Non‐canonical AUX / IAA protein IAA 33 competes with canonical AUX / IAA repressor IAA 5 to negatively regulate auxin signaling
2019; Springer Nature; Volume: 39; Issue: 1 Linguagem: Inglês
10.15252/embj.2019101515
ISSN1460-2075
AutoresBingsheng Lv, Qianqian Yu, Jiajia Liu, Xuejing Wen, Zhenwei Yan, Kongqin Hu, Hanbing Li, Xiangpei Kong, Cuiling Li, Huiyu Tian, Ive De Smet, Xiansheng Zhang, Zhaojun Ding,
Tópico(s)Plant Gene Expression Analysis
ResumoArticle16 October 2019free access Source DataTransparent process Non-canonical AUX/IAA protein IAA33 competes with canonical AUX/IAA repressor IAA5 to negatively regulate auxin signaling Bingsheng Lv orcid.org/0000-0002-6394-6229 The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Qianqian Yu The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China College of Life Sciences, Liaocheng University, Liaocheng, Shandong, China Search for more papers by this author Jiajia Liu The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Xuejing Wen The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Zhenwei Yan The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Kongqin Hu The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Hanbing Li Department of Biochemistry, University of Missouri, Columbia, MO, USA Search for more papers by this author Xiangpei Kong The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Cuiling Li The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Huiyu Tian The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Ive De Smet Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium VIB Center for Plant Systems Biology, Ghent, Belgium Search for more papers by this author Xian-Sheng Zhang State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’ an, Shandong, China Search for more papers by this author Zhaojun Ding Corresponding Author [email protected] orcid.org/0000-0003-0218-5136 The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Bingsheng Lv orcid.org/0000-0002-6394-6229 The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Qianqian Yu The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China College of Life Sciences, Liaocheng University, Liaocheng, Shandong, China Search for more papers by this author Jiajia Liu The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Xuejing Wen The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Zhenwei Yan The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Kongqin Hu The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Hanbing Li Department of Biochemistry, University of Missouri, Columbia, MO, USA Search for more papers by this author Xiangpei Kong The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Cuiling Li The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Huiyu Tian The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Ive De Smet Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium VIB Center for Plant Systems Biology, Ghent, Belgium Search for more papers by this author Xian-Sheng Zhang State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’ an, Shandong, China Search for more papers by this author Zhaojun Ding Corresponding Author [email protected] orcid.org/0000-0003-0218-5136 The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China Search for more papers by this author Author Information Bingsheng Lv1,‡, Qianqian Yu1,2,‡, Jiajia Liu1,‡, Xuejing Wen1, Zhenwei Yan1, Kongqin Hu1, Hanbing Li3, Xiangpei Kong1, Cuiling Li1, Huiyu Tian1, Ive De Smet4,5, Xian-Sheng Zhang6 and Zhaojun Ding *,1 1The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China 2College of Life Sciences, Liaocheng University, Liaocheng, Shandong, China 3Department of Biochemistry, University of Missouri, Columbia, MO, USA 4Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium 5VIB Center for Plant Systems Biology, Ghent, Belgium 6State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’ an, Shandong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 532 58630889; E-mail: [email protected] EMBO J (2020)39:e101515https://doi.org/10.15252/embj.2019101515 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The phytohormone auxin controls plant growth and development via TIR1-dependent protein degradation of canonical AUX/IAA proteins, which normally repress the activity of auxin response transcription factors (ARFs). IAA33 is a non-canonical AUX/IAA protein lacking a TIR1-binding domain, and its role in auxin signaling and plant development is not well understood. Here, we show that IAA33 maintains root distal stem cell identity and negatively regulates auxin signaling by interacting with ARF10 and ARF16. IAA33 competes with the canonical AUX/IAA repressor IAA5 for binding to ARF10/16 to protect them from IAA5-mediated inhibition. In contrast to auxin-dependent degradation of canonical AUX/IAA proteins, auxin stabilizes IAA33 protein via MITOGEN-ACTIVATED PROTEIN KINASE 14 (MPK14) and does not affect IAA33 gene expression. Taken together, this study provides insight into the molecular functions of non-canonical AUX/IAA proteins in auxin signaling transduction. Synopsis IAA33, a non-canonical AUX/IAA without typical domains I and II, negatively regulates auxin response through the competition with IAA5, a canonical AUX/IAA, and thus releases the repression of ARF10/16. Furthermore, auxin stabilizes the IAA33 protein through interaction with MPK14 without influencing its transcription. IAA33 negatively regulates auxin responses and root stem cell identity. IAA33 regulates auxin signaling through competition with IAA5 for ARF10/16 binding. Auxin activates the phosphorylation activity of MPK14. Auxin induces IAA33 accumulation through phosphorylation by MPK14. Introduction The phytohormone auxin regulates almost every aspect of plant growth and development (Mockaitis & Estelle, 2008; Lavy & Estelle, 2016; Leyser, 2018). Since the identification of the TIR1 auxin receptor, the auxin signaling pathways have been well investigated (Dharmasiri et al, 2005; Kepinski & Leyser, 2005). A canonical auxin signaling pathway starts from auxin perception by a co-receptor complex, comprised of TIR1/AFB receptors and AUX/IAA proteins, followed by AUX/IAA protein ubiquitination and degradation, and eventually derepression of AUXIN RESPONSE FACTORs (ARFs) and the transcriptional activation of auxin-induced gene expression (Peer, 2013). The domain II of AUX/IAA proteins mediates the interaction with TIR1/AFB receptors, a process which is promoted by auxin (Ramos et al, 2001; Kepinski & Leyser, 2004; Tan et al, 2007). There are 23 ARFs in Arabidopsis (Hagen & Guilfoyle, 2002). Structural studies revealed that there are three conserved regions of homology in these ARFs: an N-terminal B3-type DNA-binding domain (DBD) and two C-terminal regions, which share homology with domains III and IV of AUX/IAA proteins (Guilfoyle & Hagen, 2007; Korasick et al, 2014). Domains III and IV are responsible for homo- and heterodimerization of AUX/IAA proteins or ARFs (Tiwari et al, 2003). Between the N-terminal DBD and C-terminal domains, ARFs contain a variable middle domain, which has been proposed to confer either activation or repression properties of these transcription factors. Based on the amino acid composition of the middle regions, ARFs can be classified as activators or repressors (Tiwari et al, 2003). ARF10 and ARF16, which have been shown to regulate root stem cell identities (Ding & Friml, 2010), were characterized as transcriptional repressors (Wang et al, 2005; Bennett et al, 2014). Several recent studies suggest a high complexity of the auxin signaling pathway (Jing et al, 2015; Wang et al, 2015; Yu et al, 2015; Dezfulian et al, 2016), which can also explain how auxin modulates diverse aspects of plant growth and development. Although canonical auxin signaling has been well studied, whether the non-canonical AUX/IAA proteins, which lack the conserved domain II, take part in the auxin signaling or the mechanism of non-canonical auxin signaling still remains elusive. A recent study showed that the non-canonical AUX/IAA proteins, IAA20 and IAA30, were required for the proper vascular patterning. The double mutant iaa20/30 formed ectopic protoxylem, while overexpression of IAA30 caused discontinuous protoxylem and occasional ectopic metaxylem (Muller et al, 2016). Chen et al (2018) reported that a RING finger E3 ubiquitin ligase (SOR1) controlled root-specific ethylene responses by modulating a non-canonical AUX/IAA protein (OsIAA26) stability. Recently, Cao et al reported that auxin-mediated C-terminal cleavage of the TRANSMEMBRANE KINASE 1 (TMK1) leads to phosphorylation of two non-canonical AUX/IAA proteins (IAA32 and IAA34) and their subsequent stabilization to regulate differential growth of the apical hook (Cao et al, 2019). In this study, we investigated the role of IAA33, a non-canonical AUX/IAA protein without typical domains I and II, which are essential components to mediate the canonical auxin signaling through the TIR1-dependent pathway. The provided evidence shows that IAA33 is involved in auxin signaling through interacting with ARF10 and ARF16, which have been reported to control root distal stem cell (DSC) identity (Ding & Friml, 2010), and consequently regulates root DSC identity. IAA33 negatively regulates auxin response through the competition with IAA5, a canonical AUX/IAA protein, and thus releases the repression of ARF10/16 in this process. Furthermore, different from the up-regulation of the transcription and the destruction of canonical AUX/IAA proteins such as IAA5, auxin stabilizes the IAA33 protein through interaction with MPK14 without influencing its transcription. Results IAA33 negatively regulates auxin responses The canonical AUX/IAA proteins act as transcriptional repressors and mediate auxin signaling through interaction with TIR1 receptors (Villalobos et al, 2012; Weijers & Wagner, 2016). IAA33 has no domains I and II, which represses ARF-mediated transcription and mediates the interaction between AUX/IAA protein and TIR1, respectively (Appendix Fig S1A). Consistent with the absence of domain II, IAA33 could not interact with TIR1, while the canonical domain II-containing IAA5 could interact with TIR1 in yeast (Appendix Fig S1B). To address whether the non-canonical IAA33 could regulate auxin signaling, we examined auxin responses using DR5rev::GFP as a marker in iaa33 and IAA33OE lines. A highly increased transcriptional auxin response was observed in iaa33, which is reflected by the increased DR5rev::GFP signal (Fig 1A). Consistently, when IAA33 is overexpressed, the auxin response was repressed, which is shown by the reduced DR5rev::GFP signal (Fig 1A). Similarly, co-expression of DR5::LUC with the 35S::IAA33 construct in protoplast cells isolated from Arabidopsis leaves led to an obvious reduction in luminescence intensity (Fig 1B), further suggesting that overexpression of IAA33 represses auxin response. Our qRT–PCR results also showed that the relative expression levels of auxin-induced genes such as IAA3/4/5/11/13/18/28 were increased in iaa33 compared with Col-0 (Fig 1C). Taken together, these results suggest that IAA33 negatively regulates auxin response. Figure 1. IAA33 negatively regulates auxin responses Expression of the auxin reporter DR5rev::GFP in Col-0, iaa33, and IAA33OE roots. DR5rev::GFP crossed with iaa33 or IAA33OE seedlings, respectively. Scale bars, 50 μm. Transient expression analysis of overexpression of IAA33 on DR5::LUC activity in A. thaliana protoplasts. IAA33 was co-transfected with DR5::LUC. The LUC-to-REN ratio was shown to indicate the expression level of the DR5::LUC. LUC: firefly luciferase activity, REN: Renilla luciferase activity. Data shown are means ± standard errors (n = 9); *: means significant difference compared to control (P < 0.05) based on Duncan's test. The relative expression of IAA3/4/5/11/13/18/28 in Col-0 and iaa33. RNA was isolated from the roots of 6-day-old Col-0 and iaa33 seedlings using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Data shown are means ± standard errors (n = 3); *: means significant difference compared to Col-0 (P < 0.05) based on Duncan's test. Download figure Download PowerPoint IAA33 controls root stem cell identity Analysis of an IAA33p::GUS-GFP line showed that IAA33 is expressed in root tips (Fig 2E). Since auxin plays an essential role in the maintenance of root stem cell identity (Blilou et al, 2005; Ding & Friml, 2010), we also examined the role of IAA33, which negatively regulates auxin response, in this developmental context. The results showed that the iaa33 mutant exhibited reduced root distal stem cell (DSC) differentiation, shown by a higher rate of seedling roots with 2 layers of DSCs (Fig 2A–C, Appendix Fig S2). In contrast, the IAA33 overexpression (IAA33OE) lines displayed enhanced root DSC differentiation reflected by a higher rate of seedling roots without DSCs (Fig 2A–C, Appendix Fig S2). The allele test showed that the F1 of iaa33-cas9 crossed with iaa33 also had a higher rate of 2 layers of DSCs, which is similar to iaa33 mutant (Appendix Fig S2C). These observations are in line with IAA33 negatively regulating auxin response. Figure 2. IAA33 controls root distal stem cell (DSC) identity In Lugol-stained 5-day-old Col-0, iaa33, iaa5, IAA33OE, and IAA5OE seedling roots, root distal stem cell (DSC) differentiation is reduced in iaa33 and IAA5OE seedlings and enhanced in IAA33OE and iaa5 seedlings (red asterisk indicates QC cells, and yellow arrowheads indicate distal stem cell). Scale bars, 20 μm. Quantitative evaluation of root DSC layers in 5-day-old seedlings of Col-0, iaa33, and IAA33OE, which are grown on MS medium (n = 50). The relative expression of IAA33 in Col-0, iaa33, and IAA33OE. Data shown are means ± standard errors (n = 3), **: means of iaa33, IAA33OE differ significantly from mean of Col-0 (P < 0.01) based on Duncan's test. Quantitative evaluation of root DSC layers in 5-day-old seedlings of Col-0, iaa5, and IAA5OE, which are grown on MS medium (n = 50). IAA33 is expressed in root. Yellow asterisk indicates QC cells. Black scale bars, 1 cm. White scale bars, 50 μm. Download figure Download PowerPoint IAA33 interacts with ARF10 and ARF16 It was previously reported that among the non-canonical AUX/IAA proteins, ARF10/16 could interact with IAA32, IAA33, and IAA34 (Piya et al, 2014). Both ARF10 and ARF16 are well known to regulate auxin signaling and root stem cell identity (Wang et al, 2005; Ding & Friml, 2010; Bennett et al, 2014). The expression of IAA32p::GUS and IAA34p::GUS, which are expressed in the apical hook (Cao et al, 2019), is absent from the root tip (Appendix Fig S3). This suggested that IAA32/34 are likely not involved in the regulation of root development. Therefore, we next only focused on examining whether IAA33 could interact with root-expressed ARF10 and ARF16 (Rademacher et al, 2011). A yeast two-hybrid analysis confirmed that IAA33 could interact with both ARF10 and ARF16 (Appendix Fig S5A), which was consistent with the previous report (Piya et al, 2014). Furthermore, bimolecular fluorescence complementation (BiFC) assays also showed strong fluorescence signal in tobacco epidermal cells co-expressing IAA33 fused to the N-terminal half of YFP (NYFP) and ARF10 or ARF16 fused to the C-terminal half of YFP (CYFP), whereas no signal was observed in the empty vector control (Fig 3A). To confirm this, we performed in vitro pull-down assays with GST-tagged IAA33 in combination with His-tagged ARF10 or ARF16. The results showed that IAA33 could interact with ARF10 or ARF16 in vitro (Fig 3B). Finally, we further confirmed the interaction between IAA33 and ARF10 or ARF16, using co-immunoprecipitation assays in tobacco leaves. IAA33-GFP protein was immunoprecipitated by anti-MYC antibody from tobacco leaf cells co-expressing ARF10-MYC or ARF16-MYC (Fig 3C and D). Together, all these results indicate that IAA33 interacts with ARF10 or ARF16. Figure 3. IAA33 interacts with ARF10 and ARF16 A. BiFC analysis of interaction between IAA33 and ARF10/16. The split YFP system was used in BiFC assays. CYFP and NYFP are empty vectors. The different combinations of plasmids were transformed into tobacco epidermal cells, and the YFP signals were detected with a confocal microscope. Scale bars, 50 μm. B. Western blotting results of the GST pull-down assay of IAA33-GST and ARF10-His or ARF16-His. MBP-His was used as the negative control. C, D. In vivo Co-IP assays of IAA33 with ARF10 (C) or ARF16 (D). ARF10-MYC or ARF16-MYC was co-expressed with IAA33-GFP in tobacco leaves. Protein extracts (Input) were immunoprecipitated with anti-MYC antibody (IP). Immunoblots were developed with anti-GFP antibody to detect IAA33 and with anti-MYC to detect ARF10 and ARF16. Source data are available online for this figure. Source Data for Figure 3 [embj2019101515-sup-0002-SDataFig3.pdf] Download figure Download PowerPoint Furthermore, genetic analysis showed that the reduced DSC differentiation in iaa33 and the higher frequency of DSC differentiation in IAA33OE were both repressed by overexpression of or mutations in ARF10 or ARF16, respectively (Appendix Fig S4), indicating that IAA33 controls root stem cell identity through ARF10 and ARF16. The canonical IAA5 has also been reported to interact with ARF10 or ARF16 in yeast (Piya et al, 2014). We confirmed this interaction through BiFC assays, pull-down assays, and Co-IP analysis (Appendix Fig S5). Consistently, the overexpression of IAA5 (IAA5OE) lines exhibited reduced root DSC differentiation, shown by a higher rate of seedling roots with 2 layers of DSCs. In contrast, the iaa5 mutant displayed enhanced root DSC differentiation reflected by a higher rate of seedling roots without DSCs (Fig 2A and D). We also tested the interaction between IAA33 and other repressor ARFs or activator ARFs by yeast two-hybrid assays and found that in addition to ARF10/16, IAA33 also strongly interacted with ARF1 and ARF18 (Appendix Fig S6). In agreement with previously published data (Piya et al, 2014), this indicated that IAA33 preferentially binds to repressor ARFs. IAA33 competes with IAA5 for ARF10/16 binding IAA5 is a canonical AUX/IAA protein, which could interact with TIR1 (Shimizu-Mitao & Kakimoto, 2014) (Appendix Fig S1) and negatively regulated transcriptional auxin response (Appendix Fig S7). Furthermore, both IAA33 and IAA5 strongly interacted with ARF10 or ARF16 (Fig 3 and Appendix Fig S5), while the interaction between IAA33 and IAA5 was weak (Appendix Fig S8). This indicated that IAA33 prefers to interact with ARF10/16 rather than heterodimerize with IAA5. Therefore, we examined whether IAA33 could compete with IAA5 to interact with ARF10 or ARF16. In vitro pull-down assays indicated that increasing the level of GST-tagged IAA33 clearly reduced the interaction between IAA5 with ARF10 or ARF16, suggesting that IAA33 could compete with IAA5 to interact with ARF10 or ARF16 (Fig 4A). This result was also confirmed by yeast three-hybrid assays. In the presence of co-expressed IAA33, the interaction between IAA5 and ARF10 or ARF16 was blocked, which was shown through the inactivation of the reporter (Fig 4B). However, in the presence of co-expressed IAA5, we still observed the activation of the reporter, which showed the interaction between IAA33 and ARF10 or ARF16 (Appendix Fig S9). In addition, IAA5, IAA33, ARF10, and ARF16 all repressed transcriptional auxin response, which was shown by decreased DR5::LUC activity in transient expression assays in Arabidopsis protoplasts (Fig 4C and D). Furthermore, repression of transcriptional auxin response by ARF10 and ARF16 could be partially alleviated through co-expression of IAA5. However, this negative regulation of IAA5 on ARF10 and ARF16 is removed by IAA33 (Fig 4C and D). All these results indicate that IAA33 regulates auxin signaling through competition with IAA5. Figure 4. IAA33 interacts with ARF10 and ARF16 through the competition with IAA5 A. Immunoblot of an immunoprecipitation (IP)/protein competition assay co-expressing IAA5-MBP and ARF10-His or ARF16-His with increasing amounts of IAA33-GST. The amounts of IAA5-MBP and 1X IAA33-GST were 20 μg, respectively. The anti-MBP antibody used for IP is indicated at the top panel, while anti-GST for IP is shown at the bottom panel. B. Yeast three-hybrid assay analyzing the IAA5-ARF10/16 interaction in the presence or absence of co-expressed IAA33. Co-transformants were spotted on SD-Leu-Trp medium to check for viability and on SD-Met-His-Leu-Trp medium to test the interaction and competition, respectively. C, D. IAA33 removes the repression of IAA5 on ARF10- and ARF16-mediated auxin signaling. Values are the means ± standard errors of three biological replicates. Different letters above bars indicate a significant difference (P < 0.05) based on Duncan's test. Source data are available online for this figure. Source Data for Figure 4 [embj2019101515-sup-0003-SDataFig4.pdf] Download figure Download PowerPoint Auxin can induce the accumulation of IAA33 The canonical AUX/IAA proteins act as transcriptional repressors that are degraded through the 26S proteasome pathway in the presence of auxin and thus release the repression of ARF transcription factors and activate auxin signaling (Gray et al, 2001; Ramos et al, 2001). On the other hand, the activated auxin signaling could induce the expression of these canonical AUX/IAAs (Walker & Key, 1982). IAA5 is a canonical IAA with 4 typical domains (Appendix Fig S1). Therefore, it was not surprising that we observed the clear up-regulation of IAA5 expression with auxin treatment as shown by qRT–PCR and GUS staining of an IAA5p::GUS line (Appendix Fig S10A and B). As expected, the IAA5 protein was degraded in the presence of auxin (Appendix Fig S10C and D), a process which is dependent on the 26S proteasome pathway since MG132 co-treatment could slow down the degradation of IAA5 (Appendix Fig S10E). However, both qRT–PCR analysis and GUS staining assays in IAA33p::GUS-GFP lines suggested that the transcription of the non-canonical IAA33 is not affected by auxin (Fig 5A and B). Furthermore, though IAA33 is degraded through the 26S proteasome pathway, which is inhibited by MG132 (Fig 5D), we did not detect the auxin-induced degradation of IAA33 (Fig 5C and E, Appendix Fig S11). On the contrary, we observed that auxin stabilized the IAA33 protein, which was shown by the accumulated IAA33 upon NAA treatment (Fig 5C and E, Appendix Fig S11). All these results suggest that, compared to canonical AUX/IAA proteins such as IAA5, the non-canonical IAA33 is regulated differently in response to auxin at both transcriptional and protein levels. Figure 5. Auxin induces IAA33 protein accumulations GUS staining analysis of IAA33p::GUS-GFP seedlings when treated with or without NAA (10 μM) for 4 h. Scale bars, 50 μm. Transcript abundance of IAA33 when treated with or without NAA. Five-day-old seedlings were treated with or without NAA (10 μM) for 4 h, and used for RNA extraction and subjected to qRT–PCR analysis. Data shown are means ± standard errors (n = 3). Confocal images of 35S::GFP-IAA33 seedling roots when treated with or without NAA (10 μM) for 4 h. Scale bars, 50 μm. IAA33 protein level was examined by Western blot at different time points after CHX only or CHX and MG132 co-treatment in 5-day-old 35S::GFP-IAA33 seedlings. The relative intensity of band detected by anti-GFP antibody to that by anti-actin antibody without treatment was set to 1.0. IAA33 protein level was examined by Western blot when 5-day-old 35S::GFP-IAA33 seedlings were treated with auxin (10 μM NAA) at indicated time points. The relative intensity of band detected by anti-GFP antibody to that by anti-actin antibody without treatment was set to 1.0. Source data are available online for this figure. Source Data for Figure 5 [embj2019101515-sup-0004-SDataFig5.pdf] Download figure Download PowerPoint IAA33 interacts with MPK14 To elucidate the molecular mechanism of auxin-induced IAA33 protein accumulation, we performed a yeast two-hybrid assay to identify interacting proteins of IAA33. Through this assay, MITOGEN-ACTIVATED PROTEIN KINASE 14 (MPK14) was identified to interact with IAA33 (Fig 6B). The interaction between MPK14 and IAA33 was also confirmed by co-immunoprecipitation (Co-IP) assays using proteins extracted from Arabidopsis mesophyll protoplast transiently expressing different construct combinations (IAA33-MYC/MPK14-YFP, MPK14-YFP, IAA33-MYC). IAA33-MYC was successfully detected in the anti-GFP immunoprecipitates of cells co-expressing IAA33-MYC and MPK14-YFP (Fig 6A). Additional evidence that IAA33 interacts with MPK14 came from a bimolecular fluorescence complementation (BiFC) assay. A strong fluorescence signal was observed in tobacco epidermal cells co-expressing IAA33 fused to the N-terminal half of YFP (NYFP) and MPK14 fused to the C-terminal half of YFP (CYFP), whereas no signal was observed in the empty vector control (Fig 6C). Last, in vitro pull-down assays also confirmed the interaction between IAA33 and MPK14, since MPK14-MBP was pulled down with IAA33-GST (Fig 6D). All these results demonstrate that the IAA33 protein interacts with MPK14 in vitro and in vivo. Figure 6. IAA33 interacts with MPK14 In vivo Co-IP assays of IAA33 with MPK14. IAA33-MYC was co-expressed with MPK14-YFP in Arabidopsis mesophyll protoplast. Protein extracts (Input) were immunoprecipitated with anti-GFP antibody (IP). Immunoblots were developed with anti-GFP antibody to detect MPK14 and with anti-MYC to detect IAA33. IAA33 specifically interacts with MPK14 in yeast. IAA33 was used as bait, and MPK14 was used as prey. Empty-AD was co-transformed as negative control. BiFC analysis of interaction between IAA33 and MPK14. The split YFP system was used in BiFC assays. CYFP and NYFP are empty vectors. The different combinations of plasmids were transformed into tobacco epidermal cells, and the YFP signals were detected with a confocal microscope. Scale bars, 50 μm. Western blotting results of the GST pull-down assay of IAA33-GST and MPK14-MBP. Source data are available online for this figure. Source Data for Figure 6 [embj2019101515-sup-0005-SDataFig6.pdf] Download figure Download PowerPoint Auxin-induced IAA33 accumulation is mediated through M
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