The Plant Journal (2014)

doi: 10.1111/tpj.12712

The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants Long-Gang Cui, Jun-Xiang Shan, Min Shi, Ji-Ping Gao* and Hong-Xuan Lin* National Key Laboratory of Plant Molecular Genetics and Collaborative Innovation Center for Genetics and Development, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China Received 15 June 2014; revised 30 September 2014; accepted 20 October 2014. *For correspondence (e-mails [email protected], [email protected]).

SUMMARY Young organisms have relatively strong resistance to diseases and adverse conditions. When confronted with adversity, the process of development is delayed in plants. This phenomenon is thought to result from the rebalancing of energy, which helps plants to coordinate the relationship between development and stress tolerance; however, the molecular mechanism underlying this phenomenon remains mysterious. In this study, we found that miR156 integrates environmental signals to ensure timely flowering, thus enabling the completion of breeding. Under stress conditions, miR156 is induced to maintain the plant in the juvenile state for a relatively long period of time, whereas under favorable conditions, miR156 is suppressed to accelerate the developmental transition. Blocking the miR156 signaling pathway in Arabidopsis thaliana with 35S::MIM156 (via target mimicry) increased the sensitivity of the plant to stress treatment, whereas overexpression of miR156 increased stress tolerance. In fact, this mechanism is also conserved in Oryza sativa (rice). We also identified downstream genes of miR156, i.e. SQUAMOSA PROMOTER BINDING PROTEINLIKE 9 (SPL9) and DIHYDROFLAVONOL-4-REDUCTASE (DFR), which take part in this process by influencing the metabolism of anthocyanin. Our results uncover a molecular mechanism for plant adaptation to the environment through the miR156-SPLs-DFR pathway, which coordinates development and abiotic stress tolerance. Keywords: Arabidopsis thaliana, rice, stress tolerance, development, miRNA, anthocyanin.

INTRODUCTION The timing of the developmental stage transition largely determines the success of plant breeding. Properly timed flowering helps to ensure plant survival. Thus, plants have evolved highly sophisticated mechanisms that control the onset of flowering in response to external and endogenous signals. Previous studies have revealed four main pathways that regulate flowering time: the gibberellic acid, photoperiod, autonomous and vernalization pathways (Henderson and Dean, 2004; Srikanth and Schmid, 2011). As plants are sessile organisms, both detecting and responding to environmental perturbations are particularly important to them. Although plants normally experience only modest environmental changes during their life cycle, molecular mechanisms underlying their responses to harsh environments, especially drought and soil salinity, have been explored during the past few years. The phytohormone abscisic acid (ABA) is a central regulator of plant drought resistance. Under drought conditions, cellular ABA contents increase dramatically. Thus, the stress signaling pathways © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd

have been divided into ABA-dependent and ABA-independent pathways (Shinozaki and YamaguchiShinozaki, 1997; Shinozaki and Yamaguchi-Shinozaki, 2007; Harb et al., 2010). Elucidating the SOS pathway represents a major advance in understanding plant salt tolerance mechanisms (Liu and Zhu, 1998; Halfter et al., 2000; Ishitani et al., 2000; Liu et al., 2000; Qiu et al., 2002). Excess Na+ induces an increase in cytosolic [Ca2+]. Anthocyanins are flavonoids that are synthesized via the phenylpropanoid pathway. Anthocyanin synthesis is induced by UV-B, salt, water stress and some other conditions. Enhanced levels of anthocyanin may increase salt tolerance in Arabidopsis as well as drought tolerance in Cotinus (Chalker-Scott, 1999; Oh et al., 2011). Anthocyanins may protect plants from stress via their reactive oxygen species (ROS)-scavenging activities (Azuma et al., 2008); however, the genetic link underlying the regulation of anthocyanin synthesis during exposure to stress is largely unknown. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression in plants and animals (Ambros, 1

2 Long-Gang Cui et al. 2001; Carrington and Ambros, 2003). These small RNAs are approximately 21 nucleotides in length, single stranded and processed by DICER-LIKE1 (DCL1) in plants. MicroRNAs silence genes by degrading mRNAs in conjunction with RNA-induced silencing complexes (RISCs) or by translational inhibition (Voinnet, 2009; Rogers and Chen, 2013). Plant miRNAs affect diverse aspects of plant growth and development through regulating their target genes. Examples in Arabidopsis and other plants include miR156, which targets SPL transcription factors (Poethig, 2010; Huijser and Schmid, 2011). The abundance of miR156 decreases over the course of development, whereas SPLs are upregulated throughout development (Wu and Poethig, 2006). SPL promotes the juvenile-to-adult phase transition and flowering through activation of the expression of FRUITFULL (FUL), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and other targets (Wang et al., 2009; Wu et al., 2009). Plant miRNAs have been found to participate in plant stress responses, mainly through high-throughput analysis, including microarray analysis and miRNA sequencing (Sunkar and Zhu, 2004; Liu et al., 2008; Zhou et al., 2008; Lee et al., 2010), but few studies have investigated the role of miRNAs in these processes. Although miR169 is induced by drought, low temperature and high soil salinity, overexpression of this miRNA promotes the onset of flowering, which is inconsistent with the observation that Arabidopsis exhibits delayed flowering under drought, salt and cold stress (Xu et al., 2014). Thus, more efforts are needed to explain this phenomenon. Another study demonstrated that miR156 isoforms are induced by heat stress (HS) and are important for HS memory. Indeed, miR156 promotes the sustained expression of HS-responsive genes through SPLs, especially SPL2 and SPL11, and is critical only after exposure to HS (Stief et al., 2014). Plant development is profoundly influenced by the environment, but little is known about the interaction between these pathways. Here, we found that flowering time was delayed in Arabidopsis grown under various stress conditions, whereas miR156 abundance increased under these conditions. Plants harboring 35::MIM156 exhibited increased sensitivity to stress treatment, whereas 35S::miR156 expression increased stress tolerance. Further analysis revealed that miR156 participates in stress tolerance processes by regulating its downstream genes, SPL9 and DFR. These results suggest that a universal mechanism coordinates development and abiotic stress tolerance processes in plants. RESULTS Stress conditions delay flowering initiation by influencing miR156 expression In contrast to our detailed understanding of the gibberellic acid, photoperiod, autonomous and vernalization pathways, little is known about the contribution of stress

signals to the regulation of flowering time. To identify the component(s) that function in integrating environmental signals to regulate the flowering transition, we subjected Arabidopsis thaliana (Col-0) growing on MS medium to various conditions (Figure S1a–n), including salt and drought treatment. Under short-day (SD), low-sucrose (Yang et al., 2013; Yu et al., 2013), cold (Lee et al., 2013), heat, and salt or drought (Balasubramanian et al., 2006; Li et al., 2007) conditions, Col-0 plants exhibited a delayed onset of the flowering transition (Figure S1a–n), but under slightly elevated CO2 and sucrose levels (May et al., 2013; Yang et al., 2013; Yu et al., 2013) the plants exhibited accelerated flowering. Previous studies have shown that CONSTANS (CO) and FLOWERING LOCUS T (FT), but not FLOWERING LOCUS C (FLC), may play critical roles in mediating the effects of salt on flowering (Li et al., 2007). High-throughput data, including data from microarray analysis and microRNA sequencing, link miRNA expression and environmental conditions, which led to the notion that miR156 is involved in the response to salt, mannitol, cold and other stress treatments (Liu et al., 2008; Sunkar and Zhu, 2004; Zhou et al., 2008; Lee et al., 2010). The miR156-SPL3 module directly regulates FT expression in the leaf to control ambient temperature-responsive flowering (Kim et al., 2012). Although miRNAs were first discovered in Caenorhabditis elegans (Pasquinelli and Ruvkun, 2002), as miRNAs lin-4 and let-7 alter the developmental progress of this organism (Ambros, 2011), recent studies have shown that miR156 is necessary and sufficient for the regulation of the developmental stage transition in plants (Wang et al., 2009; Wu et al., 2009; Bergonzi et al., 2013; Zhou et al., 2013). According to the above results, we hypothesized that under stress conditions, A. thaliana increases the abundance of miR156 to maintain the plant in a relatively long juvenile state to help it withstand stress conditions until the situation becomes suitable for reproduction. To facilitate this study, we focused on salt and drought treatment. We planted Col-0 in tissue-culture bottles containing MS medium, MS medium plus 40 mM NaCl (salt stress) and MS medium plus 150 mM mannitol (to simulation drought stress) at 23°C under long-day (LD) conditions (Figure 1a, b). We measured flowering time by counting the rosette leaf number (RLN) at flowering under different treatments. Compared with normal conditions, NaCl and mannitol treatment obviously delayed the onset of the developmental transition (Figure 1a,b). The RLN increased in plants grown on MS plus NaCl (Figure 1d,f) and in plants grown on MS plus mannitol (Figure 1e,g), compared with those grown on MS (Figure 1c,f,g). Correspondingly, the abundance of mature miR156 was higher in Col-0 plants grown on MS supplemented with 100 mM NaCl or 150 mM mannitol, or under other stress conditions, versus those grown on MS (Figures 1h and S1o). To simulate improved

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

6 Long-Gang Cui et al.

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Figure 4. Expression patterns and phenotypes of miR156 downstream genes under stress conditions. (a, b) Expression levels of miR156 downstream genes SPL9, SPL10 and SOC1 in stress-recovered Col-0 plants. Nine-day-old Col0 plants on MS were used as controls. CK, N, NR, M and MR indicate seedlings of 9-day-old Col-0 on MS, 9 days of 140 mM NaCl treatment, 3 days of recovery from a 6-day 140 mM NaCl treatment, 9 days of 180 mM mannitol treatment and 3 days of recovery from a 6-day 180 mM mannitol treatment, respectively. Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (*P < 0.05, **P < 0.01). (c) Phenotypes of 17day-old Col-0, flc, 35S::MIM156, 35S::MIR172, rSPL9 and rSPL10 plants on MS (top), MS plus 120 mM NaCl (middle) and MS plus 150 mM mannitol (bottom). Scale bars: 1 cm.

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S9d). We then examined transgenic Ub::miR156 ZH11 (Oryza sativa, japonica variety) rice seedlings (Xie et al., 2006) and found that they had similar developmental phenotypes and SPL expression patterns to those of their Arabidopsis counterparts (Figure S9). Meanwhile, Ub::miR156 seedlings exhibited improved tolerance under 100 mM NaCl treatment (Figure 6c). These results suggest that miR156-dependent environmental adaption is a conserved mechanism in plants. DISCUSSION Reproduction is highly important for survival. When faced with adverse environmental conditions, animals can move to find the proper conditions for breeding, but in most cases plants have to adapt to their environment, and wait for the most appropriate time to quickly complete the process of reproduction. Plants produce different levels of miR156 in response to many types of environmental signals, and these signals are transferred to its downstream genes, SPLs. Under good conditions, e.g. slightly elevated CO2 and sucrose levels (May et al., 2013; Yang et al., 2013;

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Yu et al., 2013), miR156 levels decrease in conjunction with development, and the expression of SPL downstream genes, such as SOC1, gradually increases. Under stress conditions, however, the expression level of miR156 remains high for a relatively long period of time. In the current study, we found that anthocyanin biosynthesis increased under stress conditions, and that miR156-SPL9 may directly influence anthocyanin biosynthesis through PAP1 and DFR. Moreover, blocking anthocyanin biosynthesis increased the sensitivity of the plants to salt and drought stress. It is well known that anthocyanin can also protect plants from biotic stress, such as bacterial and insect damage. According to a previous study, JAZ interacts with PAP1 and directly regulates the expression of DFR (Qi et al., 2011). SPL9 also interacts with PAP1 and directly regulates the expression of DFR (Gou et al., 2011). Thus, it is reasonable to assume that there is a functional connection between abiotic and biotic tolerance through the miR156-SPL9 and jasmonic acid (JA) pathways. Taken together, we propose a general strategy used by plants to enable them to adjust the balance of inductive and

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

4 Long-Gang Cui et al.

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(Figures 3d,f–h and S4e,f). Taken together, these results demonstrate that when plants encounter stress, the miR156 accumulation level increases (Figure 1h) to keep the plants in a relatively longer juvenile state, which helps them withstand unfavorable environmental conditions. Conversely, when the conditions become suitable for reproduction, the miR156 accumulation level decreases (Figure 1i) to accelerate flowering. We also examined the effect of 35S::miR156 and 35S::MIM156 on flowering under stress conditions. Plants harboring 35S::miR156 exhibited delayed flowering under stress conditions, which was associated with the downregulation of SOC1, FT and FUL. By contrast, plants harboring 35S::MIM156 exhibited earlier flowering than Col-0 under stress conditions, which was associated with the upregulation of SOC1, FT and FUL (Figure S5a–c).

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Figure 2. The response of Pri-miR156s to stress signals. (a) Expression levels of miR156A-H in 6-day-old Col-0 on MS plus 100 mM NaCl and on MS plus 150 mM mannitol. Col-0 on MS was used as a control. Data represent means  SDs (n = 3). (b) Schematic representation of luciferase reporter constructs containing the 2-kb miR156C promoter. The reporter was transferred into Col-0 protoplast, and after transformation, the protoplasts were cultured with or without NaCl overnight. The ratio of firefly luciferase to Renilla luciferase represents the activity of the miR156C promoter under different conditions. Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01).

miR156-dependent stress tolerance To examine the relationship between miR156 and salinity and drought signals, we first confirmed the effect of this miRNA on stress tolerance by increasing its expression level under the control of the 35S promoter (35S::miR156), and by reducing miR156 activity through target mimicry (35S::MIM156) (Figure S3a). Unlike the wild type, 35S:: MIM156 plants were extremely sensitive to salt and drought stress, whereas 35S::miR156 plants exhibited improved salt and drought tolerance under these shortterm stress treatments (Figures 3a,b and S4a,b). We cultured Col-0 in bottles on MS, MS plus 100 mM NaCl and MS plus 150 mM mannitol media for long-term treatments. The 35S::MIM156 plants died quickly in response to stress treatment, and most Col-0 plants also died after a 32-day salt treatment or a 38-day drought treatment, whereas only the 35S::miR156 plants survived (Figures 3c,e and S4c,d). After the 35S::miR156 plants were transferred to MS medium, they recovered and completed their life cycle

Molecular mechanism of miR156-dependent stress tolerance In fact, miR156 regulates the timing of the flowering transition by repressing the expression of SPL9, which, redundantly with SPL10, promotes the transcription of miR172, an miRNA required for juvenile-to-adult determination (Wang et al., 2009; Wu et al., 2009). To further explore the molecular basis of environmental adaption, we examined the expression levels of miR156 downstream genes during salt and drought treatment, and found that the corresponding changes in expression were negatively correlated with miR156 levels. Specifically, SPL9, SPL10 and SOC1 were downregulated under stress conditions and upregulated after recovery from stress conditions (Figures 4a,b and S3b). Then, we examined the effect of salt and drought stress on plants, including Col-0, 35S::MIM156, 35S:: MIR172 and rSPL9 (expressing an miR156-insensitive SPL9 genomic sequence under the control of the SPL9 promoter), as well as rSPL10 (expressing an miR156-insensitive SPL10 genomic sequence under the control of the SPL10 promoter). To examine the specificity of this pathway, we chose an flc mutant as the control for all of these treatments, as these plants have a similar phenotype to that of Col-0 under stress conditions (Li et al., 2007). The results show that rSPL9, rSPL10, 35S::miR172 and especially rSPL9 were sensitive to salt and drought treatment (Figures 4c, S4g and S6a). Additionally, we found that rSPL9 also caused earlier flowering than that observed in Col-0 under stress conditions, as did 35S::MIM156, which was associated with the upregulation of SOC1, FT and FUL expression (Figure S5d–g). From carefully comparing Col-0 plants grown on MS, grown on MS plus NaCl and grown on MS plus mannitol, we noticed the accumulation of anthocyanin in seedlings (Figure 5a,b). We also examined the expression levels of two anthocyanin metabolism-associated genes, including DFR, a flavonoid biosynthetic gene, and PRODUCTION OF

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

Coordinates development and abiotic stress tolerance 5 MS

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ANTHOCYANIN PIGMENT 1 (PAP1), encoding a putative MYB domain-containing transcription factor involved in anthocyanin metabolism and radical scavenging, in saltand drought-treated seedlings. These genes were induced in the stress-treated seedlings (Figure 5c), whereas their expression was reduced after recovery from stress treatments (Figure 5d). Anthocyanin levels are also responsive to many other types of stress, such as UV-B, cold and biotic stress, which could protect plants from damage (Chalker-Scott, 1999; Azuma et al., 2008; Oh et al., 2011). Thus, we examined the effect of salt and drought stress on plants including Ler and tt3 (a DFR mutant with reduced anthocyanin levels in the Ler background). The results show that tt3 was sensitive to salt and drought treatment (Figures 5e, f and S6b). SPL9 interacts with PAP1 and negatively influences anthocyanin biosynthesis by directly regulating the expression of DFR (Gou et al., 2011). At the same time, SPL10 may also negatively affect anthocyanin biosynthesis by influencing the expression of DFR (Figure S7). Thus, anthocyanin may help protect plants from stress. Taken

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Figure 3. miR156 contributes to salt and mannitol tolerance. (a) Col-0, 35S::MIR156 and 35S::MIM156 plants grown on MS (left) and MS plus 120 mM NaCl (right) for 12 days. (b) Col-0, 35S::MIR156 and 35S::MIM156 plants grown on MS (left) and MS plus 150 mM mannitol (right) for 15 days. (c) Five-day-old Col-0, 35S::MIR156 and 35S::MIM156 plants grown on MS were transferred to MS plus 100 mM NaCl and grown in culture bottles for 32 days. (d) After NaCl treatment for 32 days, seedlings were transferred to MS and grown for 22 days. (e) Fiveday-old seedlings grown on MS were transferred to MS plus 150 mM mannitol and grown for 38 days. (f) After mannitol treatment for 38 days, plants were transferred to MS for 32 days. (g) Survival rates of Col-0, 35S::MIR156 and 35S::MIM156 plants after NaCl treatment (n = 3 groups, each group contained approximately 12 plants). Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01). (h) Survival rates of Col-0, 35S::MIR156 and 35S::MIM156 plants after mannitol treatment (n = 3 groups, each group contained approximately 12 plants). Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01). Scale bars: 1 cm.

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together, these results demonstrate that the miR156-SPLsDFR pathway responds to stressful environmental conditions by affecting anthocyanin metabolism, suggesting that this pathway functions as a core regulatory module that mediates coordinated development and abiotic stress tolerance processes. Functional and evolutionary conservation of miR156dependent environmental adaption in plants We analyzed the miR156 gene family in several genomesequenced species, representing algae, monocots and eudicots. We found that miR156 is present in lower and higher plants, whereas in general species considered to be less evolutionarily advanced have fewer copies of primiR156 (Figure 6a; Table S1). To investigate the functional conservation of miR156 in different species, we examined its accumulation level in Oryza sativa (rice) plants subjected to stress treatment. As observed in Arabidopsis, stress treatment also delayed flowering in rice, and miR156 abundance also increased (Figures 6b, S8 and

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

6 Long-Gang Cui et al.

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Figure 4. Expression patterns and phenotypes of miR156 downstream genes under stress conditions. (a, b) Expression levels of miR156 downstream genes SPL9, SPL10 and SOC1 in stress-recovered Col-0 plants. Nine-day-old Col0 plants on MS were used as controls. CK, N, NR, M and MR indicate seedlings of 9-day-old Col-0 on MS, 9 days of 140 mM NaCl treatment, 3 days of recovery from a 6-day 140 mM NaCl treatment, 9 days of 180 mM mannitol treatment and 3 days of recovery from a 6-day 180 mM mannitol treatment, respectively. Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (*P < 0.05, **P < 0.01). (c) Phenotypes of 17day-old Col-0, flc, 35S::MIM156, 35S::MIR172, rSPL9 and rSPL10 plants on MS (top), MS plus 120 mM NaCl (middle) and MS plus 150 mM mannitol (bottom). Scale bars: 1 cm.

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S9d). We then examined transgenic Ub::miR156 ZH11 (Oryza sativa, japonica variety) rice seedlings (Xie et al., 2006) and found that they had similar developmental phenotypes and SPL expression patterns to those of their Arabidopsis counterparts (Figure S9). Meanwhile, Ub::miR156 seedlings exhibited improved tolerance under 100 mM NaCl treatment (Figure 6c). These results suggest that miR156-dependent environmental adaption is a conserved mechanism in plants. DISCUSSION Reproduction is highly important for survival. When faced with adverse environmental conditions, animals can move to find the proper conditions for breeding, but in most cases plants have to adapt to their environment, and wait for the most appropriate time to quickly complete the process of reproduction. Plants produce different levels of miR156 in response to many types of environmental signals, and these signals are transferred to its downstream genes, SPLs. Under good conditions, e.g. slightly elevated CO2 and sucrose levels (May et al., 2013; Yang et al., 2013;

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Yu et al., 2013), miR156 levels decrease in conjunction with development, and the expression of SPL downstream genes, such as SOC1, gradually increases. Under stress conditions, however, the expression level of miR156 remains high for a relatively long period of time. In the current study, we found that anthocyanin biosynthesis increased under stress conditions, and that miR156-SPL9 may directly influence anthocyanin biosynthesis through PAP1 and DFR. Moreover, blocking anthocyanin biosynthesis increased the sensitivity of the plants to salt and drought stress. It is well known that anthocyanin can also protect plants from biotic stress, such as bacterial and insect damage. According to a previous study, JAZ interacts with PAP1 and directly regulates the expression of DFR (Qi et al., 2011). SPL9 also interacts with PAP1 and directly regulates the expression of DFR (Gou et al., 2011). Thus, it is reasonable to assume that there is a functional connection between abiotic and biotic tolerance through the miR156-SPL9 and jasmonic acid (JA) pathways. Taken together, we propose a general strategy used by plants to enable them to adjust the balance of inductive and

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

Coordinates development and abiotic stress tolerance 7

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Figure 5. Molecular mechanism of miR156mediated salt and mannitol tolerance. (a, b) Anthocyanin content in Col-0 plants under treatment with 140 mM NaCl and 300 mM mannitol. Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01). (c) DFR and PAP1 expression levels in Col-0 on MS plus 100 mM NaCl, 150 mM mannitol and MS (control). Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01). (d) DFR and PAP1 expression levels in stress-recovered Col-0. Nine-day-old Col0 plants on MS were used as controls. CK, N, NR, M and MR indicate 9-day-old Col-0 seedlings on MS and seedlings after 9 days of 140 mM NaCl treatment, 3 days of recovery from 6-day 140 mM NaCl treatment, 9 days of 180 mM mannitol treatment and 3 days of recovery from 6-day 180 mM mannitol treatment, respectively. Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01). (e) Ler and tt3 germinated on MS (top) and MS plus 100 mM NaCl (bottom) for 12 days. (f) Five-day-old Ler and tt3 grown on MS were transferred to MS (top), MS plus 120 mM NaCl (middle) and MS plus 300 mM mannitol (bottom). Photographs were taken after 7, 20 and 20 days, respectively. Scale bars: 1 cm.

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repressive signals under fluctuating conditions through the miR156-SPL modules to balance the relationship between development and abiotic stress tolerance, thereby giving plants an advantage in terms of fitness (Figure 6d). When adversity strikes, miR156 is induced and energy is assigned to the defense process through anthocyanin synthesis by the SPL9, PAP1 and DFR pathway, whereas SOC1 expression is reduced to maintain the plant in a relatively long juvenile state (Figures 1h, 4a,b, 5c and S1a–m). Conversely, when the environment is more suitable, miR156 is suppressed and development becomes more of a priority, with SPL9 and SOC1 expression enhanced and PAP1 and DFR expression reduced during this process (Figures 1i, 4a,b and 5d). We do not yet know, however, how miR156 perceives and responds to so many different environmental

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signals, and whether the SPL9-DFR mechanism represents a general abiotic stress tolerance mechanism under other adverse conditions. Further studies are needed to elucidate how plants protect themselves from adverse environmental conditions. EXPERIMENTAL PROCEDURES Genetic stocks All of the Arabidopsis genetic stock used in this study is in the Columbia background, except for tt3, which is in the Landsberg background (Wang et al., 2013). The 35S::MIM156 plants were generated by overexpressing miR156 mimicry targets (Franco-Zorrilla et al., 2007). The 35S::miR156 plants were generated via transformation with a construct produced by inserting an amplified genomic fragment containing miR156b from Col-0 genomic DNA

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

8 Long-Gang Cui et al. Figure 6. Evolutionary and functional conservation of miR156. (a) miR156 is conserved from lower to higher plants; CN, copy number. The miRNA scanning conditions were set to identify exact matches, resulting in the lack of identification of other forms of pri-miR156, such as miR156H and miR156G; thus, the copy number in Arabidopsis was reduced from 8 to 6. (b) The miR156 abundance increased in ZH11 (Oryza sativa, japonica variety) on MS plus 100 mM NaCl or 150 mM mannitol, compared with ZH11 on MS (CK). Data represent means  SDs (n = 3). Significant differences were determined by Student’s t-test (**P < 0.01). (c) Phenotypes of CK (vector in ZH11) and Ub::MIR156 before (top) and after (bottom) 100 mM NaCl treatment. (d) A simple model for the miR156dependent coordination of development and abiotic stress tolerance in plants. When adversity strikes, miR156 is induced and energy is assigned to the tolerance process; however, when the environmental conditions are appropriate, miR156 is suppressed and development becomes more of a priority.

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between the CaMV 35S promoter and the 30 -OCS terminator (Schwab et al., 2005). The 35S::miR172, pSPL9::rSPL9 (expressing an miR156-insensitive SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 sequence under the control of the SPL9 promoter) and pSPL10::rSPL10 (expressing an miR156-insensitive SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 10 sequence under the control of the SPL10 promoter) plants were described previously (Wang et al., 2009; Wu et al., 2009). The flc mutant (SALK_041126) was obtained from the Arabidopsis Biological Resource Center (http://abrc.osu.edu). The ZH11 rice used in this study is an O. sativa, japonica rice variety. Ub::MIR156s were described previously (Xie et al., 2006).

Growth conditions Arabidopsis seeds were sterilized, sown on MS medium and incubated for 2 days at 4°C in the dark before being transferred to a growth chamber. For phenotypic analysis, plants were grown on MS, or MS supplemented with NaCl, mannitol or sucrose, in Petri plates or culture bottles under SD (8-h light/16-h dark, 23°C) or LD (16-h light/8-h dark, 23°C) conditions. The humidity level in the growth chamber was 50%. For heat and cold treatment, seedlings were grown under LD conditions at 34 or 16°C, respectively. For expression analyses, rice seeds were sterilized, sown on MS medium and incubated for 2 days at 30°C in the dark before being transferred to a growth chamber. Plants were grown on MS or MS supplemented with NaCl or mannitol in culture bottles under 13-h light/11-h dark, 25°C, conditions. For stress treatments, seeds were

soaked in water at room temperature (25°C) for 2 days, followed by germination for 1 day at 37°C. The most uniformly germinated seeds were sown in a 96-well plate from which the bottom was removed. Two days later, the seedlings were cultured in Yoshida’s culture solution. Twenty-day-old seedlings were transferred to culture solution containing 100 mM NaCl.

Evolutionary analysis The genome resources used in this study are listed in Table S1. An sRNA Toolkit was used, and the genomes of these selected species were searched using the miR156 DNA sequence 50 -TGACAGAAGAGAGTGAGCAC-30 . Conditions were set to identify exact matches of this sequence that could form a hairpin structure. As this method eliminated the detection of other forms of pri-miR156, e.g. miR156H and miR156G, the copy number of this sequence in Arabidopsis was reduced from 8 to 6. The species tree was modified based on results from the National Center for Biotechnology Information (NCBI) taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html).

Luciferase assay For the transient transcriptional activity assay, the 2-kb promoter region of miR156C was cloned into the pGreenII 0800-LUC vector (Hellens et al., 2005). The reporter construct was transformed into Col-0 protoplasts, and after transformation, the protoplasts were cultured overnight with or without NaCl. The 35S::REN gene (Renilla luciferase) in the vector was used as an internal control. The

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

Coordinates development and abiotic stress tolerance 9 ratio of firefly luciferase to Renilla luciferase, which was measured using a Dual-Luciferase Reporter Assay System (Promega, http:// www.promega.com), represents the activity of the miR156C promoter under various conditions.

U6 gene as a control. The DNA probes used for small-RNA northern blotting were synthesized and biotin-labeled using a 30 -end DNA labeling method (Invitrogen).

Flowering time measurement

Expression analyses Whole shoots were used for expression analyses. Total RNA was extracted with Trizol reagent (Invitrogen, now Life Technologies, http://www.lifetechnologies.com). RNA quality was determined using a Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, http://www.nanodrop.com). One microgram of total RNA was treated with DNase I and used for cDNA synthesis using oligo(dT) primer or gene-specific primers and Superscript Reverse Transcriptase (Invitrogen). Quantitative RT-PCR was performed with FastStart Universal SYBR Green Master (Roche, http:// www.roche.com) in an ABI 7300 Real-time PCR System (Applied Biosystems, http://www.appliedbiosystems.com). The expression of SPL9, SPL10, FUL, SOC1, DRF, PAP1 and miR156A–miR156E was normalized to that of TUB2. Oligonucleotide primers are listed in Table S2. At least two biological replicates (independently harvested samples) were performed, with three technical replicates per condition. Error bars indicate the standard deviation for three technical replicates. Results from one biological replicate are shown.

Quantitative RT-PCR of mature miR156 One microgram of total RNA from whole shoots of Arabidopsis or rice was reverse transcribed using miR156-specific reverse stemloop transcription primers bound to the 30 portion of mature miR156 and the reverse primer of AtsnoR101 with Superscript III reverse transcriptase (Invitrogen). The reaction conditions were as suggested. The mixture was heated to 65°C for 5 min and incubated on ice for at least 1 min, 25°C for 5 min, 55°C for 30 min and 70°C for 15 min. Quantitative RT-PCR was performed using small RNA-specific primers and primers of AtsnoR101 and FastStart Universal SYBR Green Master (Roche) in an ABI 7300 Realtime PCR System (Applied Biosystems). The expression of mature miR156 was normalized to that of AtsnoR101 (Varkonyi-Gasic et al., 2007). Oligonucleotide primers are listed in Table S2. At least two biological replicates (independently harvested samples) were performed, with three technical replicates per sample. Error bars indicate the standard deviation for three technical replicates. Results from one biological replicate are shown.

Northern blot analysis Samples containing 30–50 lg of RNA were separated on 19% polyacrylamide denaturing gels. The RNA was transferred to a Hybond membrane (Amersham Biosciences, GE Healthcare, http:// www.gehealthcare.com) for 2 h at 200 mA. Following crosslinking by UV irradiation for 3 min, the Hybond membrane was hybridized with biotin-labeled DNA probes complementary to the predicted miRNA sequences at 42°C overnight. The membrane was washed twice with double-strength SSC and 0.1% SDS at 42°C, followed by two higher stringency washes of 0.19 SSC and 0.1% SDS at 42°C. Subsequently, the membrane was incubated with a stabilized streptavidin–horseradish peroxidase conjugate (Thermo Scientific, http://www.thermoscientific.com) in nucleic acid detection blocking buffer, and then washed five times with full-strength washing buffer. After washing with substrate equilibration buffer and adding stable peroxide solution and enhancer solution, the blots were imaged using a Tanon 5200 imaging system (http:// www.biotanon.com/index.asp). To confirm uniform loading, the blots were also probed with a DNA probe complementary to the

As plastochron length is unaffected by pFD::MIR156, leaf number is a good indicator of flowering time, according to Wang et al. (2009; Gou et al., 2011). In the current study, flowering time was measured by counting rosette leaves when the plants began to bolt. Data are expressed as means  standard deviation (SD).

Anthocyanin measurements Anthocyanin was measured as previously described (Gou et al., 2011). Seedlings were ground into a fine powder in liquid nitrogen and extracted with methanol:HCl:H2O (80:5:15) for 12 h at 4°C in the dark. After centrifugation at 16 000 g for 10 min at 4°C, the supernatants were transferred to fresh tubes and the anthocyanin levels were quantified at 520 nm (Beckman Coulter, http:// www.beckmancoulter.com).

Accession numbers The Arabidopsis Genome Initiative gene identifiers are as follows: SPL9 (At2g42200); SPL10 (At1g27370); FLC (At5g10140); BETATUBULIN-2 (At5g62690); SOC1 (At2g45660); DFR (At5g42800), PAP1 (At1g56650).

ACKNOWLEDGEMENTS We thank Dr J.W. Wang, Dr G. Wu, Dr P. Zhang and Dr L.Z. Xiong for kindly providing research materials 35S::miR156, Ub::miR156, 35S::MIM156, 35S::miR172, pSPL9::rSPL9, pSPL10::rSPL10 and tt3; Dr W.Q. Ren, Dr Y.K. He, Dr Y.J. Zhang and Dr J.F. Cheng for technical assistance. This work was supported by grants from the Ministry of Science and Technology of China (2012CB114200), National Natural Science Foundation of China (31121063 and 31130071) and Chinese Academy of Sciences.

SIGNIFICANCE STATEMENT Plant developmental processes are profoundly influenced by the environment, but little research focuses on their interplay. Our research answers a basic question in science, why plants delay flowering when met by stress. We found a conserved molecular mechanism in plants for balancing the relationship between development and abiotic stress tolerance through miR156. REFERENCES Ambros, V. (2001) microRNAs: Tiny regulators with great potential. Cell, 107, 823–826. Ambros, V. (2011) MicroRNAs and developmental timing. Curr. Opin. Genet. Dev. 21, 511–517. Azuma, K., Ohyama, A., Ippoushi, K., Ichiyanagi, T., Takeuchi, A., Saito, T. and Fukuoka, H. (2008) Structures and antioxidant activity of anthocyanins in many accessions of eggplant and its related species. J. Agric. Food Chem. 56, 10154–10159. Balasubramanian, S., Sureshkumar, S., Lempe, J. and Weigel, D. (2006) Potent induction of Arabidopsis thaliana flowering by elevated growth temperature. PLoS Genet. 2, 980–989. Bergonzi, S., Albani, M.C., Ver Loren van Themaat, E., Nordstrom, K.J., Wang, R., Schneeberger, K., Moerland, P.D. and Coupland, G. (2013) Mechanisms of age-dependent response to winter temperature in perennial flowering of Arabis alpina. Science, 340, 1094–1097.

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© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), doi: 10.1111/tpj.12712

The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants.

Young organisms have relatively strong resistance to diseases and adverse conditions. When confronted with adversity, the process of development is de...
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