2008-Nature-1.pdfTitleAuthorsAbstractMethods SummaryReferencesMethodsPlant materials and growth conditionsGeneration of transgenic Arabidopsis linesGA, MG132 and PAC treatmentsYeast two-hybrid and immunoprecipitation assaysBimolecular fluorescence complementation analysisIn vitro pull-down assaysSubcellular localization study of phyB-YFP and PIF3-CFP(cyan fluorescent protein) fusion proteinsMethods ReferencesFigure 1 Effect of GA3, MG132 and PAC on DELLA protein abundance.Figure 2 DELLA proteins and PIF3 have opposite roles in regulating Arabidopsis hypocotyl elongation.Figure 3 DELLA proteins bind PIF3 and inhibit PIF3 activity towards its target genes.Figure 4 GA-dependent interaction between GID1s and DELLA proteins.2008-Nature-1-SI.pdf3-Supplementary Note 1.pdf4-Supplementary Figures-big.pdf5-Supplementary Table 1.pdf6-Supplementary Table 2.pdfLETTERSCoordinated regulation of Arabidopsis thalianadevelopment by light and gibberellinsSuhua Feng1,2{, Cristina Martinez1*, Giuliana Gusmaroli1*, Yu Wang3*, Junli Zhou2*, Feng Wang2, Liying Chen2,Lu Yu2, Juan M. Iglesias-Pedraz4, Stefan Kircher5, Eberhard Scha¨fer5, Xiangdong Fu6, Liu-Min Fan3& Xing Wang Deng1,2,3Light and gibberellins (GAs) mediate many essential and partiallyoverlapping plant developmental processes. DELLA proteins areGA-signalling repressors that block GA-induced development1.GA induces degradation of DELLA proteins via the ubiquitin/proteasome pathway2, but light promotes accumulation ofDELLA proteins by reducing GA levels3. It was proposed thatDELLA proteins restrain plant growth largely through their effecton gene expression4,5. However, the precise mechanism of theirfunction in coordinating GA signalling and gene expressionremains unknown. Here we characterize a nuclear protein inter-action cascade mediating transduction of GA signals to the activityregulation of a light-responsive transcription factor. In theabsence of GA, nuclear-localized DELLA proteins accumulate tohigher levels, interact with phytochrome-interacting factor 3(PIF3, a bHLH-type transcription factor) and prevent PIF3 frombinding to its target gene promoters and regulating gene expres-sion, and therefore abrogate PIF3-mediated light control ofhypocotyl elongation. In the presence of GA, GID1 proteins (GAreceptors) elevate their direct interaction with DELLA proteinsin the nucleus, trigger DELLA protein’s ubiquitination andproteasome-mediated degradation, and thus release PIF3 fromthe negative effect of DELLA proteins.Light and GA interact during Arabidopsis thaliana seedlingdevelopment, regulating hypocotyl elongation, cotyledon openingand light-responsive gene expression; their pathways seem to con-verge at regulation of the abundance of DELLA proteins (GA path-way repressors)3,6. Arabidopsis has five DELLA proteins—RGA, GAI,RGL1, RGL2 and RGL3—defined by their unique DELLA domainand a conserved GRAS domain4. To analyse them in vivo, we raisedantibodies against endogenous RGA and generated transgenicArabidopsis expressing each of the five DELLA proteins with tandemaffinity purification (TAP) tags (Supplementary Fig. 1). The responseof DELLA protein levels to exogenously applied GA3(an active formof GA) or PAC (paclobutrazol, a GA biosynthesis inhibitor) wasexamined. We found that one-hour-long GA treatment eliminatesthe majority of DELLA proteins, and this GA effect can be largelyprevented by 100 mM MG132 (a 26S proteasome-specific inhibitor).PAC, on the other hand, promotes over-accumulation of DELLAproteins (Fig. 1). These results show for the first time inArabidopsis that all the DELLA proteins are under negative controlby GA and the proteasome. Next, we generated lines expressing TAP-tagged RGAD17 and GAID17, which lack a 17 amino acid motifwithin the DELLA domain that is required for GA-induced degrada-tion7,8. As expected, TAP–RGAD17 and TAP–GAID17 are completelyresistant to GA and accumulate at higher levels than wild-typeproteins, which cannot be further increased by PAC (Fig. 1, and*These authors contributed equally to this work.1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104, USA.2National Institute of Biological Sciences, ZhongguancunLife Science Park, Beijing 102206, China.3Peking–Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, and National Laboratory for Protein Engineering and PlantGenetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.4Departamento Gene´tica Molecular de Plantas, Centro Nacional de Biotecnologı´a-CSIC,Campus Universidad Auto´noma de Madrid, 28049 Madrid, Spain.5Institut fu¨r Biologie II/Botanik, Albert Ludwigs Universita¨t, Freiburg D-79104, Germany.6Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beijing 100080, China. {Present address: Howard Hughes Medical Institute, University of California at Los Angeles, LosAngeles, California 90095-1606, USA.WTAnti-RPN6Anti-RGAMG132––+–+–GA3––++––PAC+–––––35S-TAP–RGL135S-TAP–RGL235S-TAP–RGL335S-TAP–RGA35S-TAP–GAIAnti-RPN6Anti-RPN6Anti-RPN6Anti-RPN6Anti-RPN6Anti-MYCAnti-MYCAnti-MYCAnti-MYCAnti-MYCAnti-RPN6Anti-RPN6Anti-MYCAnti-MYC35S-TAP–GAI 17∆35S-TAP–RGA 17∆Figure 1|Effect of GA3, MG132 and PAC on DELLA protein abundance.Immunoblot analysis of RGA (by anti-RGA antibody) and TAP-DELLAproteins (by anti-MYC antibody) in various light-grown Arabidopsisseedlings (genotypes labelled to the left of each panel) treated with differentcombinations of GA3, MG132 and PAC. Panels on the left (four lanes) andpanels on the right (two lanes) are from two independent experiments usingdifferent protein gel systems. RPN6 immunoblotting (by anti-RPN6antibody) is used as a loading control. WT, wild type.Vol 451|24 January 2008|doi:10.1038/nature06448475Nature PublishingGroup©2008Supplementary Fig. 1b). Arabidopsis plants that overexpress theseproteins show a dominant dwarf phenotype, reflecting enhancedDELLA activity (Supplementary Fig. 2), which also suggests thatTAP–DELLA proteins retain normal DELLA function.Inhibition of hypocotyl elongation, an important characteristic ofphotomorphogenesis, is shown to be repressed by GA in the dark andpromoted by DELLA proteins in the light3,6. We further examinedthe possible mechanism of DELLA proteins in regulating photo-morphogenesis. Arabidopsis seedlings have longer hypocotyls onGA-containing medium, whereas PAC dramatically