BPM-CUL3 E3 ligase modulates thermotolerance by PNAS PLUS facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis Kyoko Morimotoa,1,2, Naohiko Ohamaa,1,3, Satoshi Kidokoroa, Junya Mizoia, Fuminori Takahashib, Daisuke Todakaa, Junro Mogamia, Hikaru Satoa, Feng Qinc,4, June-Sik Kimb, Yoichiro Fukaod, Masayuki Fujiwarad, Kazuo Shinozakib, and Kazuko Yamaguchi-Shinozakia,5 aLaboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; bGene Discovery Research Group, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan; cBiological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan; and dPlant Global Education Project, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan Edited by Julia Bailey-Serres, University of California, Riverside, CA, and approved August 22, 2017 (received for review March 13, 2017) DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A DEHYDRATION-RESPONSIVE ELEMENT-BINDING (DREB2A) acts as a key transcription factor in both drought and PROTEIN 2A (DREB2A), an APETALA2/ethylene-responsive heat stress tolerance in Arabidopsis and induces the expression of element binding factor-type (AP2/ERF) transcription factor, is a many drought- and heat stress-inducible genes. Although DREB2A key factor governing the expression of many target genes in re- expression itself is induced by stress, the posttranslational regula- sponse to drought and heat stresses via a cis-acting element known tion of DREB2A, including protein stabilization, is required for its as the dehydration-responsive element/C-repeat (DRE/CRT; transcriptional activity. The deletion of a 30-aa central region of A/GCCGAC) (7). Expression of the DREB2A gene itself is in- DREB2A known as the negative regulatory domain (NRD) trans- duced by these stresses via different cis-acting elements in its pro- forms DREB2A into a stable and constitutively active form referred moter region (8, 9); however, the expression of DREB2A alone is to as DREB2A CA. However, the molecular basis of this stabiliza- notsufficienttoactivatetheexpression of DREB2A target genes, tion and activation has remained unknown for a decade. Here we reflecting a posttranslational negative regulatory system (10). The PLANT BIOLOGY identified BTB/POZ AND MATH DOMAIN proteins (BPMs), sub- posttranslational regulation of DREB2A involves the control of strate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A- protein stability mediated by the 30-aa negative regulatory domain interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its in- Significance teraction with BPMs. BPM-knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes un- der heat and drought stress conditions. Genetic analysis indicated DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2A that the depletion of BPM expression conferred enhanced thermo- (DREB2A) is a key transcription factor for plant adaptation to tolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase drought and heat. DREB2A activity is strictly regulated via pro- is likely the long-sought factor responsible for NRD-dependent teolysis mediated by the negative regulatory domain (NRD), al- DREB2A degradation. Through the negative regulation of DREB2A though the molecular basis for this regulation has remained stability, BPMs modulate the heat stress response and prevent an unclear for a decade. We reveal that BTB/POZ AND MATH adverse effect of excess DREB2A on plant growth. Furthermore, DOMAIN proteins (BPMs), substrate adaptors for Cullin3-based we found the BPM recognition motif in various transcription fac- E3 ubiquitin ligase, are the long-sought factors responsible for tors, implying a general contribution of BPM-mediated proteolysis NRD-dependent DREB2A degradation. Through DREB2A degra- to divergent cellular responses via an accelerated turnover of dation, BPMs negatively regulate the heat stress response and transcription factors. prevent the adverse effects of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various abiotic stress response | co-IP coupled with LC-MS/MS | DREB2A-interacting transcription factors, implying a general contribution of BPM- proteins | E3 ubiquitin ligase | posttranslational regulation mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors. lants often manage to survive under various environmental Author contributions: K.M., N.O., K.S., and K.Y.-S. designed research; K.M., N.O., S.K., Pstress conditions, such as drought, high salinity, and extreme J. Mizoi, D.T., J. Mogami, H.S., and F.Q. performed research; F.T., Y.F., and M.F. con- temperatures. Transcriptional regulation is one of the most im- tributed new reagents/analytic tools; S.K., J. Mizoi, F.T., and J.-S.K. analyzed data; and portant mechanisms in the acquisition of stress tolerance (1, 2). K.M., N.O., and K.Y.-S. wrote the paper. However, in many cases, stress adaptation is exchanged for growth The authors declare no conflict of interest. and productivity; therefore, it is necessary for plants to develop a This article is a PNAS Direct Submission. resilient system to obtain the optimal trade-off for survival and Data deposition: Microarray data have been deposited in the European Bioinformatics growth. To this end, plants use elaborate mechanisms associated Institute’s ArrayExpress database, www.ebi.ac.uk/arrayexpress/ (accession no. E-MTAB- 5845). with posttranscriptional modulation (3) and posttranslational 1 regulation (4–6), as well as transcriptional regulation. In particu- K.M and N.O. contributed equally to this work. 2 lar, the appropriate control of transcription factors regulating Present address: The Plant Chemetics Laboratory, Department of Plant Sciences, Univer- sity of Oxford, Oxford OX1 3RB, United Kingdom. stress-inducible genes is important, because these transcription 3Present address: Temasek Life Sciences Laboratory, National University of Singapore, factors negatively affect plant growth and productivity while being Singapore 117604, Singapore. essential for increased stress tolerance. The environmental con- 4Present address: State Key Laboratory of Plant Physiology and Biochemistry, College of ditions surrounding plants are constantly changing; thus, post- Biological Sciences, China Agricultural University, Beijing 100193, China. translational regulation to control the protein levels of these 5To whom correspondence should be addressed. Email: [email protected]. transcription factors is considered an important mechanism to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. avoid adverse effects on plant growth and productivity. 1073/pnas.1704189114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1704189114 PNAS Early Edition | 1of9 Downloaded by guest on September 23, 2021 (NRD) adjacent to the ERF/AP2 DNA-binding domain. Removal tional regulation (SAP18) (23) as the putative regulators of of the NRD yields a constitutively stable and active form of DREB2A activity. We confirmed the interactions of candidate DREB2A known as DREB2A CA. Overexpression of DREB2A proteins and RCD1 with DREB2A using a yeast two-hybrid CA induces the expression of DREB2A target genes, even under (Y2H) assay. Yeast growth on the agar plate (Fig. 1A) and in control conditions, and enhances tolerance to drought and heat the liquid medium (Fig. S1B) indicates that BPM2 and BPM4 stresses (10, 11). At the same time, overexpression of DREB2A CA interact with DREB2A, but TCP1 and SAP18 do not. Thus, we also adversely affects plant growth, resulting in dwarfism and re- selected BPM2 and BPM4 as potential DREB2A interactors for duced reproduction. Therefore, NRD-mediated negative regula- further analysis. tion contributes to avoidance of the adverse effects of DREB2A Although BPM2 and BPM4 were predicted to be localized to induction through the strict control of its stability. However, despite the cytosol (Dataset S1), BPM2 is reportedly localized to the the importance of DREB2A in stress responses, the molecular nucleus in tobacco (24). To reveal the accurate localization of basis of NRD function has been unclear for a decade. BPM2 and BPM4 in Arabidopsis, we generated overexpressors of DREB2A-INTERACTING PROTEINS 1 and 2 (DRIP1/2) GFP-fused BPM2 and BPM4. BPM2 was localized in the nucleus are C3HC4 RING domain-containing proteins identified as under both normal and heat stress conditions, whereas BPM4 DREB2A interactors that function as E3 ubiquitin ligases (12). translocated from the cytosol to the nucleus on heat shock (Fig. Through the acceleration of 26S proteasome-mediated DREB2A 1B). The GFP-DREB2A signal was observed in the nucleus only proteolysis, DRIP1/2 negatively regulate the expression of DREB2A- after heat stress. Taken together, our findings indicate that both downstream genes. However, double knockout of DRIP1/2 was BPM2 and BPM4 are localized in the nucleus under heat stress found to only partially enhance the stability of DREB2A under conditions, suggesting that they potentially colocalize and in- stress conditions (13). Therefore, other E3 ligases have been im- teract with DREB2A in vivo. plicated in DREB2A degradation. To