Plant and Cell Physiology Advance Access originally published online on June 18, 2009
Plant and Cell Physiology 2009 50(7):1345-1363; doi:10.1093/pcp/pcp083
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This article appears in the following Plant and Cell Physiology issue: Special Issue Articles: Omics and Bioinformatics [View the issue table of contents]
Three Arabidopsis SnRK2 Protein Kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, Involved in ABA Signaling are Essential for the Control of Seed Development and Dormancy
1Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ibaraki, 305-8686 Japan
2Plant Science Center, RIKEN Yokohama Institute, Kanagawa, 230-0045 Japan
3Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 Japan
4Experimental Plant Division, Bioresource Center, RIKEN Tsukuba Institute, Ibaraki, 305-0074 Japan
*Corresponding author: E-mail, kazukoys{at}jircas.affrc.go.jp; Fax, +81-29-838-6643.
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Abstract |
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ABA is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of these protein kinases in seed development and germination. These SnRK2 proteins were mainly expressed in the nucleus during seed development and germination. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation and showed severe growth defects during seed development. It exhibited a loss of dormancy and elevated seed ABA content relative to wild-type plants. The severity of these phenotypes was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d srk2i mutant. The triple mutant had greatly reduced phosphorylation activity in in-gel kinase experiments using basic leucine zipper (bZIP) transcription factors including ABI5. Microarray experiments revealed that 48 and 30% of the down-regulated genes in abi5 and abi3 seeds were suppressed in the triple mutant seeds, respectively. Moreover, disruption of the three protein kinases induced global changes in the up-regulation of ABA-repressive gene expression, as well as the down-regulation of ABA-inducible gene expression. These alterations in gene expression result in a loss of dormancy and severe growth defects during seed development. Collectively, these results indicate that SRK2D, SRK2E and SRK2I protein kinases involved in ABA signaling are essential for the control of seed development and dormancy through the extensive control of gene expression.
Keywords: ABA signaling • ABI5 • Arabidopsis thaliana • Dormancy • Seed maturation • SnRK2 protein kinase
Abbreviations: ABI3, ABA-INSENSITIVE3; ABI5, ABA-INSENSITIVE5; ABRE, ABA-responsive element; AREB, ABRE-binding protein; bZIP, basic leucine zipper; DAF, days after flowering; DAI, days after imbibition; GFP, green fluorescent protein; GST, glutathione S-transferase; HSP, heat shock protein; LEA, late embryogenesis abundant; PP2C, protein phosphatase 2C; qRT-PCR, quantitative real-time PCR; SnRK2, SNF1-related protein kinase 2.
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Introduction |
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The phytohormone ABA plays important roles in the control of germination and in the acquisition of dehydration and desiccation tolerance in vegetative tissues and during seed development. Intensive research efforts have identified many factors involved with the synthesis, metabolism, perception, signal transduction and transcription networks of ABA (for reviews, see Finkelstein et al. 2002
, Pei and Kuchitsu 2005, Yamaguchi-Shinozaki and Shinozaki 2006, Holdsworth et al. 2008, McCourt and Creelman 2008). In order to provide a better understanding of the molecular mechanisms regulating seed development and germination, various mutants including ABA-insensitive Arabidopsis mutants (abi) were isolated and characterized. The ABI1 and ABI2 genes encode 2C-type protein phosphatases (PP2Cs) that negatively regulate ABA responses (Koornneef et al. 1984, Leung et al. 1994, Meyer et al. 1994). The ABI3 and ABI5 genes encode seed-specific B3- and basic leucine zipper (bZIP)-type transcription factors, respectively (Giraudat et al. 1992, Finkelstein and Lynch 2000, Lopez-Molina and Chua 2000). ABI3 acts as a transcriptional activator through binding to a RY/Sph sequence motif (CATGCA; Mönke et al. 2004), which is a conserved cis-acting element in the promoters of many seed-related genes. ABI5 is a protein which functions as a transcriptional activator through binding to an ABA-responsive element (ABRE; PyACGTGG/TG; Giraudat 1995, Busk and Pages 1998, Hattori et al. 2002), which is a conserved cis-acting element in the promoters of many ABA-induced genes (Yamaguchi-Shinozaki and Shinozaki 2006). ABI5 genetically and physically interacts with ABI3 (Nakamura et al. 2001) and is genetically epistatic to ABI3 (Lopez-Molina et al. 2002).
In a previous study, we isolated and identified AREB1, AREB2 and AREB3 as Arabidopsis ABRE-binding proteins (AREBs; Uno et al. 2000). Choi et al. (2000) also isolated ABF1, ABF2 (= AREB1), ABF3 and ABF4 (= AREB2) as ABRE-binding factors. All of these proteins are bZIP factors showing high similarity with ABI5. In Arabidopsis, nine AREB/ABF-type bZIP proteins containing three conserved N-terminal (C1, C2 and C3) domains and one conserved C-terminal (C4) domain have been previously described (Bensmihen et al. 2002, Jakoby et al. 2002, Furihata et al. 2006). AREB/ABF-type bZIP transcription factors bind to the ABRE element and activate their expression (Choi et al. 2000, Uno et al. 2000). Molecular analysis of AREB1 showed that transactivation of AREB1 requires ABA-dependent phosphorylation (Furihata et al. 2006). Since ABI5 contains conserved phosphorylation sites and ABA increases the transactivation of ABI5, it was suggested that phosphorylation is important for ABI5 transactivation. In fact, Lopez-Molina et al. (2001) reported that ABI5 was phosphorylated subsequent to ABA treatment.
Several SNF1 (sucrose non-fermenting 1)-related protein kinase 2 (SnRK2) proteins such as Arabidopsis SRK2E (OST1), Vicia faba AAPK and wheat PKABA1 were identified as stress- or ABA-activated protein kinases (Li et al. 2000, Mustilli et al. 2002, Yoshida et al. 2002, Zentella et al. 2002). The Arabidopsis genome contains 38 SnRKs, 10 of which are SnRK2s (Hrabak et al. 2003). Several Arabidopsis SnRK2s, including SRK2C (SnRK2.8), SRK2D (SnRK2.2), SRK2E (SnRK2.6, OST1), SRK2F (SnRK2.7) and SRK2I (SnRK2.3), are activated by ABA when expressed in Arabidopsis protoplasts (Boudsocq et al. 2004, Boudsocq et al. 2007). SRK2D, SRK2E and SRK2I, which are highly similar on the amino acid sequence level, are strongly activated by ABA. When green fluorescent protein (GFP) fusions of SRK2C, SRK2D, SRK2E, DRK2F or SRK2I were expressed in Arabidopsis T87 cells, activation by ABA and phosphorylation of glutathione S-transferase (GST)–AREB1/AREB2 fusion proteins was demonstrated (Furihata et al. 2006). Similarly, rice SAPK8, SAPK9 and SAPK10, which are homologous to Arabidopsis SRK2D, SRK2E and SRK2I, were activated by ABA in a protoplast system. It was also shown that rice SAPKs can phosphorylate TRAB1, which is a rice ortholog of the Arabidopsis AREB/ABFs (Kobayashi et al. 2004, Kobayashi et al. 2005). Moreover, the SRK2E gene is strongly expressed in stomatal guard cells, and SRK2E functions upstream of ABA-responsive genes and mediates the regulation of stomatal aperture. In contrast, SRK2D and SRK2I were shown to be likely to be involved in ABA signaling in seedlings using an srk2d srk2i double mutant (Fujii et al. 2007). We have demonstrated that unlike srk2d, srk2e and srk2i single and double mutants, the srk2d srk2e srk2i triple mutant plants exhibit vivipary, highly enhanced insensitivity to ABA and extremely reduced tolerance to drought stress in the vegetative stage (Y. Fujita et al., unpublished data). However the function of the redundant SnRK2 protein kinases during seed development and germination still remains unclear.
In this study, we focused on the expression and functional analysis of SRK2D, SRK2E and SRK2I during seed development and germination. We found that these three SnRK2s were mainly expressed in the nucleus during seed development and germination. Furthermore, we confirmed that the triple mutant showed severe growth defects during seed development and exhibited a loss of dormancy. Analysis with an in-gel kinase assay showed that these three kinases play essential roles for phosphorylation of bZIP factors, including ABI5. Moreover, transcriptome analysis of the triple mutant demonstrated that the disruption of these three protein kinases induces global changes of ABA-regulated gene expression. These alterations of gene expression result in severe growth defects during seed development and dormancy.
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Expression of SRK2D, SRK2E and SRK2I during seed development and germination
To examine the tissue and cellular localization of SRK2D, SRK2E and SRK2I during seed development, native gene promoters and the coding regions of the respective genes were fused to sGFP (ProSRK2D::SRK2D-GFP, ProSRK2E::SRK2E-GFP and ProSRK2I::SRK2I-GFP). GFP fluorescence was mainly observed in the nucleus of the embryos of these transgenic plants at 12 days after flowering (DAF) ( Fig. 1). GFP fluorescence was also observed in cytoplasm in plants containing ProSRK2E::SRK2E-GFP. GFP spots were observed in both the axes and cotyledons in the embryos containing ProSRK2D::SRK2D-GFP or ProSRK2I::SRK2I-GFP, whereas the spots were observed in cotyledons of the embryos containing ProSRK2E::SRK2E-GFP. These fluorescence images, especially those of PSRK2D:SRK2D-GFP and PSRK2I:SRK2I-GFP, were very similar to those observed in the embryos containing ProABI5::ABI5-GFP (Bensmihen et al. 2005
). These data strongly suggest that these kinases and ABI5 are co-localized in the nucleus during seed maturation. GFP fluorescence was also detected in the nucleus of cotyledons and root tips of germinating plants containing ProSRK2D::SRK2D-GFP, ProSRK2E::SRK2E-GFP or ProSRK2I::SRK2I-GFP at 2 days after imbibition (DAI) (Supplementary Fig. S1). Such fluorescence spots, as described above, were not observed in Columbia (Col) (data not shown).
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Phenotypes of srk2d, srk2e, srk2i single, double and triple mutants
The srk2d srk2e (srk2d/e), srk2e srk2i (srk2e/i), srk2d srk2i (srk2d/i) and srk2d srk2e srk2i (srk2d/e/i) mutants were generated by crossing the homozygous srk2d, srk2e and srk2i mutants to one another. Col CS60000 was used as the wild-type control (hereafter the wild type refers to CS60000 if not otherwise specified). We performed quantitative real-time PCR (qRT-PCR) analysis on the seeds of these plants and confirmed the successful isolation of the respective mutants (Supplementary Fig. S2). In this study, we analyzed silique phenotypes and assessed seed development in the mutants. Since the triple mutant was sensitive to dehydration, we grew all genotypes under high humidity conditions (80 ± 5%) by covering the pots with transparent plastic sheets. All the single and double mutants developed normal siliques and exhibited normal seed development and germination. Since Fujii et al. (2007
) reported that SRK2D and SRK2I are functionally important in ABA signaling, we mainly used srk2d/i and srk2d/e/i for further analysis in our studies. Siliques of the wild type and srk2d/i and srk2d/e/i mutants were harvested at 12 DAF and are shown in Fig. 2A. The length of siliques and number of seeds per silique of srk2d/e/i were approximately half of those measured from wild-type plants (Fig. 2B, C). The morphology of embryos was similar in the wild type and srk2d/i and srk2d/e/i mutants (data not shown). However, both siliques and ovules of the triple mutant rapidly desiccated within a few minutes of exposure to normal humidity levels (60 ± 5%; Fig. 2D). Some of the dried seeds harvested from the triple mutant were shrunken and their seed coats tended to be greenish-brown in color (Fig. 2E). Consistent with this observation, seeds of the triple mutant had higher chlorophyll content than the wild type and the srk2d/i mutant (Fig. 2F). The dry seeds of the triple mutant were collapsed and the cell-like structure of the seed coat was not distinguishable at approximately 3 months after the dry storage (Supplementary Fig. S3). Collectively, these data suggest that the srk2d/e/i seeds were unable to complete seed development effectively although the other srk2 mutants, including srk2d/i, developed seeds normally.
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A viviparous phenotype was observed in siliques of the srk2d/e/i plants when grown at high humidity (Y. Fujita et al, unpublished data). To quantify better the occurrence of vivipary for the srk2d/e/i mutant, we conducted a germination test using mature siliques at 16 DAF for the wild type, single, double and triple mutants at 100% humidity. We achieved the high humidity conditions on plates by utilizing water-saturated filter paper. After 1 week exposure to high humidity, nearly 100% of the srk2d/e/i mutant seeds germinated, whereas only approximately 2% of the srk2d/i mutant seeds germinated (Fig. 2G, H). Under the same conditions as described above, seeds from the other single and double mutants did not germinate (data not shown). In order to evaluate seed dormancy, we assessed germination rates of non-stratified seeds harvested from siliques at 16 DAF. Seeds from the srk2d/e/i mutant germinated more rapidly than seeds from the srk2d/i mutant and the wild type (Fig. 2I). The morphological phenotype of germinated seeds from all genotypes was normal. However, when we assessed the germination of seeds desiccated under low humidity condition (25 ± 5%), seeds from the srk2d/e/i mutant failed to germinate 2 weeks after harvest from siliques at 16 DAF (Fig. 2J). Conversely, seeds harvested from the wild type and the double mutant exhibited nearly 100% germination. These data suggest a diminished desiccation tolerance of the srk2d/e/i seeds. However, the ABA content in seeds of srk2d/i, and particularly srk2d/e/i, was higher than those measured for the wild type (Fig. 2K).
In-gel kinase assay with proteins extracted from seeds of srk2 mutants
In order to determine whether the respective srk2 single, double and triple mutants possess phosphorylation activity and whether ABI5 is a possible phosphorylation target, we prepared protein extracts from mature dry seeds from mutants and wild-type plants. When the recombinant ABI5b–GST fusion fragment (Ser119–Gln190, Supplementary Fig. S4) was used for in-gel kinase activity assays, a phosphorylated product was detected in a 42 kDa band. The intensity of this band was significantly reduced in the srk2d/e/i mutant ( Fig. 3A–C), suggesting that kinase activity in the triple mutant was strongly impaired. When we used the mABI5b–GST fusion fragment (Ser119–Gln190, Ser145 was substituted by alanine), the 42 kDa phosphorylation bands disappeared, suggesting that the phosphorylation occurred in Ser145 of the ABI5b fragment (Fig. 3D). We observed 60 and 33 kDa phosphorylation bands in the srk2d/e/i mutant (Fig. 3A). These bands were also observed in dehydrated vegetative tissues of wild-type plants (Fig. 3E). AREB/ABF-type bZIP proteins such as AREB3 and EEL are also expressed in nuclei of developing seeds (Bensmihen et al. 2002, Bensmihen et al. 2005) and contain conserved putative phosphorylation sites that are targeted by Ser/Thr protein kinases (Supplementary Fig. S4). In-gel kinase experiments using the AREB3 fragment showed that the triple mutant had greatly reduced 42 kDa kinase activity, resulting in diminished phosphorylation of AREB3 (Fig. 3F). The EEL fragment showed similar results (data not shown).
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Gene expression analyses in the srk2d/e/i seeds
Mature dry seeds contain large amounts of mRNAs that are not only reservoirs from embryogenesis and seed development, but also serve as a provision for germination (Nakabayashi et al. 2005
, Holdsworth et al. 2008). In order to unravel the molecular events in the srk2d/e/i seeds, we compared the mRNA profile of the srk2d/e/i seeds with that of wild-type seeds using a 44K oligoarray system (Agilent Technologies, Inc., Santa Clara, CA, USA). For microarray analysis, we used RNA extracted from seeds collected from mature siliques that were dried for approximately 1 month in plants grown under high humidity conditions (80 ± 5%) after yellowing of siliques (16–20 DAF). These seeds maintained their ability to germinate and the RNA was not degraded. By using this system, we identified 1,780 down-regulated genes and 1,907 up-regulated genes (>2-fold; Supplementary Table S1). Our preliminary experiments showed that similar up- or down-regulated genes were identified when we used the RNA extracted from mutant seeds at 16–20 DAF for microarray analysis (data not shown). Using Genevestigator (https://www.genevestigator.com/gv/index.jsp; Zimmermann et al. 2004), we obtained expression profiles of down-regulated and up-regulated genes in the srk2d/e/i seeds. The top 100 genes that were down-regulated in the srk2d/e/i seeds were mainly localized in mature siliques ( Fig. 4A), whereas genes that were up-regulated were predominately expressed throughout all plant developmental stages (Fig. 4B). The top 100 down-regulated genes appeared to be ABA responsive in seeds (Fig. 4C). As shown in Table 1, two-thirds of the down-regulated genes with expression changes of at least 20-fold in the srk2d/e/i seeds showed positive ABA responsiveness in ABA-treated seeds or germinated seeds. Promoter regions of the down-regulated genes were found frequently to contain the ABRE core sequences. Down-regulated genes also responded to paclobutrazol (PAC), a gibberellin biosynthesis inhibitor (Fig. 4C). In contrast, genes that were up-regulated in the triple mutant had a tendency to respond negatively to ABA (Fig. 4D). Approximately 70% of the up-regulated genes with expression changes of at least 20-fold in the srk2d/e/i seeds showed negative ABA-responsiveness in ABA-treated seeds or germinated seeds (Table 2). Using the Arabidopsis gene ontology database and analysis tool of PageMan (http://mapman.mpimp-golm.mpg.de/index.shtml; Usadel et al. 2006), we obtained ontological profiles of the top 100 down- and up-regulated genes in seeds of the srk2d/e/i mutant. The genes that were down-regulated in the srk2d/e/i seeds appeared to be mainly related to stress (P = 2.92E-04) and hormones (P = 9.14E-04) (Fig. 4E). In contrast, genes that were up-regulated in the triple mutant appeared to be significantly related to photosynthesis (P = 3.53E-21) and tetrapyrrole synthesis (P = 5.48E-07) (Fig. 4F).
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Among the down-regulated genes in the srk2d/e/i mutant seeds, the late embryogenesis abundant (LEA) protein gene AtEm6 was strongly down-regulated in the triple mutant seeds (Table 1, Supplementary Fig. S5, Table S2). Hundertmark and Hincha (2008
) recently reported that 34 of 51 LEA genes are expressed in seeds. Among the 34 seed-expressed LEA genes, 18 (53%) were significantly down-regulated in the triple mutant seeds (Supplementary Fig. S5A, Table S2). Genes for GASA2 and GASA3, which are structurally related to the gibberellin-regulated GAST1 gene from tomato (Roxrud et al. 2007), were down-regulated in the triple mutant seeds (Table 1). On the other hand, genes for GASA4 and GASA6 were up-regulated (Table 2, Supplementary Fig. S5B, Table S2). Many genes related to stress, especially heat shock proteins (HSPs), including HsfA9 (Kotak et al. 2007), were also down-regulated (Table 1, Supplementary Fig. S5C, Table S2). Genes encoding PP2Cs, such as At2g34740 and At2g29380, were down-regulated (Table 1). It is interesting that four out of nine ABI1/ABI2-homologous genes (Group A, Schweighofer et al. 2004), including ABI2 and AHG3/AtPP2CA, were significantly down-regulated in the triple mutant seeds (Supplementary Fig. S5D, Table S2). Genes for ABA metabolism enzymes such as CYP707A1 and CYP707A2 were down-regulated (Supplementary Fig. S5E, Table S2). Furthermore, an Arabidopsis RING-H2 gene (XERICO), conferring ABA biosynthesis (Ko et al. 2006), was slightly up-regulated in the srk2d/e/i seeds (2.1-fold; Supplementary Table S2). Genes for gibberellic acid synthesis enzymes, such as GA1 and GA20OX3, were up-regulated (Supplementary Fig. S5F, Table S2). Among genes for gibberellic acid perception and signaling (Ueguchi-Tanaka et al. 2007), genes for a gibberellic acid receptor (AtGID1) and a DELLA protein (RGA) were up-regulated (3.4- and 3.8-fold, respectively; Supplementary Table S2). Many genes for photosynthesis-related proteins and chlorophyll synthesis were up-regulated (Table 2 and Supplementary Fig. S5G, H, Table S2). We also observed an up-regulation of SRK2C and SRK2F (Supplementary Fig. S5I, Table S2) and EEL and AREB3 (Supplementary Fig. S5J, Table S2). A gene for a PYR/PYL/RCAR-type ABA receptor (PYL4; Ma et al. 2009; Park et al. 2009) was also up-regulated (Table 2). Changes were <2-fold in genes for seed-related transcription factors such as ABI3, or seed storage proteins such as 12S globulin storage protein. In an additional analysis, we searched for any 6 or 7 bp sequences that were enriched in the upstream sequences (1.0 kb upstream sequences in the TAIR database) of the top 100 down-regulated genes in the srk2d/e/i seeds. We performed this additional analysis as a means to identify putative novel cis-regulatory elements. We identified several sequences that were identical or highly similar to ABRE, Sph/RY and the MYC-responsive element (MYCR; CACATG) ( Table 3). We also found similarities to the G-box (CACGTGGC). Other enriched sequences such as ACGCAT(A) and CATGGC were also detected in the upstream sequences.
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Relationship between ABI5 and SnRK2 protein kinases, SRK2D, SRK2E and SRK2I
We subsequently characterized the functional relationship between ABI5 and the ABA-activated SnRK2s, SRK2D, SRK2E and SRK2I with mutant analysis. We first determined transcript levels of the strongly down-regulated gene AtEm6 by qRT-PCR in seeds at 16–20 DAF of the wild type, srk2 mutants, Wassilewskija (Ws), abi5-1, Col and abi5-7 ( Fig. 5A). Transcript levels were very low in the triple mutant, and lower in the abi5 mutants than in the wild type. Additionally, transcript levels of other representative strongly down-regulated genes in the triple mutant (Table 1) were examined by qRT-PCR in seeds of abi5-1. Transcript levels of genes for PP2C, LEA and AtEm1 were very low in abi5-1 seeds (<30% of the control) and mRNA levels of glycosyl hydrolase and metallothionein were approximately 50% of the control (Fig. 5B–F). We also examined the transcript level of HsfA9 by qRT-PCR and found that its level was unchanged (data not shown). Our data indicate that the expression of strongly down-regulated genes in the srk2d/e/i mutant was also suppressed in seeds of abi5-1. However, the expression of some genes was not strongly suppressed in abi5-1 seeds. Moreover, the microarray data showed that the mRNA level of ABI5 in the srk2d/e/i seeds was approximately 50% of the control (Supplementary Table S2). These data were confirmed by qRT-PCR (Fig. 5G). With the exception of AtEm1, we also found that transcript levels of these genes in srk2d/i seeds were lower than in the wild type but significantly higher than in the srk2d/e/i seeds.
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We also compared the down-regulated genes in seeds of the srk2d/e/i mutant with those from seeds of the abi5 and abi3 mutants. Using microarray analysis, we identified 213 down-regulated genes in abi5-1 and abi5-7 seeds, and 800 down-regulated genes in abi3-1 and abi3-6 seeds (>2-fold). Among the 39 down-regulated genes with expression changes of at least 20-fold in the srk2d/e/i seeds, 16 genes (about 41%) were down-regulated in abi5-1 and abi5-7 seeds, and 15 genes (about 38%) were down-regulated in abi3-1 and abi3-6 seeds (Table 1). Approximately half of the down-regulated genes in the abi5-1 and abi5-7 seeds were suppressed in srk2d/e/i seeds (102 of 213, about 48%, Fig. 5H). These observations suggest that many ABI5-dependent genes are controlled by SRK2D, SRK2E and SRK2I protein kinases in seeds. In contrast, <10% of down-regulated genes in srk2d/e/i seeds were suppressed in abi5-1 and abi5-7 seeds (102 of 1,780, about 6%, Fig. 5H), suggesting that SRK2D, SRK2E and SRK2I protein kinases may also control the expression of many genes independent of ABI5. Approximately one-third of the down-regulated genes in abi3-1 and abi3-6 seeds were suppressed in srk2d/e/i seeds (242 of 800, about 30%, Fig. 5H). To confirm whether ABI3 is phosphorylated in these seeds, we conducted in-gel kinase experiments using ABI3 and seed proteins. Since ABI3 contains two putative phosphorylation sites (L-X-R-X-X-S/T, Vlad et al. 2008
), we generated two types of ABI3–GST peptides containing each putative phosphorylation site. Phosphorylation of these peptides was not observed in seeds (Supplementary Fig. S6). No L-X-R-X-X-S/T sites were found in the other seed-related transcription factors (FUS3, LEC2 and LEC1). These data suggest that many ABI3-dependent genes are also controlled by SRK2D, SRK2E and SRK2I protein kinases in seeds indirectly. Approximately 14% of down-regulated genes in srk2d/e/i seeds were suppressed in abi3-1 and abi3-6 seeds (242 of 1,780, Fig. 5H), suggesting that SRK2D, SRK2E and SRK2I protein kinases might control the expression of many genes independently of ABI5 and ABI3. |
Discussion |
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We characterized the function of three ABA-activated protein kinases (SRK2D, SRK2E and SRK2I) using single, double and triple mutants of SRK2D, SRK2E and SRK2I. We demonstrated that the triple mutant plants exhibit viviparous germination, highly enhanced insensitivity to ABA and extremely reduced tolerance to drought stress in the vegetative stage (Y. Fujita et al. unpublished data). While our manuscript was under resubmission, Fujii and Zhu (2009
) reported similar results by using triple mutant plants which showed enhanced insensitivity to ABA and reduced tolerance to drought. In this study, we examined the role of these kinases in seed development and germination and found a near-complete loss of dormancy, a desiccation-sensitive phenotype and global changes in gene expression in the triple mutant seeds (Fig. 2). Fujii et al. (2007) previously characterized the srk2d/i double mutant and reported that the double mutant showed reduced dormancy and changes of ABA response and gene expression. However, the severity of the phenotype in seed development and dormancy of the srk2d/e/i mutant was drastic and was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d/i mutant (Fig. 2). These results indicate that the SRK2E kinase may have an important role for the control of seed development and dormancy together with SRK2D and SRK2I, although there was no visible phenotype in seeds of the srk2e mutant. Obviously, the triple mutants are severely affected in seed development and dormancy, suggesting that SRK2D, SRK2E and SRK2I may control various aspects of seed development. Since the seeds of the single and double mutants of SRK2D, SRK2E and SRK2I did not show these severe phenotypes, we concluded that at least one of these protein kinases is necessary to maintain seed development and dormancy.
A loss of dormancy sometimes indicates a low amount of ABA in seeds since ABA is well known to control germination negatively. However, when we measured the ABA content in seeds of the triple mutant, we identified particularly high levels (Fig. 2K). Arabidopsis 9-cis epoxycarotenoid diox-ygenase (NCED) genes NCED6 and NCED9 are expressed in developing seeds to promote ABA biosynthesis during seed development (Tan et al. 2003, Lefebvre et al. 2006). In our microarray experiments, no significant changes were observed in the expression of genes related to ABA synthesis such as NCED6 and NCED9 in the triple mutant seeds (Supplementary Table S2). Four members of the Arabidopsis CYP707A gene family (CYP707A1–CYP707A4) encode ABA 8'-hydroxylases. When mutants are analyzed (cyp707a1 and cyp707a2), ABA levels are elevated in dry and imbibed seeds (Kushiro et al. 2004, Okamoto et al. 2006). We observed down-regulation of genes for ABA metabolism enzymes (ABA 8'-hydrolases such as CYP707A1 and CYP707A2) in the triple mutant seeds (Supplementary Fig. S5, Table S2). We also observed up-regulation of XERICO, conferring ABA biosynthesis (Ko et al. 2006), in the srk2d/e/i seeds (Supplementary Table S2). It is possible that reduced degradation of ABA by the down-regulation of these ABA 8'-hydrolase genes and increased synthesis of ABA by the up-regulation of XERICO might be the reason for the high amount of accumulated ABA in the triple mutant seeds. Since the triple mutant is highly insensitive to ABA (Y. Fujita et al. unpublished data), ABA response mechanisms may be impaired due to the inability of the triple mutant to respond to ABA. As a result, this may cause feedback regulation of ABA biosynthesis in the mutant seeds.
Although their expression patterns were different, SRK2D, SRK2E and SRK2I were mainly localized in nuclei during seed development and germination (Fig.1, Supplementary Fig. S1). The ABI5 proteins localizes in the nucleus during seed maturation (Bensmihen et al. 2005), suggesting that these kinases have the opportunity to interact with ABI5 in nuclei. The triple mutant had greatly reduced phosphorylation activity in in-gel kinase experiments using ABI5 (Fig. 3A). Approximately half of the down-regulated genes in the abi5 seeds were significantly repressed in the triple mutant seeds (Fig. 5H). Furthermore, the transcript level of ABI5 in the triple mutant seeds was approximately 50% of that in the wild type (Fig. 5G). The promoter of the ABI5 gene contains ABRE sequences (CACGTGTC), and expression of the ABI5 gene is affected by ABI5 (Brocard et al. 2002). Taken together, these data suggest that expression of ABI5 might be suppressed due to a defect in phosphorylation of ABI5 in seeds of the triple mutant. Accordingly, ABI5 is unable to function effectively as an ABA-dependent transcriptional activator in the triple mutant seeds, suggesting that phosphorylation of ABI5 in seeds requires SRK2D, SRK2E and SRK2I. Recently, Piskurewicz et al. (2008) reported that the endogenous ABI5 phosphorylation and inhibition of germination could be recapitulated by the addition of a barley SnRK2 protein kinase PKABA1 to the ABI5 overexpression line, supporting our results. However, the phenotypes of abi5 in seed development and dormancy were weaker than those of the srk2d/e/i mutants, indicating that other factors probably participate in ABA signaling during seed development and germination. Our in-gel kinase experiments using the AREB3 and EEL fragments showed that the triple mutant had greatly reduced 42 kDa kinase activity, resulting in diminished phosphorylation of these bZIP proteins (Fig 3F). Up-regulation of AREB3 and EEL was also observed in the triple mutant seeds (Supplementary Fig. S5). Feedback regulation may possibly up-regulate the expression of these ABI5-homologous bZIP factors to complement the reduced activity of these bZIP factors including ABI5. EEL was reported to be a negative regulator of Em gene expression (Bensmihen et al. 2002). It is possible that there is a complex regulation by these bZIP proteins and that defects of the activation of these bZIP factors might have global and complex effects on the control of seed maturation and dormancy. On the other hand, recent evidence has established that proteasome degradation and SIZ1-mediated sumoylation of ABI5 regulates its stability and activity (Lopez-Molina et al. 2003, Stone et al. 2006, Zhang et al. 2007, Miura et al. 2009). Sumoylation of ABI5 negatively affects activity by modulating protein phosphorylation (Miura et al. 2009). Thus, the activation control of the bZIP factors including ABI5 through phosphorylation status by SnRK2/PP2C, and the stability control through degradation or sumoylation, appear to control the function of these transcription factors cooperatively.
According to the microarray data of the srk2d/e/i and abi5 seeds, more than half of the ABI5-regulated genes do not appear to depend on SRK2D/E/I (Fig. 5H). Although the microarray data of seeds may contain the indirect effect of ABI5, it is possible that alternative pathways exist to activate ABI5. By performing in-gel kinase experiments with bZIP transcription factors including ABI5, we observed 60 and 33 kDa phosphorylation bands instead of the 42 kDa SnRK2 phosphorylation band in the srk2d/e/i mutant (Fig. 3A). These data suggest that the other kinases are involved in the phosphorylation of ABI5. As the 60 and 33 kDa bands were also observed in dehydrated vegetative tissues of wild-type plants, phosphorylation by other protein kinases might occur in the triple mutant seeds as well as the dehydrated vegetative tissues (Fig. 3E). In fact, kinase activity appeared near 60 kDa upon the addition of calcium before the phosphorylation reaction of the AREB1 protein (Furihata et al. 2006). Furthermore, two 58 kDa protein kinases, CPK4 and CPK11, can phosphorylate both ABF1 and ABF4 (AREB2), which are homologous to ABI5 (Zhu et al. 2007). Thus, the 60 kDa protein kinase observed in the srk2d/e/i mutant seeds appears to be a calcium-dependent protein kinase (CDPK). A variety of protein kinases, including SnRK2s, CDPK2s and the 33 kDa unknown protein kinase, might phosphorylate the AREB/ABF-type proteins in the dehydrated vegetative tissues. On the other hand, it is possible that SRK2D/E/I might function mainly during seed development.
Microarray experiments revealed nearly 2,000 genes that were either down- or up-regulated in the srk2d/e/i seeds (Supplementary Table S1). These global changes in gene expression may reflect the effects of SRK2D/E/I and the developmental abnormalities of the srk2d/e/i seeds. In accordance with the role of ABA in seed germination, many of the genes that were down-regulated in the srk2d/e/i seeds appeared to be ABA inducible in the ABA-treated seeds (Fig. 4, Table 1). It is possible that suppression of these ABA-inducible genes might trigger uncompleted programs of seed maturation and dormancy. In contrast, many genes that were up-regulated in the mutant seeds appeared to be ABA repressive in the ABA-treated seeds (Fig. 4, Table 2). There might be promoter regions of the down-regulated genes frequently containing the ABRE core sequences for binding of ABI5 and the related bZIP transcription factors. Additional ABA/seed-related cis-elements were observed such as MYCR for the binding of MYC transcription factors and RY/Sph for binding of ABI3 (Table 3). These observations also support the hypothesis that ABA-responsive genes are affected in the triple mutant. Some other frequent sequences such as ACGCAT(A) and CATGGC were also detected in the upstream sequences. On the other hand, there are some down-regulated genes such as HSP genes that were not ABA responsive (Table 1). SnRK2s might be involved in an ABA-independent pathway in seed development, dormancy and germination. The novel sequences that were frequently found in the upstream promoter regions may represent cis-elements acting in ABA-independent gene expression for seed development and dormancy. Further analysis is necessary to reveal the function of these cis-element-like sequences.
Moreover, microarray experiments showed that four genes, including ABI2 and AHG3/AtPP2CA among 10 ABA-related PP2Cs (Group A, Schweighofer et al. 2004), were significantly down-regulated in the srk2d/e/i seeds (Supplementary Fig. S5). Six PP2Cs, ABI1, ABI2, HAB1, HAB2, AHG1 and AHG3/AtPP2CA, in the group A PP2Cs negatively regulate the ABA response in Arabidopsis (for reviews, see Hirayama and Shinozaki, 2007). The null mutants of these PP2Cs exhibit ABA-hypersensitive phenotypes of different strengths during germination. Recently, Yoshida et al. (2006) reported that ABI1 interacts with the C-terminus of SRK2E. It is possible that the dephosphorylation of proteins by these ABA-related PP2Cs may be coupled with the protein phosphorylation mediated by SRK2D, SRK2E and SRK2I. It is also possible that mRNA levels of the ABA-related PP2C genes were down-regulated due to a feedback regulation for the fine-tuning of phosphorylation in the srk2d/e/i mutant seeds. PP2Cs in the other groups such as At2g34740 were also severely down-regulated in the triple mutant, suggesting that these new members might have ABA- and/or seed-related functions. Further studies of these PP2Cs would clarify their functions for ABA signaling and seed maturation or germination control.
Many genes for LEA proteins and HSPs were down-regulated in the srk2d/e/i seeds (Table 1, Supplementary Fig. S5, Table S2). Two LEA genes, AtEm6 and AtEm1, which are known to be controlled by ABI5 (Finkelstein and Lynch 2000, Lopez-Molina and Chua 2000, Carles et al. 2002), were also down-regulated. LEA proteins are thought to play important roles in protection of macromolecules, such as proteins and membranes, from dehydration (Hundertmark and Hincha, 2008). In fact, seed dehydration and the establishment of desiccation tolerance during seed maturation was altered in the atem6-1 mutant (Manfre et al. 2006, Manfre et al. 2009). Similarly, HSPs also function to protect macromolecules during seed maturation (Kotak et al. 2007). It is possible that down-regulation of genes for seed proteins such as HSPs and LEA proteins including AtEm6 may result in significant damage to the mutant seeds.
Besides the down-regulated genes, vast amounts of genes encoding proteins for photosynthesis and genes for tetrapyrrole biosynthesis were up-regulated in the triple mutant seeds (Table 2, Supplementary Fig. S5, Table S2). Genes encoding enzymes for synthesis of gibberellic acid, an antagonistic phytohormone of ABA for germination, were also up-regulated in the srk2d/e/i triple mutant seeds. Substantial up-regulation of gene expression was also observed in GASA family genes such as GASA4 and GASA6 in the triple mutant seeds (Supplementary Fig. S5). Aubert et al. (1998) reported that the GASA4 gene was actively transcribed after germination and this expression was shown to be dependent on the presence of gibberellins. Recently, Wang et al. (2009) reported that OsGSR1, a member of the GASA family of rice, is a positive regulator of gibberellic acid signaling, and plays important roles in both gibberellic acid and brassinosteroid pathways. Genes for gibberellic acid perception and signaling, such as AtGID1 and RGA, were also up-regulated in the triple mutant seeds (Supplementary Table S2). A detailed analysis of gibberellic acid metabolism and signaling in the triple mutant is also necessary to reveal the dynamic phenotype in the triple mutant.
A variety of proteins have been reported to function as ABA receptors (as reviewed by McCourt and Creelman 2008). Recently, two homologous G-proteins, GTG1 and GTG2, have been characterized as plasma membrane-localized ABA receptors that are structurally related to a mammalian anion channel (Pandey et al. 2009). Moreover, the PYR/PYL/RCAR family of Bet v 1/START proteins was reported to bind ABA and control ABA signaling by inhibiting PP2Cs (Ma et al. 2009, Park et al. 2009). Park et al. (2009) also showed that the PYR/PYL/RCARs are required for normal SnRK2 kinase activity. Our microarray data revealed that the gene for PYL4 was up-regulated in the srk2d/e/i seeds (Table 2). It is possible that the mRNA level of PYL4 was up-regulated due to a feedback regulation of the ABA sensing and signaling in the srk2d/e/i mutant seeds.
ABI3 is an ortholog of maize VP1 which confers a viviparous phenotype (as reviewed by Finkelstein et al. 2002). The abi3 seeds exhibit reduced dormancy (Koorneef et al. 1989, Ooms et al. 1993) and the strongest alleles cause vivipary. Microarray analysis revealed that approximately one-third of the down-regulated genes in abi3 seeds were suppressed in the triple mutant seeds (Fig. 5H). It is possible that overlapping of the ABI3-regulated genes and the SRK2D/E/I-regulated genes is related to the loss-of-dormancy/viviparous phenotype. Transcript levels of HSP genes were reduced in seeds of the srk2d/e/i and abi3 mutants but not in abi5 seeds. Recently, ABI3 was reported to control HSP genes through the regulation of HsfA9 expression in seeds (Kotak et al. 2007). Thus, it is possible that SRK2D, SRK2E and SRK2I affect ABI3 and control ABI3-dependent gene expression. However, in-gel kinase experiments showed that ABI3 polypeptides containing putative phophorylation sites (L-X-R-X-X-S/T) were not phosphorylated in extracts of mature dry seeds. ABI3 can function through ABREs via the highly likely physical interaction with ABRE-binding bZIP factors such as ABI5 (Nakamura et al. 2001). Thus SRK2D, SRK2E and SRK2I may indirectly affect the activity of ABI3.
In conclusion, three redundant ABA-activated protein kinases (SRK2D, SRK2E and SRK2I) involved in ABA signaling were shown to control gene expression through the phosphorylation of ABI5 and the other transcription factors, such as AREB3, during seed maturation (Fig. 6). We conclude that at least one of the kinases is necessary to protect seeds during desiccation and to maintain dormancy. Recently, Park et al. (2009) suggested that PP2Cs prevent phosphorylation and activation of SnRK2s and downstream factors. Since these three kinases affect the expression of PP2Cs, it is likely that the fine-tuning of the phosphorylation status of SnRK2s, ABI5 and the related transcription factors, such as AREB3 by PP2Cs, might occur to control their activities during seed development and germination. In order to obtain a better understanding of the global effects of these kinases during seed development and germination, it is necessary to identify additional proteins that are phosphorylated by these kinases. As the triple mutant seeds showed a very severe phenotype, it is possible that these three kinases function in the most upstream step in the ABA signaling pathway close to ABA receptors. In future, identification of factors regulating the activity of these protein kinases will help us to understand ABA-related events including seed maturation and the control of germination.
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Plant materials
Arabidopsis thaliana ecotype Col and Col CS60000 seeds were obtained from the Arabidopsis Biological Resource Center (ABRC; Alonso et al. 2003
). Seeds of srk2d and srk2i T-DNA insertion lines (GABI-Kat 807G04 and Salk_096546) were obtained from the Max Planck Institute for Plant Breeding Research (Rosso et al. 2003) and the ABRC, respectively. The srk2e mutant (Salk_008068) was kindly provided by Dr. Riichiro Yoshida of RIKEN (Yoshida et al. 2002). Seeds of A. thaliana ecotype Ws, Ler, abi5-1 (Ws background) and abi3-1 (Ler background) were obtained from the ABRC. The A. thaliana abi5-7 (E74-1, Col background) and abi3-6 mutants were kindly provided by Dr. Eiji Nambara at RIKEN (Nambara et al. 2002). Seeds of wild-type and mutant plants were germinated on agar plates containing germination medium and 3% sucrose as previously described (Qin et al. 2008). Plates were maintained in the dark at 4°C for 3 d to break dormancy (stratification) and were then moved into a photoperiod of 16 h light/8 h dark (40 ± 10 µmol photons m–2 s–1) at 22°C. Three-week-old plants were transferred from plates to soil in pots (Professional Soil, Dio Chemicals, Tokyo, Japan).
Quantitative real-time PCR analysis
Total RNA was isolated from seeds at 16–20 DAF using the RNAqueous kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. cDNA was synthesized from 0.1 µg of total RNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random hexamer primers according to the manufacturer’s instructions. qRT-PCR was performed as previously described (Nakashima et al. 2007). Primers used for qRT-PCR are shown in Supplementary Table S3.
Expression analysis of transgenic plants expressing promoter::SnRK2–GFP constructs
The ProSRK2D::SRK2D-GFP, ProSRK2E::SRK2E-GFP and ProSRK2I::SRK2I-GFP plasmids were constructed as described in the Supplementary Methods. The fusion constructs were introduced into Arabidopsis (Col) by Agrobacterium-mediated transformation. T2 or T3 seeds were used for experiments. Cellular localization of SRK2D, SRK2E and SRK2I during seed development was assessed in embryos at 12 DAF and transgenic plants expressing ProSRK2D::SRK2D-GFP, ProSRK2E::SRK2E-GFP and ProSRK2I::SRK2I-GFP at 2 DAI. GFP fluorescence was visualized using a LSM5 PASAL confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Physiological assays
For germination assays, seeds at 16– 20 DAF were plated on 0.5 x MS medium containing 1% sucrose. Seeds were then stratified at 4°C for 3 d and the presence of green cotyledons was scored at the indicated times. Chlorophyll was extracted from rosette leaves with 80% acetone and quantified by the previously described methods of Arnon (1949). Photographs of the dry seeds were taken through a VK-8500 color laser three-dimensional profile microscope (Keyence, Osaka, Japan).
ABA analysis
ABA was extracted from 30 mg of dry seeds using 90% methanol. Gas chromatography–mass spectrometry (GC-MS) analysis of ABA was performed using a previously described procedure with minor modifications (Iuchi et al. 2000). The column length was modified to 60 m and oven temperature was first increased to 200°C at a rate of 30°C min–1 followed by another increase to 250°C at a rate of 2.5°C min–1.
Microarray analysis
Genome-wide expression studies by an Arabidopsis 3 Oligo Microarray (Agilent Technologies, Santa Clara, CA, USA) were performed using wild-type and mutant seeds. In all experiments, gene expression was compared between wild-type and mutant seeds. Seeds were collected from mature siliques, dried for approximately 1 month in plants grown under high humidity conditions (80 ± 5%) after yellowing of siliques (16–20 DAF), or siliques at 16–20 DAF under the same conditions. Total RNA was isolated from seeds with the RNAqueous kit (Ambion, Austin, TX, USA) from each sample (pooled from more than six plants) and used for microarray analysis. Microarray experiments of the srk2d/e/i seeds were performed by analyzing two sets of plants independently grown under the same growth conditions (both for the wild type and the mutant). Microarray experiments with extracts from the abi5 and abi3 seeds were performed by analyzing the samples obtained from two different alleles. For each experiment, two slides were analyzed for a cy3 and cy5 dye-swap. Statistical analysis of the microarray data was conducted as previously described (Qin et al. 2008). All microarray data are available in the website http://www.ebi.ac.uk/microarray-as/ae with the accession numbers E-MEXP-1939, E-MEXP-1940 and E-MEXP-1941.
In-gel kinase assay
DNA fragments of ABI5 (ABI5b, Ser119–Gln190), mABI5 (mABI5b, Ser119–Gln190, Ser145 to alanine), AREB3 (AREB3b, Gly68–Asp 114), EEL (EELb, Leu54–His 100) and ABI3 (ABI3a, Glu264–Leu318; ABI3b, Gln433–His476) were amplified with KOD DNA polymerase (TOYOBO, Osaka, Japan) for the generation of GST fusions constructs. Primers used for amplification of the fragments are listed in Supplementary Table S3 online. The DNA fragments were cloned in-frame into the pGEX4T-1 plasmid. Purification of GST fusions from Escherichia coli and the extraction of proteins from mature dry seeds at 16–20 DAF were prepared as previously described (Uno et al. 2000). Phosphorylation of the purified polypeptides was analyzed using an in-gel kinase activity assay (Uno et al. 2000, Furihata et al. 2006).
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Supplementary data |
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Supplementary data are available at PCP online.
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Funding |
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The Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid for Scientific Research (C) (No. 21570054 to K.N.); the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN) (to K.S. and K.Y.-S.); the Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan (to K.S. and K.Y.-S.).
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We are grateful for the technical support provided by E. Ohgawara, K. Murai, K. Amano, E. Kishi and K. Yoshiwara of JIRCAS, and H. Kobayashi of RIKEN. We thank M. Fujita of RIKEN for helping with the generation of plasmid constructs. We thank T. Ito of RIKEN, R. Yoshida of RIKEN (currently at the National Institute of Agrobiological Sciences, Tsukuba) and E. Nambara of RIKEN (currently at the University of Toronto) for supplying mutants.
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(Received May 18, 2009; Accepted June 4, 2009)
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