Plant Signaling & Behavior
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Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/ NEDD8-activating enzyme in Arabidopsis seedlings Yutaka Oono & Akari Nakasone To cite this article: Yutaka Oono & Akari Nakasone (2013) Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings, Plant Signaling & Behavior, 8:10, e25986, DOI: 10.4161/psb.25986 To link to this article: http://dx.doi.org/10.4161/psb.25986
Published online: 20 Aug 2013.
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Date: 13 September 2015, At: 07:35
Short Communication
Short Communication
Plant Signaling & Behavior 8:10, e25986; October 2013; © 2013 Landes Bioscience
Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/ NEDD8-activating enzyme in Arabidopsis seedlings Medical and Biotechnological Application Division; Japan Atomic Energy Agency (JAEA); Takasaki, Japan †
Current affiliation: Department of Science and Engineering; Saitama University; Saitama, Japan
Keywords: SMAP1, Related to ubiquitin, RUB, Nedd8, auxin, MLN4924
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Abbreviation: SMAP1, SMALL ACIDIC PROTEIN 1; CaMV, cauliflower mosaic virus; CSN, constitutive photomorphogenic-9 signalosome
SMALL ACIDIC PROTEIN 1 (SMAP1) functions upstream of the degradation of AUX/IAA-proteins in the response to 2,4-dichlorophenoxyacetic acid and physically interacts with the COP9 SIGNALOSOME (CSN). Also, its function is linked to RELATED TO UBIQUITIN (RUB) modification. To further investigate the relationship between SMAP1 and the RUB modification system, we examined the effect of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme, on the growth of Arabidopsis thaliana. We found that the anti-auxin resistant 1 mutants, which lack SMAP1, are more sensitive to MLN4924 than wild type and that SMAP1 is responsible for this hypersensitivity. This new evidence supports our previous speculation that SMAP1 acts in Cullin-RING ubiquitin E3 ligase regulated signaling processes via its interaction with components associated with the RUB modification system.
The small conserved protein RELATED TO UBIQUITIN (RUB, or NEDD8 in mammals) covalently binds to specific regulatory proteins such as CULLIN (CUL), a scaffold protein in CUL-RING ubiquitin E3 ligases (CRLs).1 The RUB modification system is essential for plant growth and development;1 however, only a few regulators of the RUB modification system have been identified. The Arabidopsis anti-auxin resistant 1 (aar1) mutant is resistant to root growth inhibition caused by p-chlorophenoxyisobutylic acid (PCIB), an inhibitor of auxin action, and the aar1 phenotype results from a lack of SMALL ACIDIC PROTEIN 1 (SMAP1).2-4 The roots of aar1 mutants are also resistant to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), but have no alterations in 2,4-D metabolism or transport.4 SMAP1 encodes a 62 amino acid protein with phenylalanine (F) aspartic acid (D)-rich sequence (F/D-region) in its C-terminal region. A human SMAP1 homolog is annotated as multiple myeloma overexpression gene 2, implying that SMAP genes are evolutionarily indispensable.4,5 In Arabidopsis, SMAP1 acts upstream of degradation of AUXIN /INDOLE-3-ACETIC ACID (AUX/IAA)-proteins in the 2,4-D response pathway.4 AUX/IAA proteins are degraded by the ubiquitin-dependent pathway, via the SCFTIR1/AFBs E3 ligase,
which consists of CUL1, S PHASE KINASE-ASSOCIATED PROTEIN 1, RING-BOX PROTEIN 1, and an F-box auxin receptor, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOXES (AFBs).1 SMAP1 physically binds the evolutionarily conserved CONSTITUTIVE PHOTOMORPHOGENIC-9 SIGNALOSOME (CSN) complex, through the SMAP1 F/D-region.6 CSN has metalloprotease activity, via its CSN5 subunit, and de-conjugates RUB/ NEDD8 from CULs.7 Genetic evidence indicates that SMAP1 functions with AUXIN RESISTANT 1 (AXR1).6 The aar1 mutants have longer hypocotyls and petioles in young seedlings.4 However, the axr1 aar1 double mutants exhibit severe morphological defects, including failure of root meristem formation. Also, the ectopic expression of SMAP1 from the CaMV 35S promoter restores wild-type-like morphology in the axr1 background. This evidence indicates a close functional relationship between SMAP1 and AXR1.6 A similar genetic interaction was observed for AXR1LIKE (AXL), a homolog of the AXR1; although axl mutants resemble wild-type (WT) plants, axr1 axl double mutants fail to establish root meristems and subsequently die.8 AXR1 and AXL encode subunits of RUB-activating E1 enzyme, promote posttranslational RUB modification, and regulate CRL activity.8
*Correspondence to: Yutaka Oono; Email: [email protected] Submitted: 06/24/13; Revised: 07/30/13; Accepted: 07/31/13 Citation: Oono Y, Nakasone A. Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings. Plant Signaling & Behavior 2013; 8:e25986; http://dx.doi.org/10.4161/jrn.25986 www.landesbioscience.com
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Yutaka Oono* and Akari Nakasone†
Both CSN and AXR1 have significant roles in regulation of CRL-mediated signaling including the auxin response; these findings indicate that SMAP1 may also act in CRL-mediated signaling process by interacting with RUB modification-associated components.6 However, the direct involvement of SMAP1 in RUB modification remains unclear. In our previous study, no SMAP1-dependent difference was observed in the RUB modification status of CUL1, except in a special case: the percentage
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of RUB-modified CUL1 increased slightly in the flower extracts of transgenic plants ectopically expressing SMAP1 in the axr112 background.6 In this work, to further investigate the relationship between SMAP1 and RUB modification, we examined the effects of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme,9 on growth of WT and aar1-1 Arabidopsis seedlings. MLN4924 is structurally related to adenosine monophosphate and forms a NEDD8-MLN4924 adduct that resembles the first intermediate in the reaction, NEDD8-adenylate, but cannot be used in subsequent intra-enzyme reactions.10 In plants, MLN4924 is also reported to be an efficient inhibitor of RUB/ NEDD8 conjugation.11 To test the effect of this inhibitor, we plated Arabidopsis seeds on Germination Medium (GM; half-strength Murashige and Skoog salts, 0.5 gL –1 MES (pH 5.8), 1% [w/v] sucrose, 1 × B5 vitamins and 0.8% [w/v] Bacto agar) with or without MLN4924 (from Active Biochem, Wanchai, Hong Kong, and dissolved in dimethyl sulfoxide) in rectangular plates. To synchronize germination, the plates were put in the dark for 2 d at 4 °C, then transferred to a growth room at 24 °C. Lightgrown seedlings were grown vertically under 20–30 μmol m–2 sec –1 continuous light. For dark-grown seedlings, the plates were exposed to light for 4 h to induce germination and then wrapped in aluminum foil and kept vertically in a cardboard box. To examine the gravitropic response of the roots, the plants were photographed with a digital camera and analyzed using NIH ImageJ (National Institution of Health, USA). Figure 1 shows the effects of MLN4924 on 5-d-old lightgrown Arabidopsis seedlings. We observed agravitropic root responses and a significant reduction of root growth in MLN4924-treated WT Arabidopsis (Fig. 1A), as previously reported.11 By contrast, the aar1 seedlings showed a more severe effect of MLN4924, especially on the direction of the root growth (Fig. 1A). To evaluate the effect of MLN4924 more precisely, 4-d-old light-grown seedlings were transferred onto GM containing 5 μM MLN4924 and were placed at 135° to the vertical. The positions of root tips were marked before and after the 2-d incubation and the direction of root growth was analyzed. The root growth toward the direction of gravity in WT was only slightly disturbed by MLN4924, but the roots of most of the aar1-1 seedlings did not grow in the direction of gravity (Fig. 1B), clearly suggesting that the aar1-1 seedlings are more sensitive to MLN4924 than the WT seedlings.
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Figure 1. Effects of MLN4924 on light-grown seedlings. (A) Five-day-old Arabidopsis seedlings grown vertically with or without 5 μM MLN4924. The bar indicates 0.5 cm. (B) The distributions of root growth directions of the seedlings after 2 d of incubation angled at 135° to the vertical. Four-day-old light-grown seedlings were transferred onto GM media with (black bars) or without (white bars) 5 μM MLN4924 and incubated for 2 more days under the same light condition after rotating the seedlings to 135°. The arrows at top right indicate the vector of gravity before (1) and after (2) the commencement of the rotation. The angles were grouped into 20 classes and plotted as circular histograms. The bars in the legend on top represent 10% scale. The total number of seedlings analyzed (n ≥ 36) was set to 100% for the histograms.
Dark-grown seedlings have an elongated hypocotyl, closed cotyledons, and an apical hook (Fig. 2A). However, treatment with MLN4924 induced de-etiolated seedling growth, with short hypocotyls, opened cotyledons, and no apical hook in the darkgrown WT seedlings (Fig. 2B). The aar1-1 mutants showed a stronger decrease of hypocotyl length and a visible increase in anthocyanin accumulation (Fig. 2B), suggesting again that the aar1-1 mutants are more sensitive to MLN4924 than WT. The aar1-1 mutation is an approximately 44-kbp deletion in chromosome 4. At least 10 open reading frames, including SMAP1, are located in this deleted region.4 To determine whether
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lack of SMAP1 causes the observed MLN4924 sensitivity, we tested 2 independent SMAP1 RNAi lines (line 1G and 2C; previously called 520 i lines),4 in which SMAP1 RNA expression is diminished, and 2 independent SMAP1:SMAP1-GFP/aar1 lines (line D4 and G3), which express a functional SMAP1-GFP fusion protein from the SMAP1 promoter in the aar1-1 background.6 For light-grown seedlings, the extent of the sensitivity to MLN4924 was evaluated by root bending (Fig. 1B). The root tips of the majority of SMAP1:SMAP1-GFP/aar1 seedlings moved toward the direction of gravity, suggesting that sensitivity to MLN4924 of aar1-1 mutants was restored to similar level to
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Figure 2. Effects of MLN4924 in dark-grown seedlings. (A-C) Four-day-old Arabidopsis seedlings grown vertically without MLN4924 (A) or with 10 μM MLN4924 (B and C). Bars in (A), (B), and (C) indicate 0.5 cm, 0.5 cm, and 0.05 cm, respectively. In (C), representative images are shown for classification of “no visible accumulation” (class I), “weak anthocyanin accumulation” (class II), or “strong anthocyanin accumulation” (class III) in cotyledons and cotyledon petioles. (D) Classification of seedlings by extent of anthocyanin accumulation as shown in (C), with genotype. The total number of seedlings analyzed is indicated at the tops of the bars and set to 100% for the graph.
References 1. Dreher K, Callis J. Ubiquitin, hormones and biotic stress in plants. Ann Bot 2007; 99:787-822; PMID:17220175; http://dx.doi.org/10.1093/aob/ mcl255 2. Oono Y, Ooura C, Rahman A, Aspuria ET, Hayashi K, Tanaka A, Uchimiya H. p-Chlorophenoxyisobutyric acid impairs auxin response in Arabidopsis root. Plant Physiol 2003; 133:1135-47; PMID:14526108; http:// dx.doi.org/10.1104/pp.103.027847 3. Biswas KK, Ooura C, Higuchi K, Miyazaki Y, Van Nguyen V, Rahman A, Uchimiya H, Kiyosue T, Koshiba T, Tanaka A, et al. Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots. Plant Physiol 2007; 145:773-85; PMID:17905859; http://dx.doi.org/10.1104/pp.107.104844 4. Rahman A, Nakasone A, Chhun T, Ooura C, Biswas KK, Uchimiya H, Tsurumi S, Baskin TI, Tanaka A, Oono Y. A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid. Plant J 2006; 47:788801; PMID:16923017; http://dx.doi.org/10.1111/ j.1365-313X.2006.02832.x
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experimental conditions; in both genetic backgrounds, RUB conjugated CUL1 was equally decreased by MLN4924 treatment (data not shown). Thus, the modification status of CUL1 may not be directly related to the phenotypic differences in MLN4924 sensitivity between WT and aar1-1. SMAP1 may interact with RUB modification components and regulate SCF activity, but may not affect the RUB modification of CUL1. Similarly, CULLIN ASSOCIATED AND NEDDYLATIONDISSOCIATED 1 (CAND1) binds to unmodified CUL1 and negatively regulates SCF assembly without affecting RUB modification; RUB modification status remains unchanged in cand1 mutants.12 Alternatively, undetectable differences in modification status of CUL1 between WT and aar1-1 could be enough to cause the large differences in MLN4924 sensitivity. In conclusion, our phenotypic analysis indicates that aar1 mutants are more sensitive to MLN4924 and the deficiency of SMAP1 is responsible for this hypersensitivity. This evidence supports our previously proposed speculation that SMAP1 acts with RUB modification-related components. However, uncovering the biochemical function of SMAP1 still remains as a future challenge. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Kakenhi; no. 22570056) to YO.
5. Nakasone A, Kawai-Yamada M, Kiyosue T, Narumi I, Uchimiya H, Oono Y. A gene encoding SMALL ACIDIC PROTEIN 2 potentially mediates the response to synthetic auxin, 2,4-dichlorophenoxyacetic acid, in Arabidopsis thaliana. J Plant Physiol 2009; 166:1307-13; PMID:19307045; http://dx.doi. org/10.1016/j.jplph.2009.02.005 6. Nakasone A, Fujiwara M, Fukao Y, Biswas KK, Rahman A, Kawai-Yamada M, Narumi I, Uchimiya H, Oono Y. SMALL ACIDIC PROTEIN1 acts with RUB modification components, the COP9 signalosome, and AXR1 to regulate growth and development of Arabidopsis. Plant Physiol 2012; 160:93-105; PMID:22576848; http://dx.doi.org/10.1104/pp.111.188409 7. Cope GA, Suh GSB, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 2002; 298:608-11; PMID:12183637; http://dx.doi.org/10.1126/science.1075901 8. Dharmasiri N, Dharmasiri S, Weijers D, Karunarathna N, Jurgens G, Estelle M. AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis. Plant J 2007; 52:114-23; PMID:17655650; http://dx.doi.org/10.1111/j.1365313X.2007.03211.x
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9.
Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, Brownell JE, Burke KE, Cardin DP, Critchley S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 2009; 458:732-6; PMID:19360080; http://dx.doi. org/10.1038/nature07884 10. Brownell JE, Sintchak MD, Gavin JM, Liao H, Bruzzese FJ, Bump NJ, Soucy TA, Milhollen MA, Yang X, Burkhardt AL, et al. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8AMP mimetic in situ. Mol Cell 2010; 37:102-11; PMID:20129059; http://dx.doi.org/10.1016/j.molcel.2009.12.024 11. Hakenjos JP, Richter R, Dohmann EMN, Katsiarimpa A, Isono E, Schwechheimer C. MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants. Plant Physiol 2011; 156:527-36; PMID:21527421; http:// dx.doi.org/10.1104/pp.111.176677 12. Chuang HW, Zhang W, Gray WM. Arabidopsis ETA2, an apparent ortholog of the human cullin-interacting protein CAND1, is required for auxin responses mediated by the SCF(TIR1) ubiquitin ligase. Plant Cell 2004; 16:1883-97; PMID:15208392; http://dx.doi. org/10.1105/tpc.021923
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that of WT by introducing the functional SMAP1 transgene. By contrast, many root tips of the SMAP1 RNAi plants did not grow toward the direction of gravity, resembling the response observed in aar1-1 mutants (Fig. 1B). These results suggest that the lack of SMAP1 function in the aar1-1 mutants causes the higher sensitivity to MLN4924. The dark-grown seedlings were classified into 3 classes depending on the anthocyanin accumulation in the cotyledon and its petioles (Fig. 2C and D). In WT, approximately 90% of the seedlings did not accumulate anthocyanin (class I), but more than 60% of the aar1-1 seedlings accumulated purple anthocyanin in the cotyledons (class III). The ratio of seedlings that accumulated anthocyanin increased in the SMAP1 RNAi seedlings compared with WT, and decreased in the SMAP1:SMAP1-GFP/ aar1 seedlings compared with aar1. These results confirmed that SMAP1 causes the differences in MLN4924 sensitivity between WT and aar1 seedlings. The morphological and physiological effects caused by MLN4924 are considered to be a consequence of inhibition of RUB/NEDD8 conjugation. Indeed, similar anthocyanin accumulation in cotyledons after MLN4924 treatment was reported in dark-grown axr1 mutants, which are defective in the RUB E1 enzyme.11 Together with the previous evidence that axr1 aar1 double mutants showed a severe rootless phenotype and that a GFP-SMAP1 fusion protein physically interacted with CSN,6 the observed higher MLN4924 sensitivity in SMAP1-deficient plants indicates that SMAP1 function relates to RUB/NEDD8 conjugation. However, we observed no clear difference between WT and aar1–1 in RUB modification of CUL1 in different
ISSN: (Print) 1559-2324 (Online) Journal homepage: http://www.tandfonline.com/loi/kpsb20
Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/ NEDD8-activating enzyme in Arabidopsis seedlings Yutaka Oono & Akari Nakasone To cite this article: Yutaka Oono & Akari Nakasone (2013) Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings, Plant Signaling & Behavior, 8:10, e25986, DOI: 10.4161/psb.25986 To link to this article: http://dx.doi.org/10.4161/psb.25986
Published online: 20 Aug 2013.
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Article views: 54
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kpsb20 Download by: [117.253.203.227]
Date: 13 September 2015, At: 07:35
Short Communication
Short Communication
Plant Signaling & Behavior 8:10, e25986; October 2013; © 2013 Landes Bioscience
Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/ NEDD8-activating enzyme in Arabidopsis seedlings Medical and Biotechnological Application Division; Japan Atomic Energy Agency (JAEA); Takasaki, Japan †
Current affiliation: Department of Science and Engineering; Saitama University; Saitama, Japan
Keywords: SMAP1, Related to ubiquitin, RUB, Nedd8, auxin, MLN4924
Downloaded by [117.253.203.227] at 07:35 13 September 2015
Abbreviation: SMAP1, SMALL ACIDIC PROTEIN 1; CaMV, cauliflower mosaic virus; CSN, constitutive photomorphogenic-9 signalosome
SMALL ACIDIC PROTEIN 1 (SMAP1) functions upstream of the degradation of AUX/IAA-proteins in the response to 2,4-dichlorophenoxyacetic acid and physically interacts with the COP9 SIGNALOSOME (CSN). Also, its function is linked to RELATED TO UBIQUITIN (RUB) modification. To further investigate the relationship between SMAP1 and the RUB modification system, we examined the effect of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme, on the growth of Arabidopsis thaliana. We found that the anti-auxin resistant 1 mutants, which lack SMAP1, are more sensitive to MLN4924 than wild type and that SMAP1 is responsible for this hypersensitivity. This new evidence supports our previous speculation that SMAP1 acts in Cullin-RING ubiquitin E3 ligase regulated signaling processes via its interaction with components associated with the RUB modification system.
The small conserved protein RELATED TO UBIQUITIN (RUB, or NEDD8 in mammals) covalently binds to specific regulatory proteins such as CULLIN (CUL), a scaffold protein in CUL-RING ubiquitin E3 ligases (CRLs).1 The RUB modification system is essential for plant growth and development;1 however, only a few regulators of the RUB modification system have been identified. The Arabidopsis anti-auxin resistant 1 (aar1) mutant is resistant to root growth inhibition caused by p-chlorophenoxyisobutylic acid (PCIB), an inhibitor of auxin action, and the aar1 phenotype results from a lack of SMALL ACIDIC PROTEIN 1 (SMAP1).2-4 The roots of aar1 mutants are also resistant to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), but have no alterations in 2,4-D metabolism or transport.4 SMAP1 encodes a 62 amino acid protein with phenylalanine (F) aspartic acid (D)-rich sequence (F/D-region) in its C-terminal region. A human SMAP1 homolog is annotated as multiple myeloma overexpression gene 2, implying that SMAP genes are evolutionarily indispensable.4,5 In Arabidopsis, SMAP1 acts upstream of degradation of AUXIN /INDOLE-3-ACETIC ACID (AUX/IAA)-proteins in the 2,4-D response pathway.4 AUX/IAA proteins are degraded by the ubiquitin-dependent pathway, via the SCFTIR1/AFBs E3 ligase,
which consists of CUL1, S PHASE KINASE-ASSOCIATED PROTEIN 1, RING-BOX PROTEIN 1, and an F-box auxin receptor, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOXES (AFBs).1 SMAP1 physically binds the evolutionarily conserved CONSTITUTIVE PHOTOMORPHOGENIC-9 SIGNALOSOME (CSN) complex, through the SMAP1 F/D-region.6 CSN has metalloprotease activity, via its CSN5 subunit, and de-conjugates RUB/ NEDD8 from CULs.7 Genetic evidence indicates that SMAP1 functions with AUXIN RESISTANT 1 (AXR1).6 The aar1 mutants have longer hypocotyls and petioles in young seedlings.4 However, the axr1 aar1 double mutants exhibit severe morphological defects, including failure of root meristem formation. Also, the ectopic expression of SMAP1 from the CaMV 35S promoter restores wild-type-like morphology in the axr1 background. This evidence indicates a close functional relationship between SMAP1 and AXR1.6 A similar genetic interaction was observed for AXR1LIKE (AXL), a homolog of the AXR1; although axl mutants resemble wild-type (WT) plants, axr1 axl double mutants fail to establish root meristems and subsequently die.8 AXR1 and AXL encode subunits of RUB-activating E1 enzyme, promote posttranslational RUB modification, and regulate CRL activity.8
*Correspondence to: Yutaka Oono; Email: [email protected] Submitted: 06/24/13; Revised: 07/30/13; Accepted: 07/31/13 Citation: Oono Y, Nakasone A. Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings. Plant Signaling & Behavior 2013; 8:e25986; http://dx.doi.org/10.4161/jrn.25986 www.landesbioscience.com
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Yutaka Oono* and Akari Nakasone†
Both CSN and AXR1 have significant roles in regulation of CRL-mediated signaling including the auxin response; these findings indicate that SMAP1 may also act in CRL-mediated signaling process by interacting with RUB modification-associated components.6 However, the direct involvement of SMAP1 in RUB modification remains unclear. In our previous study, no SMAP1-dependent difference was observed in the RUB modification status of CUL1, except in a special case: the percentage
e25986-2
of RUB-modified CUL1 increased slightly in the flower extracts of transgenic plants ectopically expressing SMAP1 in the axr112 background.6 In this work, to further investigate the relationship between SMAP1 and RUB modification, we examined the effects of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme,9 on growth of WT and aar1-1 Arabidopsis seedlings. MLN4924 is structurally related to adenosine monophosphate and forms a NEDD8-MLN4924 adduct that resembles the first intermediate in the reaction, NEDD8-adenylate, but cannot be used in subsequent intra-enzyme reactions.10 In plants, MLN4924 is also reported to be an efficient inhibitor of RUB/ NEDD8 conjugation.11 To test the effect of this inhibitor, we plated Arabidopsis seeds on Germination Medium (GM; half-strength Murashige and Skoog salts, 0.5 gL –1 MES (pH 5.8), 1% [w/v] sucrose, 1 × B5 vitamins and 0.8% [w/v] Bacto agar) with or without MLN4924 (from Active Biochem, Wanchai, Hong Kong, and dissolved in dimethyl sulfoxide) in rectangular plates. To synchronize germination, the plates were put in the dark for 2 d at 4 °C, then transferred to a growth room at 24 °C. Lightgrown seedlings were grown vertically under 20–30 μmol m–2 sec –1 continuous light. For dark-grown seedlings, the plates were exposed to light for 4 h to induce germination and then wrapped in aluminum foil and kept vertically in a cardboard box. To examine the gravitropic response of the roots, the plants were photographed with a digital camera and analyzed using NIH ImageJ (National Institution of Health, USA). Figure 1 shows the effects of MLN4924 on 5-d-old lightgrown Arabidopsis seedlings. We observed agravitropic root responses and a significant reduction of root growth in MLN4924-treated WT Arabidopsis (Fig. 1A), as previously reported.11 By contrast, the aar1 seedlings showed a more severe effect of MLN4924, especially on the direction of the root growth (Fig. 1A). To evaluate the effect of MLN4924 more precisely, 4-d-old light-grown seedlings were transferred onto GM containing 5 μM MLN4924 and were placed at 135° to the vertical. The positions of root tips were marked before and after the 2-d incubation and the direction of root growth was analyzed. The root growth toward the direction of gravity in WT was only slightly disturbed by MLN4924, but the roots of most of the aar1-1 seedlings did not grow in the direction of gravity (Fig. 1B), clearly suggesting that the aar1-1 seedlings are more sensitive to MLN4924 than the WT seedlings.
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Figure 1. Effects of MLN4924 on light-grown seedlings. (A) Five-day-old Arabidopsis seedlings grown vertically with or without 5 μM MLN4924. The bar indicates 0.5 cm. (B) The distributions of root growth directions of the seedlings after 2 d of incubation angled at 135° to the vertical. Four-day-old light-grown seedlings were transferred onto GM media with (black bars) or without (white bars) 5 μM MLN4924 and incubated for 2 more days under the same light condition after rotating the seedlings to 135°. The arrows at top right indicate the vector of gravity before (1) and after (2) the commencement of the rotation. The angles were grouped into 20 classes and plotted as circular histograms. The bars in the legend on top represent 10% scale. The total number of seedlings analyzed (n ≥ 36) was set to 100% for the histograms.
Dark-grown seedlings have an elongated hypocotyl, closed cotyledons, and an apical hook (Fig. 2A). However, treatment with MLN4924 induced de-etiolated seedling growth, with short hypocotyls, opened cotyledons, and no apical hook in the darkgrown WT seedlings (Fig. 2B). The aar1-1 mutants showed a stronger decrease of hypocotyl length and a visible increase in anthocyanin accumulation (Fig. 2B), suggesting again that the aar1-1 mutants are more sensitive to MLN4924 than WT. The aar1-1 mutation is an approximately 44-kbp deletion in chromosome 4. At least 10 open reading frames, including SMAP1, are located in this deleted region.4 To determine whether
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lack of SMAP1 causes the observed MLN4924 sensitivity, we tested 2 independent SMAP1 RNAi lines (line 1G and 2C; previously called 520 i lines),4 in which SMAP1 RNA expression is diminished, and 2 independent SMAP1:SMAP1-GFP/aar1 lines (line D4 and G3), which express a functional SMAP1-GFP fusion protein from the SMAP1 promoter in the aar1-1 background.6 For light-grown seedlings, the extent of the sensitivity to MLN4924 was evaluated by root bending (Fig. 1B). The root tips of the majority of SMAP1:SMAP1-GFP/aar1 seedlings moved toward the direction of gravity, suggesting that sensitivity to MLN4924 of aar1-1 mutants was restored to similar level to
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Figure 2. Effects of MLN4924 in dark-grown seedlings. (A-C) Four-day-old Arabidopsis seedlings grown vertically without MLN4924 (A) or with 10 μM MLN4924 (B and C). Bars in (A), (B), and (C) indicate 0.5 cm, 0.5 cm, and 0.05 cm, respectively. In (C), representative images are shown for classification of “no visible accumulation” (class I), “weak anthocyanin accumulation” (class II), or “strong anthocyanin accumulation” (class III) in cotyledons and cotyledon petioles. (D) Classification of seedlings by extent of anthocyanin accumulation as shown in (C), with genotype. The total number of seedlings analyzed is indicated at the tops of the bars and set to 100% for the graph.
References 1. Dreher K, Callis J. Ubiquitin, hormones and biotic stress in plants. Ann Bot 2007; 99:787-822; PMID:17220175; http://dx.doi.org/10.1093/aob/ mcl255 2. Oono Y, Ooura C, Rahman A, Aspuria ET, Hayashi K, Tanaka A, Uchimiya H. p-Chlorophenoxyisobutyric acid impairs auxin response in Arabidopsis root. Plant Physiol 2003; 133:1135-47; PMID:14526108; http:// dx.doi.org/10.1104/pp.103.027847 3. Biswas KK, Ooura C, Higuchi K, Miyazaki Y, Van Nguyen V, Rahman A, Uchimiya H, Kiyosue T, Koshiba T, Tanaka A, et al. Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots. Plant Physiol 2007; 145:773-85; PMID:17905859; http://dx.doi.org/10.1104/pp.107.104844 4. Rahman A, Nakasone A, Chhun T, Ooura C, Biswas KK, Uchimiya H, Tsurumi S, Baskin TI, Tanaka A, Oono Y. A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid. Plant J 2006; 47:788801; PMID:16923017; http://dx.doi.org/10.1111/ j.1365-313X.2006.02832.x
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experimental conditions; in both genetic backgrounds, RUB conjugated CUL1 was equally decreased by MLN4924 treatment (data not shown). Thus, the modification status of CUL1 may not be directly related to the phenotypic differences in MLN4924 sensitivity between WT and aar1-1. SMAP1 may interact with RUB modification components and regulate SCF activity, but may not affect the RUB modification of CUL1. Similarly, CULLIN ASSOCIATED AND NEDDYLATIONDISSOCIATED 1 (CAND1) binds to unmodified CUL1 and negatively regulates SCF assembly without affecting RUB modification; RUB modification status remains unchanged in cand1 mutants.12 Alternatively, undetectable differences in modification status of CUL1 between WT and aar1-1 could be enough to cause the large differences in MLN4924 sensitivity. In conclusion, our phenotypic analysis indicates that aar1 mutants are more sensitive to MLN4924 and the deficiency of SMAP1 is responsible for this hypersensitivity. This evidence supports our previously proposed speculation that SMAP1 acts with RUB modification-related components. However, uncovering the biochemical function of SMAP1 still remains as a future challenge. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Kakenhi; no. 22570056) to YO.
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that of WT by introducing the functional SMAP1 transgene. By contrast, many root tips of the SMAP1 RNAi plants did not grow toward the direction of gravity, resembling the response observed in aar1-1 mutants (Fig. 1B). These results suggest that the lack of SMAP1 function in the aar1-1 mutants causes the higher sensitivity to MLN4924. The dark-grown seedlings were classified into 3 classes depending on the anthocyanin accumulation in the cotyledon and its petioles (Fig. 2C and D). In WT, approximately 90% of the seedlings did not accumulate anthocyanin (class I), but more than 60% of the aar1-1 seedlings accumulated purple anthocyanin in the cotyledons (class III). The ratio of seedlings that accumulated anthocyanin increased in the SMAP1 RNAi seedlings compared with WT, and decreased in the SMAP1:SMAP1-GFP/ aar1 seedlings compared with aar1. These results confirmed that SMAP1 causes the differences in MLN4924 sensitivity between WT and aar1 seedlings. The morphological and physiological effects caused by MLN4924 are considered to be a consequence of inhibition of RUB/NEDD8 conjugation. Indeed, similar anthocyanin accumulation in cotyledons after MLN4924 treatment was reported in dark-grown axr1 mutants, which are defective in the RUB E1 enzyme.11 Together with the previous evidence that axr1 aar1 double mutants showed a severe rootless phenotype and that a GFP-SMAP1 fusion protein physically interacted with CSN,6 the observed higher MLN4924 sensitivity in SMAP1-deficient plants indicates that SMAP1 function relates to RUB/NEDD8 conjugation. However, we observed no clear difference between WT and aar1–1 in RUB modification of CUL1 in different
