Plant Physiology Preview. Published on September 11, 2017, as DOI:10.1104/pp.17.00946
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Title: Transcription factor OsWRKY53 positively regulates brassinosteroid signaling and plant architecture
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Running title: OsWRKY53 regulates rice brassinosteroid signaling
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Authors: Xiaojie Tian 1, 2 , Xiufeng Li 1, Wenjia Zhou 1, 2, Yuekun Ren 1, 2, Zhenyu
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Wang 1, Zhiqi Liu 3, Jiaqi Tang1, 2, Hongning Tong 4, Jun Fang 1 and Qingyun Bu 1,*
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1
Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean
Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China 2
Graduate University of Chinese Academy of Sciences, Beijing 100049, China
3
College of Life Science, Northeast Forestry University, Harbin 150040, China
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Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing
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100081, China
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Author contributions:
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Q.B. conceived and supervised the project. X.T. performed most of experiments. X. L.,
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W. Z., Y. R., Z. W., Z. L., and J. T. assisted the experiments. Q.B. and X.T. analyzed
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the data and wrote the article with the contribution from H. T. and J. F.
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Funding information:
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This study was supported by National Natural Science Foundation of China (Grant No.
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31671653), the Strategic Priority Research Program of Chinese Academy of Sciences
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(Grant No. XDA08040101), the Natural Science Foundation of Heilongjiang (Grant
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No. ZD2015005), and the Hundred–Talent-Program of Chinese Academy of Sciences
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to Q.Y. Bu.
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One sentence summary: OsWRKY53 is a novel positive regulator of rice BR
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signaling and its phosphorylation by OsMAPK6 is indispensable for its function in rice
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*
Corresponding author:
E-mail:
[email protected]
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Copyright 2017 by the American Society of Plant Biologists
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Abstract
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Brassinosteroids (BRs) are a class of steroid hormones regulating multiple
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aspects of plant growth, development, and adaptation. Compared with extensive
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studies in Arabidopsis, the mechanism of BR signaling in rice is less understood. Here,
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we identified OsWRKY53, a transcription factor involved in defense responses, as an
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important regulator of rice BR signaling. Phenotypic analyses showed that
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OsWRKY53 overexpression led to enlarged leaf angles and increased grain size, in
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contrast to the erect leaves and smaller seeds in oswrky53 mutant. In addition, the
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oswrky53 exhibited decreased BR sensitivity, whereas OsWRKY53 overexpression
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plants were hypersensitive to BR, suggesting that OsWRKY53 positively regulates
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rice BR signaling. Moreover, we show that OsWRKY53 can interact with and be
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phosphorylated by OsMAPKK4-OsMAPK6 cascade, and the phosphorylation is
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required for the biological function of OsWRKY53 in regulating BR responses.
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Furthermore, we found that BR promotes OsWRKY53 protein accumulation but
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represses OsWRKY53 transcript level. Taken together, this study revealed the novel
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role of OsWRKY53 as a regulator of rice BR signaling, and also suggested a potential
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role of OsWRKY53 in mediating the crosstalk between the hormone and other
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signaling pathways.
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Key words: OsWRKY53, Rice, Brassinosteroids, Phosphorylation.
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Introduction
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Brassinosteroids (BRs) are a group of plant-specific steroidal hormones that play
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various roles in plant growth, development, and stress responses. In the past decades,
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extensive studies have identified numerous BR-signaling components to establish a
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signaling network, and further provided a global view of BR function in the model
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plant Arabidopsis (Kim and Wang, 2010). Briefly, BR is recognized by the
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membrane-localized receptor BRI1 (BR insensitive 1) and its coreceptor BAK1
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(BRI1-associated receptor kinase 1), and form BRI1–BR–BAK1 complex (Li and
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Chory, 1997; Li et al., 2002; Sun et al., 2013). Transphosphorylation between BRI1
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and BAK1 activates BRI1 which then phosphorylates cytoplasmic kinases BSKs (BR
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signaling kinases) and CDG1 (Constitutive differential growth 1), and activation of
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BSKs/CDG1 leads to phosphorylation and activation of the protein phosphatase
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BSU1 (bri1-suppressor 1) (Tang et al., 2008; Wang et al., 2008; Kim et al., 2011).
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BSU1 dephosphorylates and inactivates the GSK3/Shaggy-like kinase BIN2 (BR
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insensitive 2) (Kim et al., 2009).Therefore, in the presence of BR, BIN2 is inhibited,
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which allows BZR1 (Brassinazole-resistant 1) and BES1 (BRI1 EMS suppressor 1) to
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be dephosphorylated by PP2A (Protein phosphatase 2A) (Tang et al., 2011). Finally,
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dephosphorylated BZR1 and BES1 regulate the expression of numerous
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BR-responsive genes through binding to BR response element or E-box cis-element
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(Yin et al., 2005; Sun et al., 2010; Yu et al., 2011).
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In contrast to the tremendous progress of BR signaling in Arabidopsis, relatively
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fewer components have been characterized in rice (Zhang et al., 2014). So far, many
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known rice BR signaling components (e.g., OsBRI1, OsBAK1, OsGSK2, OsBZR1
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and OsBSK) have orthologues in Arabidopsis and served
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(Yamamuro et al., 2000; Bai et al., 2007; Li et al., 2009; Tong et al., 2012; Zhang et
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al., 2016). However, some components including OsDLT (Dwarf and low tillering),
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OsTUD1 (Taihu dwarf1), and OsLIC (Leaf and tiller angle increased controller) have
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no counterpart identified in Arabidopsis, implying that a rice specific BR signaling
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pathway might exist (Tong et al., 2009; Zhang et al., 2012; Hu et al., 2013). In
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addition, Helix-Loop-Helix (HLH) proteins such as OsBU1 (Brassinsteroid
conserved functions
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upregulated 1), OsBUL1 (Brassinsteroid upregulated like 1), and ILI1 (Increased
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laminar inclination 1) can positively regulate rice BR signaling (Tanaka et al., 2009;
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Zhang et al., 2009; Jang et al., 2017). RLA1 (Reduced leaf angle 1)/SMOS1 (Small
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organ size 1) was characterized as a positive regulator of BR signaling, which can
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form complex with OsBZR1 and OsDLT to co-regulate the expression of downstream
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genes (Hirano et al., 2017; Qiao et al., 2017). Very recently, ELT1 (Enhanced leaf
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inclination and tiller number 1), a receptor-like protein, was shown to promote BR
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signaling through interacting with and suppressing the degradation of OsBRI1 (Yang
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et al., 2017).
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Plants WRKY transcription factors contain one or two conserved WRKYGQK
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sequence followed by a C2H2 or C2HC zinc finger motif. Accumulating evidence
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revealed that the WRKY proteins play diverse roles in responses to biotic and abiotic
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stresses, and are involved in various processes of plant growth and development by
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regulating the expression of target genes via binding to the W-box cis-element
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(Rushton et al., 2010). In rice, WRKY family has at least 102 members and only a few
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members have been functionally characterized (Xie et al., 2005; Sun et al., 2014).
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Most of the identified rice WRKY members are involved in plant biotic stress
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response, including OsWRKY70, OsWRKY53, OsWRKY13, OsWRKY45, and
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OsWRKY28 (Chujo et al., 2007; Qiu et al., 2007; Shimono et al., 2007; Tao et al.,
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2009; Chujo et al., 2013; Hu et al., 2015; Ma et al., 2015). Recently, OsWRKY70 and
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OsWRKY53 were proposed to function in trade-off mechanism between biotic stress
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response and growth; however, the mechanism by which they regulate plant growth
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remains elusive (Hu et al., 2015; Ma et al., 2015).
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MAPK (Mitogen-activated protein kinase) cascade is comprised of three
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components: MEKKs (MAPK kinase kinases), MEKs (MAPK kinases), and MAPKs,
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and has pivotal roles in plant innate immunity (Ishihama et al., 2011; Adachi et al.,
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2015). Several Group Ia members of WRKY proteins have been shown as direct
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downstream targets of MAPK cascade. MAPK can interact with and phosphorylate
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Group Ia WRKYs via five conserved Ser in SP cluster (Clustered Pro-directed Ser
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residues), and phosphorylation of WRKY proteins by MAPK can enhance the DNA 4 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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binding activity or transcriptional activity of WRKY proteins (Qiu et al., 2008;
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Ishihama et al., 2011; Shen et al., 2012; Chujo et al., 2014; Yoo et al., 2014). In rice,
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the mutation of either OsMAPKK4 or OsMAPK6 leads to BR-deficient phenotypes
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(dwarfism, erect leaf, and smaller grain size), decreased BR sensitivity, and disrupted
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expression of BR-related genes, which suggested that OsMAPKK4-OsMAPK6
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cascade is involved in rice BR signaling (Duan et al., 2014; Liu et al., 2015). However,
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their downstream targets remain elusive.
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Here, we show that OsWRKY53 plays a positive role in rice BR signaling and
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acts downstream of OsMAPKK4-MAPK6 cascade. Overexpression and knockout of
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OsWRKY53 led to contrasting BR-related phenotypes. Genetic analysis showed that
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OsWRKY53 acts downstream of OsBRI1 receptor. In addition, we show that the
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phosphorylation of OsWRKY53 by OsMAPKK4-MAPK6 is critical for its biological
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function. Moreover, OsWRKY53 protein is enhanced by BR, and can repress its own
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expression. Taken together, our results suggest that OsWRKY53 is a novel fine tuner
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of rice BR signaling.
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Results
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Characterization of OsWRKY53 Overexpression Lines
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We previously generated a rice mutant library overexpressing different
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transcription factors directed by the maize (Zea mays) Ubiquitin promoter. A number
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of lines carrying OsWRKY53 (Os05g0343400) showed obviously increased leaf
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angles (described in detail below), and were chosen for further study.
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Generally, OsWRKY53-OEs (OsWRKY53 overexpression lines) showed greatly
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enlarged leaf angles and dwarfism (Figure 1A to 1C). At 3-leaf stage, the angles of the
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first leaf in WT (wild type) were about 30 degree, while the angles of the parallel leaf
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in OsWRKY53-OE reached almost 60 degree (Supplemental Figure 1A, 1B). With the
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growth of plant, the larger leaf angles of OsWRKY53-OE appeared to be more evident.
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At heading stage, the angles of flag leaf in OsWRKY53-OE were about -120 degree,
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which is significantly larger than that of WT at the same stage (Figure 1B, 1C). It has
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been shown that enlarged leaf inclination is mainly associated with abnormal
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development of lamina joint (Sun et al., 2015). Morphological observation revealed
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that the collar length of adaxial surface in OsWRKY53-OE is significantly increased
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compared with that in WT (Supplemental Figure 1C). Scanning electron microscopic
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observation of lamina joint showed that the adaxial surface in OsWRKY53-OE is not
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smooth and contain some protuberance compared with that in WT (Supplemental
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Figure 1D). Further cytological observation showed that the cell length of adaxial
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surface in OsWRKY53-OE is markedly longer than that in WT (Supplemental Figure
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1E, 1F), which contributed to the enlarged leaf angles of OsWRKY53-OE.
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In addition, compared with WT, both the grain length and grain width of
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OsWRKY53-OE increased significantly (Figure 1D to 1F, Supplemental Figure 11A to
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11C). Scanning electron microscopic observation of the spikelet hull showed that the
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epidermal cells of both palea and lemma in the OsWRKY53-OE are much longer than
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that in WT, indicating that increased grain size is mainly attributed to the cell
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enlargement (Figure 1G to 1I). In agreement with this result, a number of genes
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associated with cell expansion were up-regulated in the panicles of the
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OsWRKY53-OE (Supplemental Figure 2). Moreover, compared with WT, the leaves 7 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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of OsWRKY53-OE appeared to be pale green (Supplemental Figure 3), and the plant
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height was also reduced (Supplemental Figure 4D).
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Most of the independent OsWRKY53-OEs showed similar phenotypes as
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described above (Supplemental Figure 4A, 4B), thus excluding the possibility that the
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phenotypes are caused by mutations of other endogenous genes coming from T-DNA
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insertion or tissue culture. We also showed the expression of OsWRKY53 is indeed
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over-expressed in three representative OsWRKY53-OE plants (Supplemental Figure
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4B). Further immunoblotting analyses confirmed the accumulation of OsWRKY53
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proteins in these OsWRKY53-OEs (Supplemental Figure 4C). Taken together, we
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concluded that the overexpression of OsWRKY53 leads to enlarged leaf angles and
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increased grain size in rice.
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OsWRKY53 Positively Regulates BR Signaling in Rice
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The increased leaf angles and enlarged grain size in OsWRKY53-OEs resembled
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many of the typical enhanced-BR-signaling mutants, such as osbzr1-D, GSK2-RNAi,
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OsBU1-OE, and mRLA1-OE (Tanaka et al., 2009; Tong et al., 2012; Qiao et al., 2017).
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Therefore, we hypothesized that OsWRKY53 might be involved in rice BR signaling.
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We then performed the BR-induced lamina inclination assay to test the BR sensitivity
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of OsWRKY53-OE in response to different concentrations of 24-epibrassinolide
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(24-epiBL), an active form of BRs (Tanabe et al., 2005). The results showed that
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lamina bending of OsWRKY53-OE was obviously much more sensitive than that of
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WT (Figure 2A, 2B). After incubation in 10 nM 24-epiBL for 3 days, leaf angles in
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OsWRKY53-OE reached around ~140 degree, dramatically higher than those in WT
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which were about ~70 degree (Figure 2A, 2B). This result strongly suggested that
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overexpression of OsWRKY53 leads to enhanced BR responses.
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It is well known that expressions of BR biosynthesis genes are usually negatively
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feedback regulated by BR signaling in both rice and Arabidopsis (Tong et al., 2012;
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Zhang et al., 2016; Qiao et al., 2017). The enhancement of BR signaling in
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OsWRKY53-OE prompted us to investigate whether OsWRKY53 is involved in this
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process. We analyzed the expression of BR biosynthesis genes including D2,
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OsDWF4, and D11, and found all of them have significantly decreased expression in
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OsWRKY53-OE plants compared with WT (Figure 2C), indicating that OsWRKY53 is
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involved in feedback inhibition of BR biosynthesis genes. In addition, we checked the
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expression of several BR responsive genes, and found that the expression of OsBU1
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and OsXTR1 was indeed increased in OsWRKY53-OE plants (Figure 2D). Taken
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together, these results provided strong evidence that OsWRKY53 positively regulates
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BR signaling in rice.
191 9 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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Characterization of oswrky53 Mutant
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To further confirm the function of OsWRKY53 in regulating BR signaling, we
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generated oswrky53 mutant via CRISPR/Cas9 mediated genome editing technology.
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Two independent oswrky53 mutant allele, oswrky53-1 and oswrky53-2, were
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identified and the mutation sites were characterized by DNA sequencing (Figure 3A
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and Supplemental Figure 5). In contrast to OsWRKY53-OE, the leaves of oswrky53
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were more erect than those of WT (Figure 3B to 3D). The plant height of oswrky53
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was also slightly decreased (Figure 3E). In addition, the mutants produced obviously
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smaller grains, with reduced seed length and seed width (Figure 3F to 3H,
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Supplemental Figure 11D to 11F). Moreover, oswrky53 were dark green compared
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with WT (Supplemental Figure 6). These phenotypes of oswrky53 were similar to
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those of typical BR-deficient or BR defective mutants, such as d11, d61, and 10 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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GSK2-OE (Yamamuro et al., 2000; Tanabe et al., 2005; Tong et al., 2009; Tong et al.,
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2012; Qiao et al., 2017). Furthermore, lamina bending assay showed that oswrky53 is
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hyposensitive to BR, which is opposite to OsWRKY53-OE (Figure 3I, 3J). Together,
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oswrky53 exhibited BR deficient phenotypes and decreased BR response, which
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further supported our hypothesis that OsWRKY53 plays positive roles in regulating
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BR signaling in rice.
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OsWRKY53 Acts Downstream of OsBRI1
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To analyze whether OsWRKY53 functions in the primary BR signaling pathway,
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we crossed OsWRKY53-OE with d61-2, a weak allele of the BR receptor OsBRI1
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mutant (Yamamuro et al., 2000), and identified the double mutant (Figure 4A).
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Compared with WT, OsWRKY53-OE exhibited larger leaf angles and increased seed
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size, while d61-2 showed smaller leaf angles and decreased seed size (Figure 1A)
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(Yamamuro et al., 2000). However, for both leaf angles and seed size, d61-2
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OsWRKY53-OE double mutant was remarkably larger than d61-2 (Figure 4B to 4F,
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Supplemental Figure 11G to 11I), suggesting that overexpression of OsWRKY53 can
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largely rescue BR signaling deficiency phenotypes of d61-2. This result suggested
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that OsWRKY53 is involved in BR signaling and might act downstream of BR
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receptor.
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OsWRKY53 Interacts with and Is Phosphorylated by OsMAPK6
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OsWRKY53 belongs to the group Ia subset of WRKY family, and contains two
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WRKY domains followed by C2H2-type zinc finger motif (Xie et al., 2005; Chujo et
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al., 2007; Hu et al., 2015). It has been shown that group Ia WRKY proteins can be
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phosphorylated by the MAPK cascade, and the phosphorylation can affect the DNA
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binding activity or transcription activity of WRKY proteins (Ishihama et al., 2011;
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Chujo et al., 2014; Adachi et al., 2015). Interestingly, previous studies also showed
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that OsWRKY53 can interact with and be phosphorylated by OsMAPK3 and
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OsMAPK6, and the phosphorylated sites were located in the SP cluster at the amino
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terminal domain of OsWRKY53 (Chujo et al., 2014; Hu et al., 2015). Consistent with
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these results, we confirmed that OsWRKY53 can interact with OsMAPK6 in yeast
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two hybrid assays (Figure 5A). Further BiFC (Bimolecular Fluorescence
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Complementation) and LCI (LUC Complementation Imaging) assays confirmed that
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OsWRKY53 can interact with OsMAPK6 in planta system (Figure 5B, 5C). In 12 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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addition, we showed that the OsWRKY53 was weakly phosphorylated by OsMAPK6,
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whereas this phosphorylation was greatly enhanced in the presence of constitutively
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active form of OsMAPKK4 (Figure 5E). However, the phosphorylation of
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OsWRKY53 by OsMAPKK4-OsMAPK6 was markedly decreased when the five
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conserved Ser residues in the SP cluster were mutated to Ala, indicating that the five
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conserved Ser residues are the critical phosphorylation sites of OsWRKY53 by
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OsMAPKK4-OsMAPK6 cascade (Figure 5D, 5E).
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To examine the effect of OsWRKY53 phosphorylation by OsMAPK6, EMSA
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(Electrophoretic mobility shift assay) was t performed, and the result indicated that
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phosphorylation of OsWRKY53 by OsMAPKK4-OsMAPK6 cascade can markedly 13 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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enhance the DNA binding activity of OsWRKY53 protein to the W-box containing
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DNA sequence (Supplemental Figure7A). In addition, we co-expressed OsWRKY53
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and OsMAPKK4-OsMAPK6 cascade in rice protoplast transient expression assay,
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and found that the addition of OsMAPKK4 and OsMAPK6 does not change the
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protein
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OsMAPKK4-OsMAPK6 cascade cannot affect OsWRKY53 stability (Supplemental
254
Figure 7B).
level
of
OsWRKY53,
implying
that
phosphorylation
by
255 256
Phosphorylation of OsWRKY53 by OsMAPKK4-OsMAPK6 Cascade Is Critical
257
for the Function of OsWRKY53 in Regulating BR Response
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To test biological significance of phosphorylation of OsWRKY53 by
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OsMAPKK4-OsMAPK6 cascade, we mutated the five conserved Ser in SP cluster to
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Ala (generating OsWRKY53 (SA)) or Asp (generating OsWRKY53 (SD)) to mimic the
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inactive or active phosphorylated forms of OsWRKY53, respectively (Figure 5D). We
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then generated the transgenic lines OsWRKY53 (SA)-OEs and OsWRKY53 (SD)-OEs
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in which OsWRKY53 (SA) and OsWRKY53 (SD) were overexpressed, respectively.
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Interestingly, we found that OsWRKY53 (SD)-OEs showed much stronger phenotypes
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than OsWRKY53-OEs, including larger leaf angles, increased grain size and dwarfism
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(Figure 6A to 6E, Supplemental Figure 11J, 11K). In addition, OsWRKY53 (SD)-OEs
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were more hypersensitive to BR than OsWRKY53-OEs as shown by lamina bending
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assay (Figure 6F, 6G). Furthermore, compared with that in OsWRKY53-OEs,
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expression of the three BR biosynthesis genes including D2, OsDWF4, and D11 was
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decreased more significantly in OsWRKY53 (SD)-OEs (Figure 6H). Together, these
271
results
272
OsMAPKK4-OsMAPK6 cascade can enhance the function of OsWRKY53 in BR
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signaling. In contrast, OsWRKY53 (SA)-OEs failed to produce any obvious
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phenotypes, even the expression level of OsWRKY53 was much higher than that in
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OsWRKY53-OE (Supplemental Figure 8, Supplemental Figure 11L, 11M), suggesting
276
that these phosphorylation sites are critical for OsWRKY53 functions in regulating
277
BR-related
278
OsMAPKK4-OsMAPK6-mediated phosphorylation of OsWRKY53 is indispensable
279
for function of OsWRKY53 in regulating BR signaling.
suggested
that
phenotypes.
constitutive
phosphorylation
Collectively,
these
of
results
OsWRKY53
indicated
by
that
280 281
OsWRKY53 Is Promoted by BR and Negatively Feedback Regulated by Itself
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The fact that OsWRKY53 is involved in BR responses prompted us to test
283
whether expression of OsWRKY53 is regulated by BR. For this purpose,
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two-week-old seedlings of WT were treated with 1 μM 24epi-BL, and then the
285
transcript levels of OsWRKY53 were examined by RT-qPCR assay. As shown in
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Figure 7A, OsWRKY53 transcripts were decreased upon BR treatment, similar to the
287
expression of OsDWF4 gene. We also investigated the BR effects on OsWRKY53 15 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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protein stability using MYC-OsWRKY53-OE plants in which OsWRKY53 fused with
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MYC tag was overexpressed. MYC-OsWRKY53-OE lines were characterized and
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show similar phenotype to OsWRKY53-OE (Supplemental Figure 9). Interestingly,
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unlike OsWRKY53 transcript, OsWRKY53 protein level was enhanced by BR
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treatment (Figure 7B). By contrast, when the MYC-OsWRKY53-OE plants were
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grown on medium containing brassinozole (BRZ), a BR biosynthesis inhibitor, the
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MYC-OsWRKY53 protein level decreased (Figure 7C), whereas the RNA level of
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OsWRKY53 did not change (Figure 7D). These results indicated that BR can promote
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the OsWRKY53 protein stability. It has been reported that the transcript and protein 18 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
297
levels of several key components in BR signaling (e.g., DLT, BZR1, RLA1) also show
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a similar BR responsive pattern, which tends to represent a common negative
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feedback regulation mechanism in fine-tuning BR signaling (Tong et al., 2009; Qiao
300
et al., 2017).
301
Considering that BR oppositely regulates the expression pattern of OsWRKY53
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transcript and OsWRKY53 protein level (Figure 7A, 7B), we asked if OsWRKY53
303
can regulate its own expression. RT-qPCR assay was performed to check the
304
expression of native OsWRKY53 in oswrky53 mutants. We showed that the expression
305
of OsWRKY53 in both of these two mutants was increased (Figure 7F). Then, we
306
checked the expression of native OsWRKY53 in OsWRKY53-OE lines using the
307
5`-UTR and 3`-UTR specific primers respectively (Figure 7E). As shown in Figure
308
7G, in OsWRKY53-OE lines with increased expression of ectopic OsWRKY53, the
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native OsWRKY53 expression was reduced. This result was further supported by the
310
transient expression assay in rice protoplast, in which 35Spro:OsWRKY53 can suppress
311
the expression of LUC fused with the native OsWRKY53 promoter (Figure 7H, 7I).
312
There are two W-box elements in OsWRKY53 promoter which could be bound by
313
WRKY transcription factors (Figure 7E). We then performed EMSA to test if
314
OsWRKY53 binds to its native promoter. As shown in 7J, MBP-OsWRKY53 fusion
315
protein bound to its own promoter in a W-box-dependent manner. Furthermore, ChIP
316
(Chromatin immunoprecipitation) assay also indicated that OsWRKY53 was
317
associated with W-box region of its own promoter (Figure 7K). Taken together, these
318
results suggested that OsWRKY53 protein can directly suppress its own expression,
319
which partially explained the negative feedback regulation of OsWRKY53, and
320
implied that OsWRKY53 might be a fine tuner in rice BR signaling pathway.
321
In addition, we analyzed the expression of OsWRKY53 in different tissues by
322
RT-qPCR. We showed that OsWRKY53 transcripts can be detected in various tissues,
323
and have the highest expression in lamina joint (Supplemental Figure 10A). In
324
addition, we also generated OsWRKY53 pro:GUS transgenic plants. In agreement with
325
RT-qPCR results, GUS staining of transgenic plants showed that activity of the
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promoter could be detected in different organs, preferentially higher in lamina joint 19 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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compared with other tissues (Supplemental Figure 10B). Altogether, the spatial
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expression pattern of OsWRKY53 was correlated with one of its physiological
329
functions that controlled the leaf angles.
330 331
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Discussion
333
Function of OsWRKY53 in Regulating BR Signaling
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In the present study, we provide substantial physiological, genetic and
335
biochemical evidence for the involvement of OsWRKY53 in rice BR signaling
336
pathway.
337
including enlarged leaf angles, increased seed size (Figure 1, Supplemental Figure 1
338
to 4), whereas oswrky53 mutant exhibited BR-deficient phenotypes, such as erect
339
leaves, smaller seed size, and dark green leaves (Figure 3, Supplemental Figure 6).
340
Second, BR sensitivity assay of lamina bending in OsWRKY53-OE and oswrky53
341
demonstrated that OsWRKY53 is a positive regulator of BR signaling (Figure 2,
342
Figure 3). Third, genetic analysis revealed that the OsWRKY53 overexpression can
343
largely rescue BR deficiency phenotypes of BR receptor mutant d61-2 (Figure 4).
344
Forth, OsWRKY53 interacts with and is phosphorylated by OsMAPK6, and the
345
phosphorylation is required for BR-related function of OsWRKY53 (Figure 5, Figure
346
6). Finally, we showed that the protein level of OsWRKY53 is promoted by the BR
347
treatment, and the transcript level of OsWRKY53 is suppressed by BR possibly
348
through a negatively auto-feedback regulation (Figure 7). Taken together, these results
349
demonstrated an important biological role of OsWRKY53 in regulating rice BR
350
signaling (Supplemental Figure 12).
351
Functional Diversity of OsWRKY53
First,
OsWRKY53-OE
showed
enhanced-BR-signaling
phenotypes,
352
WRKY family transcription factors play a variety of developmental and
353
physiological roles in plants. Rice genome contains more than 102 WRKY genes
354
which were divided into four subgroups (Xie et al., 2005; Sun et al., 2014). Up to now,
355
several rice WRKY genes have been functionally characterized, and most of them
356
were involved in stress responses. For instance, OsWRKY31, OsWRKY33 and
357
OsWRKY53 can positively regulate pathogen infection response (Chujo et al., 2007;
358
Zhang et al., 2008; Koo et al., 2009), while OsWRKY53 and OsWRKY70 negatively
359
regulate herbivore resistance (Hu et al., 2015; Li et al., 2015). Besides, OsWRKY30
360
and OsWRKY42 play roles in drought response and senescence process respectively
361
(Shen et al., 2012; Han et al., 2014). By contrast, a role of WRKY genes in controlling 21 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
362
growth and development is less known. Similar to OsWRKY53-OE plants,
363
overexpression of OsWRKY70 led to a severe reduction in plant height (Li et al.,
364
2015). In addition, a decreased expression of OsWRKY78 resulted in semi-dwarf and
365
small grain due to the reduced cell length (Zhang et al., 2011), which is similar to
366
oswrky53 mutant. Interestingly, OsWRKY53, OsWRKY70 and OsWRKY78 belong
367
to the same subgroup Ia of WRKY genes and show high sequence similarity (Xie et
368
al., 2005), implying that this subgroup of WRKY genes may play common roles in
369
regulating growth and development. However, the underlying mechanisms might be
370
diverse. OsWRKY70 overexpression lines showed strong dwarfism, but no observable
371
effect on leaf angles and seed size, while OsWRKY70 RNAi lines were similar to WT,
372
and OsWRKY70 was suggested as growth suppressor by inhibiting GA biosynthesis
373
(Li et al., 2015). In contrast, in this study, both OsWRKY53-OE and oswrky53 showed
374
opposite BR-related phenotypes and BR sensitivities. Therefore, we proposed that
375
OsWRKY53 is a positive regulator of rice BR signaling. It is worthy to note that, two
376
previous studies also generated the OsWRKY53 overexpression lines; however, while
377
one study showed that OsWRKY53-OE plants have dwarfism and larger leaf angles
378
(Hu et al., 2015), another study showed the normal growth of the OsWRKY53-OE
379
plants (Chujo et al., 2007). We speculated that these differences may either result from
380
different rice varieties used for the analyses or different expression levels of
381
OsWRKY53 in transgenic lines.
382
Interestingly, it had been shown that OsWRKY53 can enhance the defense
383
response to pathogen (Chujo et al., 2007; Chujo et al., 2014), and our data show that
384
OsWRKY53 positively regulates BR signaling as well. It is reasonable to speculate
385
that active OsWRKY53 can positively regulate both BR signaling and defense
386
response, which greatly support the previous conclusion that BR can enhance the
387
pathogen response in rice (Nakashita et al., 2003), and also suggest that OsWRKY53
388
might be a new node mediating the crosstalk between the growth vs defense response.
389
Moreover, OsWRKY53 transcription is induced by pathogen infection, wounding
390
and herbivore attack (Chujo et al., 2007; Hu et al., 2015), whereas is repressed by BR
391
(this study). Nevertheless, OsWRKY53 positively regulated BR (this study) and 22 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
392
pathogen response (Chujo et al., 2007; Chujo et al., 2014), but negatively regulated
393
herbivore-induced defenses (Hu et al., 2015), suggesting that OsWRKY53 might
394
mediate crosstalk among diverse signaling pathway, and play diverse roles in
395
context-dependent conditions (Supplemental Figure 12).
396
MAPK Module in Regulating Rice BR Signaling
397
Plant MAPK plays crucial roles in multiple signal transduction pathways, such as
398
plant immunity response and hormone signaling (Tena et al., 2001; Ishihama et al.,
399
2011; Shen et al., 2012; Chujo et al., 2014; Adachi et al., 2015). Rice dwarf and small
400
grains1 (dsg1) and small grains1 (smg1) were caused by mutations in OsMAPK6 and
401
OsMAPKK4 respectively, and both showed small grain phenotype (Duan et al., 2014;
402
Liu et al., 2015). OsMAPK6 interacts with OsMAPKK4, implying that
403
OsMAPKK4-OsMAPK6 cascade was also involved in regulating seed development
404
(Liu et al., 2015). Further studies indicated that the mechanism of OsMAPK6 and
405
OsMKK4 in controlling seed size is through regulating BR response and expression
406
of BR-related genes (Duan et al., 2014; Liu et al., 2015). However, the downstream
407
target of OsMAPKK4-OsMAPK6 cascade involved in controlling seed size and BR
408
signaling has not been identified. In rice, OsMAPK6 interacts with and can
409
phosphorylate OsWRKY53, and OsMAPKK4-OsMAPK6-OsWRKY53 module plays
410
key roles in pathogen, wounding and herbivore-induced defenses response (Chujo et
411
al., 2007; Chujo et al., 2014; Yoo et al., 2014; Hu et al., 2015). In this study, we
412
showed that OsMAPK6 can phosphorylate OsWRKY53 in OsMAPKK4-dependent
413
manner, and this phosphorylation is required for the biological function of
414
OsWRKY53 in regulating BR-related responses. Collectively, we proposed that
415
OsWRKY53 is one of the downstream targets of OsMAPKK4-OsMAPK6 cascade in
416
mediating BR signaling as well (Supplemental Figure 12). Very interestingly, during
417
preparation of this manuscript, it was reported that the Arabidopsis WRKY46/54/70
418
are positively involved in BR regulated growth and negatively involved in drought
419
responses, WRKY54 can be phosphorylated by BIN2 and interact with BES1 to
420
cooperatively regulate the expression of target genes (Chen et al., 2017). This findings
421
in Arabidopsis greatly support our conclusion that OsWRKY53 can positively 23 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
422
regulate BR signaling in rice, which implied that the some WRKY family members
423
may play conserve roles in regulating plant BR signaling to some extent. However,
424
their function mechanisms might not be similar. First, AtWRKY46/54/70 are not the
425
OsWRKY53’s homologs with the highest sequence similarity. AtWRKY46/54/70
426
belongs to group III WRKY proteins which contains one WRKY domain, whereas
427
OsWRKY53 contains two WRKY domains and belongs to group I WRKY proteins.
428
Second, we showed that phosphorylation of OsWRKY53 by OsMAPKK4/OsMAPK6
429
is required for function of OsWRKY53, and the five Ser residues in SP cluster of
430
OsWRKY53 are the critical phosphorylation sites. But there is no SP cluster in
431
AtWRKY46/54/70, and whether AtWRKY46/54/70 can be phosphorylated by MAPK
432
is not known. Third, we need to test the physical and genetic interaction of
433
OsWRKY53 with rice BR signaling components (OsBZR1 and OsGSK2) to further
434
investigate the function mechanism of OsWRKY53 in rice BR signaling pathway.
435
Collectively, there is still much work to be done to prove whether function
436
mechanisms of OsWRKY53 and AtWRKY46/54/70 are similar or not.
437
WRKY family genes function as transcriptional activator or repressor by binding
438
W-box motif in promoters of target genes (Han et al., 2014; Adachi et al., 2015).
439
Previously, it was reported that OsWRKY53 can function as transcriptional activator
440
in pathogen response (Chujo et al., 2007; Chujo et al., 2014), but as a suppressor in
441
herbivore response (Hu et al., 2015), although the direct target genes and the
442
underlying mechanism remained unknown. In this study, we showed that OsWRKY53
443
could function as a transcription repressor to inhibit its own expression via directly
444
binding to W-box elements in promoter. Considering the functional diversity of
445
OsWRKY53, it is reasonable to speculate that OsWRKY53 might regulate diverse
446
target genes in response to differential environmental stimuli, and it is of interest to
447
identify the direct target genes that are involved in specific functions of OsWRKY53.
24 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
448
Materials and Methods
449
Plant Materials and Growth Conditions
450
Rice cultivar Longjing 11 (Oryza sativa ssp. japonica) was used for generate
451
OsWRKY53 transgenic plants. d61-2 (Yamamuro et al., 2000) was used to develop
452
double mutant. Plants were grown in the field (natural long day condition) or growth
453
chamber at 30°C for 14 h (day) and 24°C for 10 h (night). For transient induction
454
analysis, two-week-old seedlings were sprayed with 1 μM 24-epiBL (Sigma, E1641),
455
and then whole plants were harvested at various time points. For BRZ treatment, the
456
3-day-old plants were moved to medium with 5 μM BRZ for 7 days (Sigma, SML
457
1406). For BR-related gene expression, two-week-old seedlings were sampled.
458
Materials were collected and frozen in liquid nitrogen immediately or stored at -80°C
459
for RNA extraction.
460 461
Lamina Joint Assay
462
The lamina joint assay using excised leaf segments was performed as described
463
previously (Tong et al., 2009). Simply, uniform germinated seeds were selected and
464
grown in the dark for 8 days at 30°C. The entire segments comprising 1 centimeter of
465
the second leaf blade, the lamina joint and 1 centimeter of the leaf sheath were
466
incubated in various concentrations of 24-epiBL for 48 h in dark. The angles of
467
lamina
468
(http://rsbweb.nih.gov/ij/).
joint
bending
were
measured
using
ImageJ
software
469 470
Generation of Transgenic Plants and Double Mutant
471
To generate the OsWRKY53 overexpression plant, the coding regions of
472
OsWRKY53 (Os05g0343400) was amplified by PCR, and cloned into p1390U, and
473
Ubiquitinpro: OsWRKY53 construct in pCAMBIA1300 backbone was made. For
474
creating 35Spro: MYC-OsWRKY53, OsWRKY53 in pENTRY vector was cloned into
475
pGWB18 through LR reaction (Nakagawa et al., 2007). For the generation of
476
oswrky53 mutant, the target sequence (Supplemental Table 1) was synthesized and
477
ligated with respective sgRNA catastases, and then were sequentially ligated into the 25 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
478
CRISPR/Cas9 binary vectors pYLCRISPR/Cas9Pubi-H as described (Ma et al., 2015).
479
To generate OsWRKY53(SA) and OsWRKY53(SD), the mutation was introduced by
480
point mutation kit followed the manufacture’s instruction. Primers used to generate
481
the above-mentioned constructs are listed in Supplemental Table 1, and all of the
482
constructs were confirmed by sequencing. The constructs were introduced into
483
Agrobacterium tumefaciens strain EHA105. Cultivar Longjing11 was used as the
484
recipients for Agrobacterium-mediated transformation as described previously (Hiei
485
et al., 1994). Homozygous T2 transgenic rice seedlings were used for the phenotype
486
analysis.
487
To generate d61-2 OsWRKY53-OE, we cross d61-2 (Yamamuro et al., 2000) with
488
OsWRKY53-OE#1 (Figure 1 A). The mutation site and expression level of
489
corresponding genes was examined by DNA sequence and RT-qPCR.
490 491
Total RNA Isolation and RT-qPCR Analysis
492
Total RNA was extracted using TRIzol (Invitrogen) and treated with DNaseI.
493
cDNA was synthesized from 2 µg of total RNA using SuperscriptII Reverse
494
Transcriptase (Invitrogen). Real-time PCR was performed with SYBR Green PCR
495
master mix (Takara). Data were collected using Bio-Rad chromo 4 real-time PCR
496
detector. All expressions were normalized against the Actin gene (Os03g0718100).
497
The primers used are listed at Supplemental Table 1. Three biological repeats were
498
performed for each analysis. Values are means±SE of three biological repeats.
499 500
Yeast Two Hybrid Assay
501
The full-length coding sequence of OsWRKY53 and the OsMAPK6 were cloned
502
into pGADT7 and pGBKT7, respectively. These two vectors and corresponding empty
503
vectors were co-transformed into the yeast strain Y2H gold. The yeast two hybrid
504
assays was performed as the kit’s instructions.
505 506 507
BIFC (Bimolecular Fluorescence Complementation) Assay For BiFC assays, the OsMAPK6 and OsWRKY53 were fused with partial GFP, 26 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
508
and generated nGFP-OsMAPK6, cGFP-OsWRKY53. These vectors were transformed
509
into Agrobacterium strain GV3101 and co-injected into young leaves of N.
510
benthamiana. The fluorescence was observed by confocal microscopy (Leica) after 2
511
days growth.
512 513
LUC Complementation Imaging (LCI) Assay
514
The LCI assays for the interaction between OsMAPK6 and OsWRKY53 were
515
performed in N. benthamiana leaves. The full-length OsMAPK6 and OsWRKY53
516
coding region were fused with the N-terminal part and C-terminal part of the
517
luciferase reporter gene respectively. Agrobacteria harboring nLUC-OsMAPK6 and
518
cLUC-OsWRKY53 constructs were coinfiltrated into N. benthamiana, and the
519
infiltrated leaves were analyzed for LUC activity 48h after infiltration using
520
Chemiluminescence imaging (Tanon 5200).
521 522
Electrophoretic Mobility Shift Assay (EMSA)
523
Full-length coding sequence of OsWRKY53 was cloned into PVP13 vector via a
524
LR recombination reaction to generate MBP-OsWRKY53 fusion protein, and
525
transformed into E. coli strain BL21 (DE3). The recombinant proteins were affinity
526
purified using Amylose Resin (BioLabs, Cat No E8021S). About 40bp length
527
oligonucleotide probes containing wide type W-box (TTGACC) or mutated W-box
528
(AAAAAA)
529
EMSA Probe Biotin Labeling Kit (Beyotime, Cat No GS008). For nonlabeled probe
530
competition, nonlabeled probe was added to the reactions. EMSA was performed
531
using a Chemiluminescent EMSA kit (Beyotime, Cat No GS009). Probe sequences
532
are shown in Supplemental Table 1.
motifs
were
synthesized
and
labeled
with
biotin
using
533 534
Transient Transcription Dual-Luciferase (Dual-LUC) assay
535
Full-length coding sequence of OsWRKY53 was cloned into KpnⅠand BamHⅠ
536
site of pRT107, and effector (35Spro:OsWRKY53) was generated. The promoter
537
regions (upstream of the ATG) of OsWRKY53 were amplified and cloned into SalⅠ 27 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
538
and BamHⅠsite of pGreenII 0800-LUC vector and used as reporter (Hellens et al.,
539
2005). The resulting effector and reporter constructs were co-transfected into
540
protoplasts prepared from two-week-old rice seedlings. Protoplast isolation and PEG
541
transformation was carried out as described (Huang et al., 2015). The Renilla
542
luciferase (REN) gene directed by 35S promoter in the pGreenII 0800-LUC vector
543
was used as an internal control. Firefly LUC and REN activities were measured with a
544
Dual-Luciferase reporter assay kit using a GloMax 20/20 luminometer (Promega).
545
The LUC activity was normalized to REN activity and LUC/REN ratios were
546
calculated. For each plasmid combination, five independent transformations were
547
performed. Values are means ± SE of five biological repeats. Primers used for these
548
constructs are listed in Supplemental Table 1.
549 550
ChIP (Chromatin Immunoprecipitation) Assay
551
OsWRKY53-OE was used for ChIP assay as previously described (Zhu et al.,
552
2012). Briefly, approximately 2 g of rice seedlings was cross-linked in 1%
553
formaldehyde under a vacuum, and cross-linking was stopped with 0.125 M glycine.
554
The sample was ground to a powder in liquid nitrogen, and used to isolate nuclei.
555
Anti-OsWRKY53 (1:150 dilutions) was used to immunoprecipitate the protein-DNA
556
complex, and the precipitated DNA was recovered and analyzed by quantitative PCR.
557
Chromatin precipitated without antibody was used as a control. The data are presented
558
as means±SE of three biological repeats. Anti-OsWRKY53 (AbP80050-A-SE) was
559
ordered from Beijing Protein innovation. Primers used for ChIP-qPCR are listed in
560
Supplemental Table 1.
561 562
In Vitro Kinase Assay
563
The full length OsMAPK6 and OsMAPKK4DD was cloned into pDEST15 vector
564
via an LR recombination reaction and transformed into E. coli strain BL21 (DE3).
565
OsMAPKK4DD was generated as described (Chujo et al., 2014). Primers used for
566
these constructs are listed in Supplemental Table 1. The recombinant proteins were
567
affinity purified using Glutathione Sepharose 4B beads (GE Healthcare). For each 28 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
568
kinase assay, 0.5 μg MBP-OsWRKY53 or mutated fusion protein MBP-OsWRKY53
569
(SA) and 0.3 μg GST-OsMAPK6, GST-OsMAPKK4DD or GST were incubated with
570
phosphorylation buffer (25 mM Tris (pH 7.4), 12 mM MgCl2, 1 mM DTT, and 1 mM
571
ATP). The reactions were incubated at 30°C for 45min and boiled with 5×SDS
572
loading buffer then separated by SDS-PAGE. The protein was transferred on PVDF
573
membrane (Millipore, MA, USA), and phosphorylation signal was detected by
574
Phos-tag Biotin BTL-104 (Wako, Japan) according to the manufacturer’s instruction.
575 576
Transient Expression Assay in Rice Protoplast
577
The coding region of OsMAPKK4 DD, OsMAPK6, and OsWRKY53 fused with
578
MYC tag were ligated into the pRT107 vector to generate the 35Spro: OsMAPKK4 DD,
579
35Spro: OsMAPK6 and 35Spro: MYC-OsWRKY53 constructs. Rice protoplast was
580
isolated from stem and sheath tissues of wild type young seedlings as described
581
previously (Chen et al., 2006). Different combination of plasmid DNAs (about 10 μg
582
DNA of each construct) were transiently expressed in protoplasts by PEG-mediated
583
transfection procedure.
584
After overnight incubation in dark at 28℃, total proteins were isolated from rice
585
protoplasts with extraction buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.2%
586
NP-40, 0.1% Triton X-100, 5 mM EDTA, Complete protease inhibitor cocktail, and
587
50 μM MG132), and 5×SDS buffer was added and boiled for 5 minutes. The samples
588
were loaded on a 10% SDS-PAGE for immunoblotting with anti-MYC antibody
589
(Abmart, M20002L) and anti-HSP antibody (BGI Tech, AbM51099), respectively.
590 591
Accession Numbers
592
Sequence data from this article can be found in the GenBank/EMBL database
593
under the following accession numbers: OsWRKY53 (Os05g0343400); OsD2
594
(Os01g0197100);
595
OsMAPKK4 (Os02g0787300); OsMAPK6 (Os06g0154500); EXPA2 (Os01g0823100);
596
EXPA4 (Os05g0477600); EXPB3 (Os10g0555900); OsBU1 (Os06g0226500);
597
OsXTR1 (Os11g0539200); OsACTIN1 (Os03g0718100).
OsDWARF4
(Os03g0227700);
OsD11
(Os04g0469800);
29 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
598 599
Supplemental Data
600 601
Supplemental Figure 1. Enlarged leaf angle of OsWRKY53 overexpression lines
602
Supplemental Figure 2. Expression of cell enlargement-related gene in
603
OsWRKY53-OE
604
Supplemental Figure 3. Pale green phenotype of OsWRKY53-OE plant
605
Supplemental Figure 4. Identification of OsWRKY53 overexpression lines
606
Supplemental Figure 5. Identification of oswrky53 mutant
607
Supplemental Figure 6. The leaf of oswrky53 is dark green
608
Supplemental Figure 7. OsWRKY53 phosphorylation by OsMAPK6 enhance the
609
DNA binding activity of OsWRKY53 protein without affecting its stability.
610
Supplemental Figure 8. Phenotypic analysis of OsWRKY53(SA)-OE
611
Supplemental Figure 9. Characterization of MYC-OsWRKY53-OE
612
Supplemental Figure 10. Spatial expression pattern of OsWRKY53
613
Supplemental Figure 11. Phenotype and quantification of unhusked grain of
614
diverse lines.
615
Supplemental Figure 12. Schematic diagram of working model of OsWRKY53
616
Supplemental Table. List of Primers
617 618
Supplemental Figure 1. Enlarged leaf angle of OsWRKY53 overexpression lines
619
(A) Enlarged leaf angle of OsWRKY53-OE at young seedlings stage.
620
(B) Quantification of leaf angle in (A). Data are means ±SE (n=20).
621
(C) Phenotype of collar in WT and OsWRKY53-OE.
622
(D) Morphology observation of adaxial surface of the lamina joint in WT and
623
OsWRKY53-OE by scanning electron microscope (Scale bar=100 μm). The boxed
624
regions were enlarged to highlight.
625
(E) Cytological observation of adaxial in WT and OsWRKY53-OE (Scale bar=50 μm).
626
(F) Quantification of cell length and width of adaxial surface in WT and
30 Downloaded from on September 13, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.
627
OsWRKY53-OE. Data are means ±SE (n=50).
628
P values were calculated by Student’s t test, ** is P