators in plants and animals are integrated into this framework remains to be discovered.

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Materials and Methods Plant materials and treatments Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild type except where noted. The seeds for upb1-1, 35S::UPB1, and 35S::Per57-GFP were kindly provided by Prof. Philip N Benfey. The seeds for WUSpro::WUS:GFP were kindly provided by Prof. Jan Lohmann. The seeds for ndufv1 and ndufs4 mutants were kindly provided by Prof. Etienne H. Meyer. The seeds for atrbohD/F were kindly provided by Dr. Miguel Angel Torres. All seeds were sterilized with 70% ethanol and 0.5% Tween for 10 min except in the ethanol induction assay, followed by washing two times with 95% ethanol and air-drying; the seeds were then placed at 4°C for 2 days before being planted. For the ethanol induction assay, 0.16 M NaClO was used to sterilize the seeds, followed by washing several times with sterilized water. Seedlings were grown in 1/2 MS media plates supplemented with 1% sucrose under 16-h light/8-h dark, in an identical manner to growth in nutrient soil. For the ethanol inductions, we cut the inflorescence apexes and placed them into 1/2 MS medium with 1% ethanol for 12 h in the light. In situ hybridization Templates of RNA probes were amplified from cDNAs with genespecific primers containing T7 or T3 promoter sequences at the 50 end. A complete list of primer sequences is shown in Appendix Table S1. The RNA probes were synthesized with T7/T3 RNA polymerase, and in situ hybridization was performed with standard protocols (Weigel & Glazebrook, 2002; Andersen et al, 2008). For the miR398 probe, LNA oligonucleotides were endlabeled with the DIG oligonucleotide 30 -end labeling kit (Roche) as previously described (Wang et al, 2008). The SAM size was measured by the maximum width between the primordia. Quantitative RT–PCR Whole seedlings or inflorescence apices were dissected and immediately transferred to liquid nitrogen. The Tripure Isolation Reagent (Roche) was used to isolate total RNA from the plant samples. The PrimeScriptTM RT Reagent Kit (TaKaRa) was used for cDNA synthesis. Primers used for qRT–PCR were designed to amplify products that were 100–300 bp in length, and the gene-specific primer sequences are listed in Appendix Table S1. Quantitative PCR was performed with the Thermo PIKO REAL96 Real-Time PCR system using the GoTaq qPCR Master Mix (Promega) with the following PCR conditions: 95°C for 5 min and 40 cycles of 95°C for 10 s, 57°C for 30 s, and 72°C for 30 s, followed by 72°C for 10 min and 20°C for 10 s. TUBULIN was used to normalize the mRNA levels. ROS staining 3,30 -diaminobenzidine (DAB) and 20 ,70 -dichlorofluorescin diacetate (H2DCF-DA) were used for hydrogen peroxide (H2O2) staining. Nitroblue tetrazolium (NBT) and dihydroethidium (DHE) were used for superoxide (O
 2 ) staining. Histochemical staining of H2O2 and O
 2 was performed using DAB (Sigma-Aldrich) and NBT (Sigma-Aldrich), respectively,

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according to previous studies (Thordal Christensen et al, 1997; Huckelhoven et al, 2000; Jiao et al, 2011). Inflorescences of the plants without dissection were infiltrated with 1/2 liquid MS containing either 1 mg/ml NBT and 50 mM potassium dihydrogen phosphate (pH 7.6) (for O
 2 detection) or 1 mg/ml DAB and 10 mM disodium hydrogen phosphate (pH 6.5) (for H2O2 detection) and were incubated in the dark for < 12 h at room temperature. The staining was terminated by transferring the plants into boiling ethanol (ethanol:glycerin:glacial acetic acid = 3:1:1). The plants were then fixed and embedded as previously described (Weigel & Glazebrook, 2002; Andersen et al, 2008). Staining of H2O2 and O
 2 was performed with H2DCF-DA (SigmaAldrich) and DHE (Sigma-Aldrich), respectively, as previously described (Lee et al, 1999; Pei et al, 2000; Tsukagoshi et al, 2010; Leterrier et al, 2012). For visualizing H2O2 and O
 2 with H2DCF-DA and DHE, respectively, seedlings or inflorescences of plants without dissection were incubated in 10 mm K+-MES (pH 6.1) and 50 mm KCl solutions containing either 25 lM H2DCF-DA or 20 lM DHE for 1 h in the dark.

CLV3::MSD1, UBQ10::FSD2, and MP::FSD2. The 1.4 kb upstream and 1.3 kb downstream of the CLV3 coding sequence were used as the promoter and terminator. Artificial microRNAs were created as previously described (Schwab et al, 2006) using the Gateway tailed primers listed in Appendix Table S1 and then subcloned into an ethanol-inducible vector to construct CLV3::AlcR;AlcA::amiRNANDUFS4. The same promoter and terminator of CLV3 were also used in ethanol induction system. All those constructs were transformed into the Col-0.

Confocal microscopy

Statistical analysis

Images for H2DCF-DA (excitation 465–495 nm, barrier 515–555 nm) and DHE (excitation 450–490 nm, emission at 525 nm) staining procedures were acquired using a Zeiss LSM710 microscope. Stained samples were washed twice with 10 mm K+-MES (pH 6.1) and 50 mm KCl, and then fixed and sectioned as previously described (Wang et al, 2014); all sectioning procedures were performed at low temperatures. For imaging of WUS-GFP, the seedlings of the WUSpro:: WUS-GFP plants were fixed and sectioned as previously described (Wang et al, 2014), and visualized under a confocal microscope (Zeiss LSM710). GFP was excited by an argon laser at 488 nm, and the emission spectra were collected between 500 and 550 nm.

Differences between two groups were evaluated by Student’s t-test, and the P-value level was set to 5%.

ROS-related treatments For long-term chemical treatments, the wild-type Col-0 was grown on a 1/2 MS medium plate with/without the different chemicals for 7 days except noted. For short-term chemical treatments, the 11-day-old seedlings of Col-0 were transferred from a 1/2 MS medium plate to a 1/2 MS liquid medium containing the different chemicals for 16 h. The concentrations were 1 mM for KI, PG, and AT, 100 lM for H2O2, and 10 mM DMTU, except where noted.

Expanded View for this article is available online.

Acknowledgements This study was supported by grants to Z.Z. from the Ministry of Science and Technology of China (2013CB967300), the National Natural Science Foundation of China (31270325, 91317310, and 31570273), and the Chinese Academy of Sciences (Hundred Talents Program). The authors thank Prof. Jan Lohmann, Prof. Philip N Benfey, Prof. Etienne H. Meyer, and Dr. Miguel Angel Torres for sharing mutants and transgene seeds.

Spectrophotometric analysis of O
 2 production

Author contributions O
 2 production was measured using a previously described procedure (Jiao et al, 2011). Fresh inflorescence samples from plants (approximately 300 mg), 3 ml of 65 mM phosphate buffer (pH 7.8), 0.2 ml of 0.5 M EDTA-Na (pH 8.0), and 1 ml of 10 mM hydroxylammonium chloride were homogenized using an ice-cold mortar and pestle and then centrifuged at 15,000 × g for 15 min at 4°C. To remove the pigment, 1 ml of the supernatant was mixed with 1 ml of chloroform and centrifuged at 15,000 × g for 10 min at room temperature. Then, 1 ml of the supernatant was mixed with 1 ml of 7 mM 1-naphthylamine and 1 ml of 17 mM 4-aminobenzenesulfonic acid and incubated at 37°C for 20 min. The samples were measured using a spectrophotometer at 530 nm with phosphate buffer as a blank. The amount of O
 2 was expressed as lM/g/min.

Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs)

Plasmid construction

Andersen SU, Buechel S, Zhao Z, Ljung K, Novak O, Busch W, Schuster C,

ZZ and JZ designed the experiments, analyzed the data, and wrote the paper. ZD, HW and ZT conducted the superoxide measurements and plasmid construction. JZ performed all other experiments.

Conflict of interest The authors declare that they have no conflict of interest.

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