OsBRXL4 Regulates Shoot Gravitropism and Rice Tiller Angle through Affecting LAZY1 Nuclear Localization

Molecular Plant Research Article

OsBRXL4 Regulates Shoot Gravitropism and Rice Tiller Angle through Affecting LAZY1 Nuclear Localization Zhen Li1,2,4,7, Yan Liang1,7, Yundong Yuan1, Lei Wang1, Xiangbing Meng1, Guosheng Xiong5, Jie Zhou1, Yueyue Cai1,2, Ningpei Han1,2, Lekai Hua6, Guifu Liu1, Jiayang Li1,2,* and Yonghong Wang1,2,3,* 1

State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

2

University of Chinese Academy of Sciences, Beijing 100039, China

3

CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China

4

Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100081, China

5

Plant Phenomics Research Center, Nanjing Agriculture University, Nanjing 210095, China

6

Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China

7These

authors contributed equally to this article.

*Correspondence: Jiayang Li ([email protected]), Yonghong Wang ([email protected]) https://doi.org/10.1016/j.molp.2019.05.014

ABSTRACT Rice tiller angle is a key agronomic trait that contributes to ideal plant architecture and grain production. LAZY1 (LA1) was previously shown to control tiller angle via affecting shoot gravitropism, but the underlying molecular mechanism remains largely unknown. In this study, we identified an LA1-interacting protein named Brevis Radix Like 4 (OsBRXL4). We showed that the interaction between OsBRXL4 and LA1 occurs at the plasma membrane and that their interaction determines nuclear localization of LA1. We found that nuclear localization of LA1 is essential for its function, which is different from AtLA1, its Arabidopsis ortholog. Overexpression of OsBRXL4 leads to a prostrate growth phenotype, whereas OsBRXLs RNAi plants, in which the expression levels of OsBRXL1, OsBRXL4, and OsBRXL5 were decreased, display a compact phenotype. Further genetic analysis also supported that OsBRXL4 controls rice tiller angle by affecting nuclear localization of LA1. Consistently, we demonstrated that OsBRXL4 regulates the shoot gravitropism through affecting polar auxin transport as did LA1. Taken together, our study not only identifies OsBRXL4 as a regulatory component of rice tiller angle but also provides new insights into genetic regulation of rice plant architecture. Key words: OsBRXL4, LA1, nuclear localization, shoot gravitropism, tiller angle, rice Li Z., Liang Y., Yuan Y., Wang L., Meng X., Xiong G., Zhou J., Cai Y., Han N., Hua L., Liu G., Li J., and Wang Y. (2019). OsBRXL4 Regulates Shoot Gravitropism and Rice Tiller Angle through Affecting LAZY1 Nuclear Localization. Mol. Plant. 12, 1143–1156.

INTRODUCTION In rice (Oryza sativa), tillers are specialized branches that bear grains and originate from the unelongated basal internodes of the stem. The angle between the tillers and the main culm (the tiller angle) significantly influences rice grain production by not only determining the growth and productivity of individual plants but also affecting planting density (Wang and Li, 2005; Teichmann and Muhr, 2015). Tiller angle formation is attributed to the growth of the unelongated internodes of tiller base. The asymmetric growth, through which the length of near-ground

part of the basal internodes is longer than that of the far-ground part, leads to the compact status (smaller tiller angle), whereas symmetric growth whereby the length of near-ground and farground parts is similar leads to the prostrate growth (larger tiller angle) (Supplemental Figure 1). Much more space is needed for rice varieties with larger tiller angles, leading to reduced grain production within a given area. Smaller tiller angles likely make

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019. 1143

Molecular Plant rice plants more susceptible to pathogen attack and also reduce their capacity in light capture. Therefore, optimal tiller angle is vital for ideal plant architecture and, thus, grain yield. Gaining insight into the molecular mechanisms that control rice tiller angle will improve our ability to rationally modify rice plant architecture. Over the past decade, major quantitative trait loci and key genes controlling tiller angle have been identified in rice, including LAZY 1 (LA1) (Li et al., 2007; Yoshihara and Iino, 2007), PROSTRATE GROWTH 1 (PROG1) (Jin et al., 2008; Tan et al., 2008), PROG7 (Hu et al., 2018), RICE PLANT ARCHITECTURE DOMESTICATION (RPAD) (Wu et al., 2018), Tiller Angle Control 1 (TAC1) (Yu et al., 2007), TAC3 (Dong et al., 2016), Loose Plant Architecture 1 (LPA1) (Wu et al., 2013), PLANT ARCHITECTURE AND YIELD 1 (PAY1) (Zhao et al., 2015), DWARF 2 (D2) (Dong et al., 2016), and TILLER INCLINED GROWTH 1 (TIG1) (Zhang et al., 2019). However, the molecular mechanisms underlying tiller angle regulation and the relationships among known regulatory genes in rice remain to be elucidated. Gravitropism, the gravity-mediated growth process, ensures shoot growth upward and root growth downward (Chen et al., 1999). Previous studies have shown that shoot gravitropism largely contributes to the establishment of rice tiller angle (Li et al., 2007; Yoshihara and Iino, 2007; Chen et al., 2012; Wu et al., 2013; Sang et al., 2014; Sakuraba et al., 2015; Harmoko et al., 2016; Zhang et al., 2018). The molecular mechanisms of gravitropic response are different in various organs, such as roots, hypocotyls, and inflorescences (Chen et al., 1999; Tasaka et al., 1999; Baldwin et al., 2013). Nevertheless, the overall process of gravitropism is conserved and comprises four sequential steps: gravity perception, signal transduction, auxin redistribution, and growth response (Strohm et al., 2012; Baldwin et al., 2013). Molecular genetic studies on dicot Arabidopsis mutants have enriched our knowledge about gravitropism and branch angle through the identification of SHOOT GRAVITROPISM (SGR) genes (Hashiguchi et al., 2013; Kolesnikov et al., 2016). The identification of gravitropismrelated genes in monocot rice is also helping us to elucidate the mechanisms of gravitropism involved in tiller angle regulation. For instance, altered expression of genes involved in starch accumulation or amyloplast sedimentation causes deficiencies in shoot gravitropism and rice tiller angle (Wu et al., 2013; Okamura et al., 2014; Morita et al., 2015; Sakuraba et al., 2015). It has been shown that several genes, including LA1, PIN-FORMED 1a (PIN1a), PIN2, PAY1, and a1,3flucosyltransferase (FucT), regulate auxin transport to control rice tiller angle (Li et al., 2007; Chen et al., 2012; Zhao et al., 2015; Harmoko et al., 2016; Sun et al., 2019). Recently, a largescale transcriptome analysis in response to gravitropic stimulation was used to explore the key components involved in rice tiller angle regulation via shoot gravitropism, through which a core regulatory pathway controlling rice tiller angle mediated by LA1dependent asymmetric distribution of auxin was identified (Zhang et al., 2018). In addition, strigolactones (SLs), a group of recently identified plant hormones, are also reported to be involved in shoot gravitropism and rice tiller angle by inhibiting auxin biosynthesis (Sang et al., 2014). The sleeping stature of corn was portrayed in the early 1930s as ‘‘lazy.’’ Subsequently, this key trait has drawn considerable inter1144 Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019.

OsBRXL4 Regulates Rice Tiller Angle est of plant biologists and breeders. In rice, the ‘‘lazy 1,’’ a classical shoot gravitropic deficient mutant that displays a prostrate phenotype, was reported in 1938, but the corresponding gene ‘‘LA1’’ was not cloned until 2007 (Jones and Adair, 1938; Abe et al., 1996; Godbole et al., 1999; Li et al., 2007; Yoshihara and Iino, 2007). Recently, orthologs of LA1 have been identified in other plant species, e.g., ZmLA1 in maize (Dong et al., 2013; Howard et al., 2014) and AtLA1 in Arabidopsis (Yoshihara et al., 2013; Sasaki and Yamamoto, 2015; Taniguchi et al., 2017; Yoshihara and Spalding, 2017). The function of LA1 genes is conserved in regulating shoot gravitropism, even though the gravitropism-responsive organs are not always the same due to the diversity of shoot structures in different species. In Arabidopsis, five AtLAZY1-like (AtLZY) genes that possess conserved regions with sequence similarity to LA1 and AtLA1/ AtLZY1 have been identified (Yoshihara et al., 2013). AtLZY2 and AtLZY3 have redundant functions with AtLA1 but contribute less to shoot gravitropism than AtLA1 (Taniguchi et al., 2017). Unlike AtLA1, AtLZY2 and AtLZY3 also function redundantly in root gravitropism regulation (Ge and Chen, 2016; Guseman et al., 2017; Taniguchi et al., 2017; Yoshihara and Spalding, 2017). DEEPER ROOTING 1 (DRO1), the homolog of AtLZY3/AtDRO1 in rice, is also reported to control root architecture (Uga et al., 2013). Overexpression of AtLZY3/ AtDRO1 resulted in smaller lateral root angles and branch angles. Removing the ethylene response factor-associated amphiphilic repression (EAR) motif of AtLZY3/AtDRO1, however, did not lead to obvious changes in roots and branches in AtDRO1-overexpressing plants, indicating that the EAR motif is critical for the function of AtLZY3/AtDRO1 (Guseman et al., 2017). Although the identification and characterization of LA1 and its homologs have increased our understanding of the role of gravitropism in shaping plant architecture, the biochemical functions of LA1 genes in plants still remain largely unclear. Here, we report the identification and characterization of the LA1-interacting protein OsBRXL4, a member of the plantspecific Brevis Radix (BRX) family. We demonstrated that OsBRXL4 can interact with LA1 and affect its nuclear localization, which is essential for the biological function of LA1 in controlling rice tiller angle. BRXL1, BRXL4, and BRXL5 act redundantly in generating the rice tiller angle, and downregulation of their expression results in a compact plant phenotype. By contrast, OsBRXL4-overexpressing plants exhibit a prostrate growth phenotype. Like la1, overexpression of OsBRXL4 results in enhanced polar auxin transport (PAT) and decreased lateral auxin transport (LAT), leading to reduced shoot gravitropism and, thus, enlarged tiller angle.

RESULTS OsBRXL4 Physically Interacts with LA1 Previously, we showed that LA1 plays an important role in regulating shoot gravitropism whereby the rice tiller angle is controlled (Li et al., 2007). However, the underlying mechanism is still unknown. To elucidate the molecular mechanism by which LA1 controls rice tiller angle, we performed a yeast two-hybrid assay to screen for the LA1-interacting proteins. Among several candidate LA1-interacting proteins, OsBRXL4 showed a strong interaction with LA1 in yeast based on a b-galactosidase activity

OsBRXL4 Regulates Rice Tiller Angle

Molecular Plant 2006; Li et al., 2009; Rodrigues et al., 2009; Beuchat et al., 2010a; Marhava et al., 2018). In contrast to LA1 that is specifically expressed in vascular cells at the adaxial parts of the junctions of young leaves and stems, OsBRXL4 was not only expressed in the peripheral cylinder of vascular bundles but also in the leaf primordia, young leaves, axillary meristems, and tiller buds (Supplemental Figure 2), implying that the interaction between LA1 and OsBRX4 might occur in vascular cells at the adaxial parts of the junctions of young rice leaves and stems and the peripheral cylinder of vascular bundles. To further verify the interaction between LA1 and OsBRXL4, we carried out a coimmunoprecipitation (CoIP) assay in rice protoplasts transiently expressing LA1-GFP and Myc-OsBRXL4. As shown in Figure 1B, Myc-OsBRXL4 could be precipitated by LA1-GFP, but not by GFP alone. In addition, a bimolecular fluorescence complementation (BiFC) assay was performed in rice protoplasts, showing that LA1 and OsBRXL4 interacted at the plasma membrane in rice cells (Figure 1C).

Three BRX Domains Cooperatively Affect the OsBRXL4– LA1 Interaction

Figure 1. LA1 Interacts with OsBRXL4 In Vitro and In Vivo. (A) LA1 interacts with OsBRXL4199–397 in yeast and no interaction is detected using an empty-vector control (pAD). -L-T denotes the medium lacking Leu and Trp. -L-T-H-A denotes the medium lacking Leu, Trp, His, and Ade. 3AT indicates 3-amino-1,2,4-triazole used to inhibit the background activation. Blue color in an X-Gal assay indicates the two proteins can interact in this system. (B) OsBRXL4 can be precipitated by LA1 in rice protoplasts. The total proteins cotransfected with LA1-GFP and 6Myc-OsBRXL4 in rice protoplasts were immunoprecipitated with anti-GFP monoclonal antibody and the immunoprecipitated proteins were detected with anti-Myc antibody. GFP protein was used as a negative control. (C) LA1 interacts with OsBRXL4 at the plasma membrane of rice protoplasts in the BiFC assay. LA1 was fused with the N terminus of CFP in the pSCYNE vector and OsBRXL4 was fused with the C terminus of CFP in the pSCYCE vector. The THIONIN protein in rice was used as a negative control. The fluorescence signals indicate the interaction between LA1 and OsBRXL4. BF, bright field. Scale bars, 5 mm.

assay (Figure 1A). OsBRXL4 belongs to the BRX family that comprises BRX and BRX-like (BRXL) genes in different species and is a conserved plant-specific gene family (Briggs et al., 2006; Liu et al., 2010). However, the physiological and biochemical functions of most of the members in the BRX family remain unknown, except that the AtBRX is shown to be involved in crosstalk of signaling pathways mediated by various hormones to regulate root development (Mouchel et al., 2004,

Like all members of the BRX family (Briggs et al., 2006; Liu et al., 2010), OsBRXL4 contains three highly conserved tandem repeat domains (BRX domains). One short domain is located at the N terminus between amino acids 34 and 71, whereas another two domains are located between amino acids 146 and 205 and between 338 and 397 in the C-terminal region (Figure 2A). The BRX domain has been demonstrated to regulate the subcellular localization of AtBRX protein (Scacchi et al., 2009). To characterize the subcellular localization of OsBRXL4, we fused a GFP protein in-frame to the C terminus of OsBRXL4 controlled by the 35S promoter. The results showed that OsBRXL4-GFP was localized to plasma membrane and cytoplasm (Figure 2A and 2B; Supplemental Figure 3A). Furthermore, we developed a series of OsBRXL4-GFP truncated derivatives, in which individual or different combinations of BRX domains were deleted (Figure 2A). Through a transient expression assay in rice protoplasts, we found that deletion of either or both the second and third BRX domains—i.e., OsBRXL4D2, OsBRXL4D3, and OsBRXL4D23—did not affect the localization of OsBRXL4 (Figure 2B). However, OsBRXL4-GFP truncated derivatives lacking the first BRX domain—i.e., OsBRXL4D1, OsBRXL4D12, OsBRXL4D13, and OsBRXL4D123—were not only detected at the plasma membrane and cytoplasm but also in the nucleus (Figure 2B and Supplemental Figure 3A), indicating that the first BRX domain may be essential for OsBRXL4 subcellular localization. The BRX domain is a protein–protein interaction domain (Briggs et al., 2006). Therefore, we attempted to identify the domains of OsBRXL4 responsible for the interaction with LA1 via a BiFC assay. When LA1 was cotransfected with OsBRXL4 derivatives and the first BRX domain was deleted—i.e., OsBRXL4D1, OsBRXL4D12, and OsBRXL4D13—strong signals were not only observed at the plasma membrane but also in the nucleus (Figure 2C). Deletion of either the second or the third BRX domain—i.e., OsBRXL4D2 and OsBRXL4D3—did not obviously affect the OsBRXL4–LA1 interaction. However, OsBRXL4D23, Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019. 1145

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OsBRXL4 Regulates Rice Tiller Angle

Figure 2. Domain Mapping for the OsBRXL4–LA1 Interaction. (A) Schematic diagram of OsBRXL4 and its truncated derivatives. The orange, blue, and purple colors indicate the first, second, and third BRX domain, respectively. The folded lines indicate the deleted BRX domains. (B) Subcellular localization analysis of GFP-tagged OsBRXL4 and OsBRXL4 truncated derivatives in rice protoplasts. The Hoechst signals indicate the nucleus. BF, bright field. Scale bars, 5 mm. (C) BiFC assays of pSCYNE-LA1 and pSCYCE-OsBRXL4 or OsBRXL4 truncated derivatives in rice protoplasts. BF, bright field. Scale bars, 5 mm. (D) Schematic diagram of LA1 and its truncated derivatives. The blue, purple, and orange colors indicate the predicted transmembrane (TM) domain, nuclear localization signal (NLS) domain, and EAR motif, respectively. The folded lines indicate the deleted regions. (E) Subcellular localization analysis of mCherry-tagged LA1 and LA1 truncated derivatives. The Hoechst signals indicate the nucleus. BF, bright field. Scale bars, 5 mm. (F) BiFC assays of pSCYCE-OsBRXL4 and pSCYNE-LA1 or LA1 truncated derivatives in rice protoplasts. Scale bars, 5 mm.

1146 Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019.

OsBRXL4 Regulates Rice Tiller Angle where the two BRX domains were simultaneously removed, reduced their interaction intensity (Figure 2C). In addition, when all the three BRX domains were deleted—OsBRXL4D123—the interaction between LA1 and OsBRXL4 was completely abolished (Figure 2C). These results indicated that all three BRX domains of OsBRXL4 cooperatively determine its interaction with LA1 at the plasma membrane.

The 96 C-Terminal Amino Acids of LA1 Are Essential for the OsBRXL4–LA1 Interaction In addition to the transmembrane domain and nuclear localization signal (NLS) domain, LA1 has a C-terminal domain comprising an EAR motif that is unique for most LA1 and LA1-like proteins (Figure 2D) (Yoshihara et al., 2013). To define the domains of LA1 interacting with OsBRXL4, we generated a series of LA1mCherry truncated forms. Removal of the 100 N-terminal amino acids (LA1DN100) including the transmembrane domain significantly eliminated the plasma membrane localization of LA1 in rice protoplasts (Figure 2D and 2E), consistent with our previous results observed in onion epidermal cells (Li et al., 2007). The prediction from cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/ cgi-bin/NLS_Mapper_form.cgi) and the PredictNLS (http://cubic. bioc.columbia.edu/predictNLS) programs indicated that the NLS domain of LA1 is made of 281–312 amino acid residues (Figure 2D). Compared with the full-length LA1, the signals of the LA1DNLS localized in the nucleus almost disappeared (Figure 2E and Supplemental Figure 3B). Deletion of a 96-aminoacid fragment at the C terminus abolished the plasma membrane localization of LA1 (Figure 2D and 2E; Supplemental Figure 3B). However, only lacking the EAR motif (LA1DEAR) within this 96amino-acid fragment did not affect the subcellular localization of LA1 (Figure 2D and 2E; Supplemental Figure 3B). To assess the requirement of these regions for the OsBRXL4–LA1 interaction, we then cotransfected LA1 truncated forms with OsBRXL4 in rice protoplasts. Deletion of the 100 N-terminal amino acids (DN100), NLS domain (DNLS), or the EAR motif (DEAR) of LA1 did not obviously affect their interaction (Figure 2F), but removal of the 96 C-terminal amino acids (DC96) significantly eliminated the ability of LA1 to interact with OsBRXL4 (Figure 2F). Collectively, these results suggested that the 96 C-terminal amino acids are essential for the OsBRXL4– LA1 interaction.

OsBRXL4 Significantly Affects Nuclear Localization of LA1 The fact that both OsBRXL4 and LA1 showed multiple subcellular distributions but their interaction exclusively occurred at the plasma membrane prompted us to investigate whether the interaction between OsBRXL4 and LA1 could affect their subcellular localization. To this end, LA1-mCherry and OsBRXL4-GFP were transiently coexpressed in rice protoplasts. We found that in about 85% of the coexpressed protoplasts, LA1 was clearly absent from the nucleus and exclusively localized to the plasma membrane. However, this alteration of LA1 subcellular distribution was not observed in a parallel experiment coexpressing LA1-mCherry with GFP. In contrast, almost all coexpressed protoplasts exhibited normal OsBRXL4 subcellular distribution (Figure 3A and 3B; Supplemental Figure 3C). These results indicated that

Molecular Plant OsBRXL4 might be able to determine the nuclear localization of LA1, but LA1 has no effect on the subcellular distribution of OsBRXL4. To further illustrate the effect of OsBRXL4 on LA1 subcellular localization, we transiently transfected rice protoplasts with equal amounts of LA1-mCherry plasmids but increasing amounts of OsBRXL4-GFP plasmids. The ratio of cells exhibiting LA1 nuclear localization was gradually decreased along with increasing quantity of OsBRXL4-GFP plasmids (Figure 3C). Meanwhile, another assay was performed with equal amounts of OsBRXL4-GFP plasmids but increasing amounts of LA1mCherry plasmids. The percentage of cells showing LA1 nuclear localization was significantly increased along with the increasing amount of LA1-mCherry plasmids (Figure 3D). To assess the contribution of each BRX domain of OsBRXL4 to LA1 nuclear localization, we transiently cotransfected LA1mCherry with a series of truncated OsBRXL4-GFP derivatives in rice protoplasts. When cotransfected with the full-length OsBRXL4, the ratio of LA1 nuclear distribution was only 15% (Figure 3E and Supplemental Figure 4). Deleting the second or third BRX domain—i.e., OsBRXL4D2 or OsBRXL4D3 truncated forms—about 23% and 49% of protoplasts displayed LA1 nuclear localization, respectively (Figure 3E and Supplemental Figure 4), indicating that either the second or third BRX domain may partially affect the nuclear localization of LA1. When deleting the first BRX domain of OsBRXL4, namely OsBRXL4D1, 76% of protoplasts exhibited LA1 nuclear distribution (Figure 3E and Supplemental Figure 4), indicating that the first BRX domain significantly contributes to LA1 nuclear localization. Moreover, when deleting any combinations of two BRX domains or all three BRX domains—i.e., OsBRXL4D12, OsBRXL4D13, OsBRXL4D23, and OsBRXL4D123—LA1 nuclear localization was almost normal (Figure 3E and Supplemental Figure 4).

Nuclear Localization of LA1 Is Essential for Its Function in Regulating Rice Tiller Angle The above results suggest that the nuclear localization of LA1 may be essential for its function. Therefore, we further investigated the role of LA1 nuclear localization in regulating the rice tiller angle. The domain-deleted LA1 (LA1DNLS) construct was introduced into the la1 mutant and the independent transgenic lines still displayed a prostrate phenotype, whereas the full-length LA1 rescued the phenotype of la1 (Figure 4A; Supplemental Figures 5A and 6A). These results suggested that the nuclear localization of LA1 is essential for rice tiller angle regulation. However, it has been reported that the nuclear localization of AtLA1 appears to be unnecessary for its function in the control of branch angle in Arabidopsis (Yoshihara et al., 2013). To further confirm the role of LA1 nuclear localization in rice, we replaced the two critical lysine residues within the NLS region of LA1 with alanine (K286A/K287A) as in Arabidopsis (Supplemental Figure 6B). A transient expression assay in rice protoplasts showed that the K286A/K287A mutations resulted in significantly reduced fluorescence signals in the nucleus (Figure 4B and Supplemental Figure 6C), similar to LA1DNLS (Figure 2D and 2E). We then introduced the Ubi:LA1K286A/K287A into the la1 mutant and found that the independent transgenic lines displayed a prostrate phenotype, nearly identical to those Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019. 1147

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OsBRXL4 Regulates Rice Tiller Angle Figure 3. OsBRXL4 Significantly Affects Nuclear Localization of LA1. (A) Subcellular localization and coexpression of LA1-mCherry and OsBRXL4-GFP in rice protoplasts. The empty vector GFP was cotransfected with LA1-mCherry as a negative control. Scale bars, 5 mm. (B) Quantification data of (A). Numbers indicate the ratios of cells displaying the indicated localization pattern of LA1 and OsBRXL4. Data are presented as mean ± SEM (n = 3 independent experiments with 100 cells analyzed for each assay). **P < 0.01, Student’s t-test. (C) The ratios of LA1 nuclear localization were gradually reduced along with increasing amount of OsBRXL4 plasmids. Data are presented as mean ± SEM (n = 3 independent experiments with 100 cells analyzed for each assay). (D) The ratios of LA1 nuclear localization were increased along with increasing amount of LA1 plasmids. Data are presented as mean ± SEM (n = 3 independent experiments with 100 cells analyzed for each assay). (E) Subcellular localization of LA1-mCherry was analyzed in the protoplasts coexpressing differential OsBRXL4 derivatives. Numbers indicate the ratio of cells displaying the indicated localization pattern of LA1. Data are presented as mean ± SEM (n = 3 independent experiments with 100 cells analyzed for each assay).

lines exhibited a prostrate phenotype with significantly enlarged rice tiller angles (Figure 5A and Supplemental Figure 7A). Similar to the full-length OsBRXL4, the overexpressed truncated OsBRXL4D2 or OsBRXL4D3 that partially affected LA1 nuclear localization also resulted in the prostrate phenotype (Figure 5A and Supplemental Figure 7A). In contrast, the transgenic lines overexpressing, for example, OsBRXL4D1, OsBRXL4D23, or OsBRXL4D123, significantly or fully failed to affect LA1 nuclear localization, and displayed an erect status similar to that of wild-type plants (Figure 5A and Supplemental Figure 7A). These results suggested that all the three BRX domains of OsBRXL4 are essential for the function of OsBRXL4 involved in regulating rice tiller angle. of la1 (Figure 4A; Supplemental Figures 5A and 6D), indicating that, unlike AtLA1, nuclear localization is essential for LA1 function in rice.

OsBRXL4 Is Involved in Tiller Angle Regulation To elucidate the function of OsBRXL4 in planta, we generated overexpression transgenic lines transformed with full-length OsBRXL4 and its truncated derivatives in Nipponbare background, respectively. The full-length OsBRXL4 overexpression 1148 Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019.

To further explore the role of OsBRXL4 in regulating rice tiller angle, we generated transgenic plants expressing an OsBRXLs RNAi construct targeting three OsBRXLs (OsBRXL1, OsBRXL4, and OsBRXL5) in the Nipponbare background. Compared with the wild-type, the expression levels of OsBRXL1, OsBRXL4, and OsBRXL5 were obviously decreased in the OsBRXL RNAi transgenic lines (Supplemental Figure 7B). The OsBRXLs RNAi transgenic plants exhibited a more compact phenotype compared with the wild type (Figure 5A), suggesting that the three BRXL genes may be functionally redundant to control

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OsBRXL4 Regulates Rice Tiller Angle

Figure 4. Nuclear Localization of LA1 Is Required for Its Function. (A) The phenotypes of the wild-type (Shiokari), la1, and the transgenic plants of overexpressed LA1, LA1DNLS, and LA1K286A/K287A under the la1 background. Hash mark (#) denotes the independent transgenic line. Scale bars, 10 cm. (B) Subcellular localization analysis of mCherrytagged LA1 and LA1K286A/K287A derivative. The Hoechst signals indicate the nucleus. BF, bright field. Scale bars, 5 mm.

rice tiller angle. Furthermore, LA1-mCherry was transiently expressed in protoplasts derived from Ubi:OsBRXL4, OsBRXLs RNAi transgenic lines, and Nipponbare. Compared with the wild-type, the inhibitory effect on LA1 nuclear localization was significantly enhanced in the Ubi:OsBRXL4 background and mildly reduced in the OsBRXLs RNAi background (Figure 5B and 5C). These results demonstrated that OsBRXL4 participates in controlling rice tiller angle by affecting LA1 nuclear localization.

OsBRXL4 Acts Upstream of LA1 in Controlling Rice Tiller Angle LA1 controls rice tiller angle via affecting shoot gravitropism (Li et al., 2007). To verify the involvement of OsBRXL4 in regulating shoot gravitropism, we examined the shoot gravitropic response of seedlings of Ubi:OsBRXL4 and OsBRXLs RNAi transgenic plants. Compared with the wildtype, the Ubi:OsBRXL4 seedlings displayed a reduced gravitropic response, whereas the gravitropic response was enhanced in OsBRXLs RNAi seedlings (Figure 6A and 6B). These results suggested that OsBRXL4 negatively regulates shoot gravitropism and thus affects the tiller angle. It has been shown that shoot gravitropic response is largely determined by auxin redistribution to the lower side of the responding stem. Loss of function of LA1 enhances PAT and disrupts LAT, leading to shoot gravitropism deficiency (Li et al., 2007). To characterize whether OsBRXL4 also affects auxin redistribution, we detected the expression of auxinresponsive marker gene IAA20 in the upper and lower sides of seedling shoot bases upon gravistimulation. Compared with the wild-type, the lower part of Ubi:OsBRXL4 shoot bases had reduced expression of IAA20, whereas OsBRXLs RNAi displayed increased IAA20 transcripts in the lower sides

(Figure 6C). To further confirm whether the reduced shoot gravitropism and the spreading phenotype of Ubi:OsBRXL4 result from alteration of PAT and LAT as in those in la1, we assessed the PAT and LAT in etiolated coleoptiles of Ubi:OsBRXL4 plants by monitoring 3HIAA (indole-3-acetic acid) movement. Consistent with the Ubi:OsBRXL4 seedlings, the Ubi:OsBRXL4 coleoptiles also showed reduced gravitropic response (Supplemental Figure 8). Compared with the wild-type, PAT was significantly enhanced in Ubi:OsBRXL4 coleoptiles and LAT was disrupted in Ubi:OsBRXL4 coleoptiles, like those in la1 (Figure 6D). Our previous study showed that the LA1-dependent auxin redistribution induces the asymmetric expression of WUSCHEL RELATED HOMEOBOX 6 (WOX6) and WOX11 to regulate the rice tiller angle (Zhang et al., 2018). In this study, we found that the expression levels of WOX6 and WOX11 were reduced in the lower part of the Ubi:OsBRXL4 seedling shoot bases but enhanced in that of OsBRXLs RNAi plants (Figure 6E), suggesting that OsBRXL4 and LA1 may regulate rice tiller angle through the same downstream genes, such as WOX6 and WOX11. To further elucidate the genetic relationship between OsBRXL4 and LA1, we introduced Ubi:OsBRXL4 to Ubi:LA1/la1 background by crossing. The Ubi:OsBRXL4 Ubi:LA1 plants identified from the progeny of the crossing displayed compact phenotype similar to the wild-type (Figure 6F and 6G), suggesting that extra LA1 could compensate the effect of OsBRXL4 on LA1 nuclear localization. The above results together indicate that OsBRXL4 acts upstream of LA1 in the control of rice tiller angle.

DISCUSSION Rice tiller angle is one of the most important agronomic traits contributing to plant architecture and grain yield (Wang and Li, 2005). Shoot gravitropism has long been regarded as the main factor that controls rice tiller angle. To date, several key components that control shoot gravitropism and tiller angle have been cloned and characterized (Li et al., 2007; Chen et al., 2012; Wu et al., 2013; Okamura et al., 2014; Morita et al., 2015; Sakuraba et al., 2015; Zhao et al., 2015; Harmoko et al., 2016), but the detailed functions and the underlying molecular Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019. 1149

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OsBRXL4 Regulates Rice Tiller Angle Figure 5. OsBRXL4 Regulates Rice Tiller Angle by Affecting Nuclear Localization of LA1. (A) Phenotypes of the wild-type (Nipponbare), la1, and the transgenic plants of overexpressed OsBRXL4 and its truncated derivatives in Nipponbare background and OsBRXLs RNAi plants. Hash mark (#) denotes the independent transgenic line. Scale bars, 10 cm. (B) Nuclear localization of LA1-mCherry was analyzed in the protoplasts derived from Nipponbare and transgenic plants of OsBRXL4 overexpression and OsBRXLs RNAi. Scale bars, 5 mm. (C) Quantification data of (B). Numbers indicate the ratio of cells displaying dual localization or exclusive plasma membrane localization of LA1. Data are presented as mean ± SEM (n = 3 independent experiments with 50 cells analyzed for each assay). Means with different letters are significantly different (P < 0.05, ANOVA and Tukey’s honest significant difference).

anism of OsBRXL4 involved in tiller angle regulation by affecting the nuclear localization of LA1.

Nuclear Localization Is Required for LA1 Function in Rice LA1 is a key regulator that controls rice shoot gravitropism and tiller angle (Li et al., 2007; Yoshihara and Iino, 2007). Loss of function of LA1 results in enhanced PAT and decreased LAT in the shoot, leading to a deficient shoot gravitropic response and thus an enlarged tiller angle (Li et al., 2007). Recently, a core pathway controlling rice tiller angle mediated by the LA1dependent asymmetric distribution of auxin was explored using a dynamic transcriptome of rice shoots in response to gravistimulation (Zhang et al., 2018). However, although it is a key regulator of rice tiller angle, the action of LA1 still remains largely unknown.

mechanisms of these genes remain largely unknown. In this study, we identified an LA1-interacting protein, OsBRXL4, and further elucidated its regulatory mechanism in controlling rice tiller angle. LA1 nuclear localization is essential for its function, and OsBRXL4 regulates shoot gravitropism and rice tiller angle by affecting LA1 nuclear localization. Therefore, our results not only identify a novel component that regulates rice tiller angle, OsBRXL4, but also further shed light on a new molecular mech1150 Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019.

In this study, we demonstrated that LA1 nuclear localization is essential for its biological function in rice. First, deletion of the predicted NLS domain or mutation of the critical lysine to alanine within the NLS region significantly affected LA1 nuclear localization (Figures 2E and 4B; Supplemental Figures 3B and 6C). Second, the full-length LA1 was able to rescue the prostrate phenotype of la1, whereas deleting LA1 from the NLS region or a mutation harboring two key residues within the NLS region could not (Figure 4A). However, unlike rice LA1, the nuclear localization of AtLA1, a functional ortholog of LA1 in Arabidopsis, is reported to be unnecessary for its function in regulating branch angle in Arabidopsis (Yoshihara et al., 2013). This discrepancy indicates

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Figure 6. OsBRXL4 Acts Upstream of LA1 in Controlling Rice Tiller Angle. (A) Comparison of shoot curvature in the wild-type (Nipponbare), la1, OsBRXL4 overexpression, and OsBRXLs RNAi seedlings at different time points after gravitropic stimulation. The #1 transgenic seedlings were grown under light for 4 days and subsequently reoriented by 90 from the vertical for the gravitropism assay. The arrow marked with G represents the direction of gravity. (B) Quantitative data of (A). Data are presented as mean ± SEM (n = 35). Multiple comparisons are performed at each time point. Means with different letters are significantly different (P < 0.05, ANOVA and Tukey’s honest significant difference). (C) The expression levels of IAA20 at the lower and upper sides of the shoot bases in the wild-type (Nipponbare), OsBRXL4 overexpression, and OsBRXLs RNAi seedlings. G, gravistimulation treatment; US, upper side; LS, lower side. Data are presented as mean ± SEM (n = 3). **P < 0.01 and *P < 0.05, Student’s t-test. (D) Comparison of PAT and LAT among the wild-type (Nipponbare), la1, and OsBRXL4 overexpression in dark-grown coleoptiles. Data are presented as mean ± SEM (n = 3 for the PAT and n = 10 for the LAT). **P < 0.01 and *P < 0.05, Student’s t-test. (legend continued on next page)

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Molecular Plant that LA1 and AtLA1 exert a similar function in regulating branch/ tiller angle, but their more detailed mechanisms might be distinct from each other, mostly because there are only limited regions of similarity including the NLS region between these two orthologs (Yoshihara et al., 2013). Moreover, functional differentiation, i.e., LA1 in coleoptile circumnutation (Yoshihara and Iino, 2007) and ZmLA1 in inflorescence development (Dong et al., 2013), suggests that distinct functions might have evolved in orthologs of LA1 in different species. The different patterns of branches in these two species also support the possibility that rice tiller angle and Arabidopsis lateral branch angle might be regulated by distinct mechanisms to some extent. Notably, in Arabidopsis, lateral branches are revealed to maintain nonvertical growth at specific angles with respect to gravity, known as gravitropic setpoint angle, which is determined by the extent of antigravitropic offset and its interaction with gravitropic response (Roychoudhry et al., 2013; Roychoudhry and Kepinski, 2015). In rice, tiller angle is closely related to gravitropic response of shoot because some reported rice tiller angle mutants are defective in shoot gravitropic response and knockout of shoot gravitropic response genes leads to altered tiller angle (Li et al., 2007; Yoshihara and Iino, 2007; Chen et al., 2012; Wu et al., 2013; Sang et al., 2014; Sakuraba et al., 2015; Harmoko et al., 2016). So far, there is no evidence showing the involvement of antigravitropic mechanism as well as other factors in the control of rice tiller angle. Identification of rice tiller angle mutant with normal shoot gravitropism will help to elucidate the possible alternative mechanism underlying tiller angle independent of gravitropism.

OsBRXL4 Controls Shoot Gravitropism and Tiller Angle by Affecting Nuclear Localization of LA1 Although LA1 has been extensively studied in different species, we still do not know its underlying mechanism in tiller angle regulation. In this study, we aimed to dissect the mechanism of LA1 by screening for the interacting proteins. OsBRXL4, an LA1interacting protein, was identified by a yeast two-hybrid assay. The interaction between OsBRXL4 and LA1 was verified through yeast two-hybrid, Co-IP, and BiFC assays (Figure 1). Compared with the wild-type, OsBRXL4 overexpression and OsBRXLs RNAi transgenic plants displayed prostrate and compact phenotypes, respectively (Figure 5A). Consistent with the prostrate and compact phenotypes, the seedlings of Ubi:OsBRXL4 and OsBRXLs RNAi displayed decreased and enhanced gravitropic response, respectively (Figure 6A and 6B). The auxin-responsive marker gene IAA20 exhibited reduced expression in the lower part of Ubi:OsBRXL4 seedling shoot bases, but increased in that of OsBRXLs RNAi plants (Figure 6C). Moreover, our data showed that overexpressed OsBRXL4 resulted in an enhanced PAT and thus altered IAA distribution (Figure 6D). Therefore, OsBRXL4 is a novel

OsBRXL4 Regulates Rice Tiller Angle component controlling tiller angle via shoot gravitropism in rice, not only facilitating the dissection of the regulatory mechanisms of LA1 but also providing a new useful gene to achieve ideal plant architecture in the future. In addition, we attempted to determine how OsBRXL4 controls shoot gravitropism to affect tiller angle by interacting with LA1. A transient coexpression assay revealed that OsBRXL4 significantly affected LA1 nuclear localization, but LA1 has no effect on the localization of OsBRXL4 (Figure 3A and 3B; Supplemental Figure 3C). Combined with the importance of LA1 nuclear localization for its function, we raised the possibility that OsBRXL4 might affect the LA1 nuclear localization and thus control shoot gravitropism and tiller angle. The evidence that overexpression of OsBRXL4 could result in prostrate growth similar to that of la1 and that overexpression of LA1 could rescue the prostrate phenotype of overexpression of OsBRXL4 further supports this possibility (Figures 5A, 6F, and 6G). OsBRXL4D2 or OsBRXL4D3 has partial effects on the LA1 nuclear localization, and overexpressing them resulted in the prostrate stature similar to that of OsBRXL4 overexpression plants (Figures 3E and 5A). However, due to having no effect on the LA1 nuclear localization, OsBRXL4D1, OsBRXL4D23, and OsBRXL4D123 overexpression plants displayed the erect stature similar to that of the wild-type (Figure 5A). Therefore, all data demonstrated that OsBRXL4 controls shoot gravitropism and tiller angle through affecting LA1 nuclear localization (Figure 7). In line with this, the genetic evidence clarified that OsBRXL4 functions upstream of LA1 to regulate downstream genes WOX6 and WOX11 in controlling rice tiller angle (Figure 6E–6G).

OsBRXL4 and AtBRX Exhibit Functional Differentiation Brevis Radix (BRX), latin for ‘‘short root,’’ is a highly conserved plant-specific gene family, named after the phenotype of lossof-function mutant of the first identified member AtBRX (Mouchel et al., 2004). As previously reported, AtBRX is also involved in embryo development and shoot growth (Scacchi et al., 2009; Beuchat et al., 2010b). In Arabidopsis, there are four other BRXL members in addition to AtBRX. These four AtBRXLs do not act redundantly with AtBRX in root growth regulation (Briggs et al., 2006). However, AtBRXL1 and AtBRX have functional redundancy during embryogenesis (Scacchi et al., 2009). It appears that AtBRXL proteins are functionally diversified in Arabidopsis. By contrast, nearly all tested monocot BRXLs could rescue the short root phenotype of the atbrx null mutant (Briggs et al., 2006; Beuchat et al., 2010a), indicating that these monocot BRXLs are functionally equivalent to AtBRX. However, these BRXLs of monocot plants are most likely involved in developmental processes different from those of AtBRX. For instance, except OsBRXL6, the other five OsBRXLs are responsive to major stresses to different degrees, of which OsBRXL1, OsBRXL4, and OsBRXL5 show similar

(E) The expression levels of WOX6 and WOX11 at the lower and upper sides of the shoot bases in the wild-type (Nipponbare), OsBRXL4 overexpression, and OsBRXLs RNAi seedlings. #1 denotes the transgenic line numbered 1. G, gravistimulation treatment; US, upper side; LS, lower side. Data are presented as mean ± SEM (n = 3). **P < 0.01, Student’s t-test. (F) Overexpressed LA1 rescued the prostrate growth of OsBRXL4 overexpression plants. Scale bars, 10 cm. (G) The expression levels of LA1 and OsBRXL4 in the plants simultaneously overexpressed LA1 and OsBRXL4. Data are presented as mean ± SEM (n = 3). **P < 0.01, Student’s t-test.

1152 Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019.

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Figure 7. A Working Model for LA1 and OsBRXL4 in the Regulation of Rice Tiller Angle. OsBRXL4 affects nuclear localization of LA1 by physical interaction. In normal conditions, the lower ratio of OsBRXL4/LA1 maintains a smaller tiller angle. However, accompanied by increased OsBRXL4, the tiller angle will become enlarged gradually owing to the reduced nuclear localization of LA1. The arrow indicates the direction of gravity.

BRX domain (OsBRXL4D1, OsBRXL4D12, OsBRXL4D13, and OsBRXL4D123) were detected not only at the plasma membrane but also in the nucleus (Figure 2A and 2B), leading to the alteration of interaction patterns between these truncated OsBRXL4 derivatives and LA1 (Figure 2C). Such alteration of subcellular localization and interaction patterns caused these truncated OsBRXL4 derivatives to lose their ability to affect LA1 nuclear localization (Figure 3E). Deletion of either or both the second and third BRX domains (OsBRXL4D2, OsBRXL4D3, and OsBRXL4D23) did not affect the subcellular localization of OsBRXL4. The interaction intensity of OsBRXL4D23 with LA1 was dampened, which resulted in the loss of ability to affect LA1 nuclear localization (Figures 2 and 3E). However, removal of either the second or third BRX domain (OsBRXL4D2, OsBRXL4D3) had a partial effect on LA1 nuclear localization (Figure 3E), indicating that the second and third BRX domains have redundant functions. Based on these data, we proposed that unlike AtBRX, the first BRX domain is essential for OsBRXL4 subcellular localization, and that all three BRX domains cooperatively determine its interaction with LA1 to affect LA1 nuclear localization. In addition, genetic evidence further demonstrated that all three BRX domains are required for the function of OsBRXL4. Specifically, OsBRXL4D1 and OsBRXL4D23 overexpression plants displayed the erect phenotype, which is different from the prostrate phenotype of the full-length OsBRXL4 overexpression plants (Figure 5A). Therefore, the corresponding BRX domains of AtBRX and OsBRXL4 have different contributions to their functions, indicating that AtBRX and OsBRXL4 have diverse functional differentiation.

METHODS expression patterns after cold treatment (Liu et al., 2010). Our results suggested that OsBRXL1, OsBRXL4, and OsBRXL5 also have functional redundancy in tiller angle regulation (Figure 5A). However there is, as yet, no evidence that AtBRX or AtBRXLs also controls branch angle and abiotic stresses. The root length of OsBRXL4 overexpression seedlings is significantly longer than that of the wild type, but no such phenotype exists in AtBRX overexpression plants (Liu et al., 2010). In addition, PROTEIN KINASE ASSOCIATED WITH BRX (PAX) could recruit AtBRX to the plasma membrane, which inhibits PIN-mediated auxin efflux at lower auxin level in Arabidopsis root tip (Marhava et al., 2018). Along with the cellular auxin increases, AtBRX translocates from plasma membrane to the nucleus and subsequently degrades by the proteasome pathway, which activates PAX and thus promotes the PIN-mediated auxin efflux (Scacchi et al., 2009; Marhava et al., 2018). However, the transcription of OsBRXL4 was induced by auxin in rice seedlings (Liu et al., 2010), implying that OsBRXL4 and AtBRX may have differential functions in regulating plant development in monocots and dicots. The BRX fragments of AtBRX have different contributions to its subcellular localization and function. The first BRX domain located at the N terminus promotes BRX membrane association and is not crucial for its function, whereas the two BRX domains at the C terminus, localized in the plasma membrane and nucleus, have an indispensable role in root growth (Scacchi et al., 2009). Our results revealed that the truncated OsBRXL4-GFP proteins lacking the first

Plant Materials The la1-Shiokari used in this work for LA1 domain analyses was described previously (Li et al., 2007). OsBRXL4 and its truncated derivatives were introduced to Nipponbare for morphological analyses. For field experiments, the plants were spaced 30 cm apart and grown in paddy fields under natural conditions.

Yeast Two-Hybrid Assays For yeast two-hybrid screening, the full-length open reading frame of LA1 was amplified by the primers LA1_BD_F and LA1_BD_R and subcloned into pLexA-C bait vectors of the Dualhybrid system as bait (Supplemental Table 1). cDNA libraries from rice seedlings were introduced to pGAD-HA prey vectors. The combinatory constructs were transformed simultaneously into the NMY51 yeast strain and tested for Leu, Trp, His, and Ade auxotrophs in the presence of 20 mM 3-aminotriazole. LacZ (b-galactosidase assay) reporter activity was monitored using the HTX b-galactosidase assay kit (Dualsystems Biotech) according to the manufacturer’s protocols.

BiFC Assays in Rice Protoplast For BiFC assays, LA1, OsBRXL4, and their corresponding truncated derivatives were amplified with gene-specific primers (Supplemental Table 1), including overhangs necessary for recombination in fusion with pSCYNE and pSCYCE vectors. The THIONIN protein in rice was used as a negative control. The generated plasmids were transformed into rice leaf mesophyll protoplasts by PEG (polyethylene glycol)-mediated transformation as described previously (Bart et al., 2006). After 12 h of incubation in the dark, the CFP fluorescence was analyzed with a confocal laser scanning microscope at an excitation wavelength of 405 nm (FluoView 1000; Olympus).

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Coimmunoprecipitation Assays

Constructs for Genetic Transformation

For construction of tag-fused transient expression plasmids for Co-IP, LA1 was amplified with primers LA1_GFP_F and LA1_GFP_R and constructed into the pBI221 vector (Supplemental Table 1). For generation of MYC-tagged OsBRXL4, 63MYC was amplified with primers 63MYC_F and 63MYC_R and the SCFPC155 of pSCYCE-OsBRXL4 was replaced with 63MYC (Supplemental Table 1). For Co-IP, MYC-tagged OsBRXL4 was cotransfected with GFP-tagged LA1 to rice leaf mesophyll protoplasts as described above, and incubated for 12 h to allow protein expression. The Co-IP assay was carried out according to a method described previously with some minor modifications (Xu et al., 2012). In briefly, total protein was extracted from the transfected protoplasts using immunoprecipitation buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 10% glycerol, 0.1% Nonidet P-40, and 13 complete protease inhibitor cocktail [Roche]). The lysate was centrifuged at 14 000 g for 20 min at 4 C and the supernatant was used for Co-IP experiments. Following the supplier’s instructions, 20 ml of the agarose-conjugated anti-GFP monoclonal antibody (MBL) was added into total extracted protoplast protein and incubated at 4 C for 3 h under gentle rotation. The beads were washed three times with the extraction buffer (without 0.1% Nonidet P-40) and eluted with 30 ml of SDS–PAGE sample buffer for the western blotting analysis. Mouse anti-GFP monoclonal antibody (cat. no. 11814460001, Roche) was used at a 1:10 000 dilution and mouse antic-MYC monoclonal antibody (cat. no. 11667149001, Roche) was used at a 1:5000 dilution.

To generate Ubi:LA1 and Ubi:OsBRXL4, we amplified the full-length cDNAs by primer pairs LA1_OE_F/LA1_OE_R and OsBRXL4_OE_F/ OsBRXL4_OE_R and cloned them into the pTCK303 vector. The 243-bp region between nucleotides 136 and 378 of OsBRXL4 cDNA was amplified by the primers OsBRXLs_RNAi_F and OsBRXLs_RNAi_R and subcloned into the pBb7GW-I-WG-UBIL vector to generate the OsBRXLs RNAi construct. The domain deletion or point mutation constructs were generated in a similar approach as described previously (Shyu et al., 2012). The primers for these constructs to generate transgenic plants are provided in Supplemental Table 1.

Subcellular Localization and Colocalization Analysis To determine the subcellular localization of OsBRXL4 and LA1, we constructed 35S:LA1-mCherry and 35S:OsBRXL4-GFP. To generate 35S:LA1-mCherry, we amplified an mCherry fragment with primers mCherry_F and mCherry_R and replaced the SCFP3AN173 of pSCYNE-LA1 with mCherry. OsBRXL4 was amplified with primers OsBRXL4_GFP_F and OsBRXL4_GFP_R and cloned into pBI221GFP. To further confirm the contribution of each domain to subcellular localization, we also constructed the corresponding truncated or point mutation derivatives based on the 35S:LA1-mCherry and 35S:OsBRXL4-GFP. The primers for these constructs are listed in Supplemental Table 1. These plasmids were transformed to rice leaf mesophyll protoplasts, as described above. After incubation at 28 C overnight, the GFP and mCherry fluorescence were analyzed with confocal laser scanning microscopy at excitation wavelengths of 488 and 559 nm, respectively (FluoView 1000; Olympus). To indicate the nucleus, we simultaneously used Hoechst 33342 (0.2 mg/ml, Beyotime) to stain the nuclei and analyzed the Hoechst fluorescence at excitation wavelengths of 405 nm (FluoView 1000; Olympus). To monitor the impact of OsBRXL4 on LA1 subcellular localization, we cotransfected 35S:LA1-mCherry with 35S:OsBRXL4-GFP or 35S:GFP to rice leaf mesophyll protoplasts, as described above. We further confirmed the effect of each domain of OsBRXL4 on LA1 subcellular localization by cotransfecting 35S:LA1-mCherry with 35S:OsBRXL4GFP truncated derivatives. The signal intensities of mCherry or GFP in the nucleus or non-nucleus were quantitatively determined with FluoView 1000 software. All transient transfection experiments were repeated at least three times.

Real-Time PCR Assays Total RNAs from examined organs were extracted using TRIzol according to the user manual (Invitrogen). Genomic contaminations were eliminated using the TURBO DNA-free kit (Ambion) following the supplier’s instructions. First-strand cDNA was prepared using SuperScriptIII reverse transcriptase enzyme (Invitrogen) according to the manufacturer’s protocol. Real-time PCR experiments were performed using genespecific primers (Supplemental Table 2) in the reaction system of SosoFast EvaGreen supermix (Bio-Rad) on the CFX96 Real-time system (Bio-Rad) following the manufacturer’s instructions. All quantifications were normalized to ubiquitin cDNA fragments.

1154 Molecular Plant 12, 1143–1156, August 2019 ª The Author 2019.

Shoot Gravitropism Assay Rice gravity response under light was measured using 5-day-old seedlings planted in plates containing half-strength Murashige and Skoog (MS) medium (pH 5.8). Plates were kept in a vertical position at 28 C. After reorienting the plates by 90 , the shoot curvature was recorded every 12 h over a 48-h time period. The angles of curvature were measured using the ImageJ program (downloaded from the following website: https//imagej. nih.gov/ij/index.html). Averages and standard deviations were calculated from 35 seedlings.

Polar Auxin Transport Assay PAT was assayed according to a method described previously with some minor modifications (Li et al., 2007). In brief, three groups of 15-mm excised coleoptile segments from 5-day-old dark-grown seedlings were used for the assay. First, the segments were deprived of endogenous IAA by preincubation in half-strength MS (pH 5.8) liquid medium for 2 h with gentle shaking. The apical or basal ends of the segments (for basipetal or acropetal transport assays, respectively) were then incubated in 10 ml of half-strength MS liquid medium containing 0.35% Phytogel and 0.1 mM 3H-labeled IAA (American Radiolabeled Chemicals) in 1.5-ml Eppendorf tubes in the dark at room temperature. After 3 h, 5-mm sections from the nonsubmerged ends of segments were excised and transferred into Eppendorf tubes containing 2 ml of scintillation liquid. After 18 h of incubation in 2 ml of scintillation liquid, the radioactivity of each tube was counted using a liquid scintillation counter (1450 MicroBeta TriLux; PerkinElmer).

Lateral Auxin Transport Assay Four-day-old coleoptiles of seedlings grown in the dark were used for the assay of auxin lateral transport as described previously, with some modifications (Li et al., 2007). Coleoptiles (10 mm) were harvested and deprived of endogenous IAA as mentioned above. The coleoptiles were laid horizontally in 0.6-ml tubes with their apical ends contacting 10-ml agar blocks that contained 500 nM 3H-IAA or 500 nM BA. After transport in the dark at room temperature for 2.5 h, sections of the 5-mm segments away from the apex were evenly split into upper and lower halves. After 18 h of incubation in 2 ml of scintillation liquid, the radioactivity of each half (n = 15) was counted by the liquid scintillation counter (1450 MicroBeta TriLux; PerkinElmer).

In Situ Hybridization RNA in situ hybridization was performed according to Xu et al. (2012) with minor modifications. Shoot bases of 14-day-old rice seedlings were put into the vacuum tissue processor (ASP200, Leica) to fix, dehydrate, clear, and embed, and were subsequently embedded in paraffin (Paraplast Plus; Sigma). The shoot bases were sliced into 10-mm sections with a microtome (Leica RM2145). The OsBRXL4 cDNA was amplified with primer pairs OsBRXL4_in situ_F/OsBRXL4_in situ_R and subcloned into the pSK vector (Supplemental Table 1). The pSK-OsBRXL4 was used as the template to generate sense and antisense RNA probes. Digoxigenin-labeled RNA probes were prepared using a DIG Northern Starter Kit (Roche) according to the manufacturer’s instruction. Slides were observed under bright field through a microscope (Leica DMR)

OsBRXL4 Regulates Rice Tiller Angle and photographed with a Micro color charge-coupled device camera (Apogee Instruments).

ACCESSION NUMBERS Sequence data from this article can be found in the Rice MSU Genome Annotation Project under the following accession numbers: LA1 (LOC_Os11g29840), OsBRXL1 (LOC_Os02g47230), OsBRXL2 (LOC_Os03g63650), OsBRXL3 (LOC_Os04g51172), OsBRXL4 (LOC_ Os08g36020), OsBRXL5 (LOC_Os09g27220/LOC_Os09g27230), and OsBRXL6 (LOC_Os12g09080).

SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

FUNDING This work was supported by grants from the National Natural Science Foundation of China (91635301; 91535204; 31601276), the Ministry of Agriculture of China (2016ZX08009-003), and the Strategic Priority Research Program ‘‘Molecular Mechanism of Plant Growth and Development’’ of CAS (XDPB0401).

AUTHOR CONTRIBUTIONS Z.L. and Y.L. designed experiments, analyzed data, and wrote the article; Y.Y., L.W., X.M., G.X., J.Z., Y.C., N.H., L.H., and G.L. performed the experiments; Y.W. and J.L. supervised the project, designed research, analyzed data, and wrote the paper.

ACKNOWLEDGMENTS We thank Chengcai Chu (Institute of Genetics and Developmental Biology) for providing the allelic mutant la1-Nipponbare. No conflict of interest declared. Received: January 8, 2019 Revised: May 7, 2019 Accepted: May 31, 2019 Published: June 11, 2019

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