Jasmonate signaling involves the abscisic acid receptor PYL4 to regulate metabolic reprogramming in Arabidopsis and tobacco Petri Lackmana,1, Miguel González-Guzmánb,1,Sofie Tillemanc,d, Inês Carqueijeiroc,d,e, Amparo Cuéllar Pérezc,d, Tessa Mosesc,d,f,g, Mitsunori Seoh, Yuri Kannoh, Suvi T. Häkkinena, Marc C. E. Van Montagud,2, Johan M. Theveleinf,g, Hannu Maaheimoa, Kirsi-Marja Oksman-Caldenteya, Pedro L. Rodriguezb, Heiko Rischera, and Alain Goossensc,d,2 aVTT Technical Research Center of Finland, FIN-02044 VTT, Espoo, Finland; bInstituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, E-46022 Valencia, Spain; cDepartment of Plant Systems Biology, VIB, B-9052 Ghent, Belgium; dDepartment of Plant Biotechnology and Genetics, Ghent University, B-9052 Ghent, Belgium; eInstituto de Biologia Molecular e Celular and Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4150-180 Porto, Portugal; fDepartment of Molecular Microbiology, VIB, B-3001 Leuven, Belgium; gLaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, B-3001 Leuven-Heverlee, Belgium; and hRIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan Contributed by Marc C. E. Van Montagu, February 23, 2011 (sent for review May 19, 2010) The phytohormones jasmonates (JAs) constitute an important class linolenic acid, they are converted into the biologically active of elicitors for many plant secondary metabolic pathways. How- form (+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile), which is sub- ever, JAs do not act independently but operate in complex sequently bound to its receptor, the SCFCOI1 complex that con- networks with crosstalk to several other phytohormonal signaling tains the CORONATINE INSENSITIVE1 (COI1) F-box protein pathways. Here, crosstalk was detected between the JA and (12, 13). This hormone–receptor interaction causes degradation abscisic acid (ABA) signaling pathways in the regulation of tobacco of the JAZ repressor proteins by the 26S proteasome, which in (Nicotiana tabacum) alkaloid biosynthesis. A tobacco gene from the turn releases activator proteins such as MYC2, MYC3, and PYR/PYL/RCAR family, NtPYL4, the expression of which is regulated MYC4 in Arabidopsis to activate distinct JA response genes (14– by JAs, was found to encode a functional ABA receptor. NtPYL4 16). In the absence of bioactive JAs, the JAZ repressor proteins inhibited the type-2C protein phosphatases known to be key neg- bind to the MYC factors and recruit the NINJA-TOPLESS co- ative regulators of ABA signaling in an ABA-dependent manner. repressor complex to inhibit MYC activity (17). Overexpression of NtPYL4 in tobacco hairy roots caused a reprog- In Arabidopsis, the MYC, JAZ, and COI1 proteins compose the ramming of the cellular metabolism that resulted in a decreased core JA signaling machinery and have been shown to serve as alkaloid accumulation and conferred ABA sensitivity to the produc- a point of crosstalk with other signaling pathways. For instance, the tion of alkaloids. In contrast, the alkaloid biosynthetic pathway was DELLA proteins, the central repressors of the GA pathway, can not responsive to ABA in control tobacco roots. Functional analysis manipulate MYC2 activity through competitive binding to the of the Arabidopsis (Arabidopsis thaliana) homologs of NtPYL4, JAZ proteins, thereby modulating the JA signaling (18). The PYL4 PYL5 Arabidopsis PYL and , indicated that also in altered ex- central regulators of the auxin signaling pathway, the auxin/indole- pression affected the JA response, both in terms of biomass and 3-acetic acid (AUX/IAA) and auxin response transcription factors, fi fi anthocyanin production. These ndings de ne a connection be- have been shown to control the expression of the JAZ1 gene, en- tween a component of the core ABA signaling pathway and the abling molecular interplay between auxin and JA signaling (19). JA responses and contribute to the understanding of the role of JAs Furthermore, the auxin, the JA, and the ABA pathways all can in balancing tradeoffs between growth and defense. recruit the TOPLESS corepressor proteins through the interaction with specific adaptor proteins: the AUX/IAA, NINJA, and the nicotine | phenylpropanoid | primary metabolism | secondary metabolism | PLANT BIOLOGY AFP proteins, respectively (17). Crosstalk between the ABA and stress response JA signaling pathways can also occur through MYC2 because this transcription factor was originally described as an activator of lant hormones operate in a complex framework of interacting ABA signaling (20) before being found to play a critical role in JA Presponses rather than through isolated linear pathways. This signaling (21). Furthermore, besides the recruitment of MYC2 and hormonal “crosstalk” network can be modulated by a multitude TOPLESS, that of other signaling proteins, such as the MAP ki- of signals from developmental or environmental origins. The nase MPK6 (22, 23) and the AUXIN-RESISTANT 1, a subunit of integration of these signals defines the amplitude and the spec- the RUB1-activating enzyme that regulates the protein degrada- ificity of the responses in different cellular contexts (1–6). tion activity of Skp1–Cullin–F-box complexes (24), might possibly Plant responses to several (a)biotic stresses involve the jasm- enable crosstalk between the ABA and JA signaling pathways. onates (JAs) as one of the key hormonal components but also Similarly to the JAs, ABA plays an important role in several implicate the action of salicylic acid, ethylene, abscisic acid stress responses (2, 4, 25–27). ABA affects the biosynthesis of (ABA), auxin, and gibberellic acid (GA), the signaling pathways several osmocompatible solutes and secondary metabolites, such of which interact with that of the JAs to ensure a timely, spatially, and fitness-cost correct response (2, 3, 6, 7). JAs are particularly renowned for their prominent and universal role in the regulation Author contributions: M.C.E.V.M., H.M., K.-M.O.-C., H.R., and A.G. designed research; P.L., of plant metabolism, which is typically manifested as the elicita- M.G.-G., S.T., I.C., A.C.P., T.M., M.S., Y.K., S.T.H., and P.L.R. performed research; P.L., M.G.-G., S.T., P.L.R., H.R., and A.G. analyzed data; and P.L., M.G.-G., J.M.T., P.L.R., H.R., and A.G. tion of secondary metabolite biosynthesis when plants face par- wrote the paper. ticular environmental stresses (6, 8). For instance, in common The authors declare no conflict of interest. tobacco (Nicotiana tabacum), JAs, of which the synthesis is in- 1P.L. and M.G.-G. contributed equally to this work. duced in planta by herbivore attack (9), can stimulate the pro- 2To whom correspondence may be addressed. E-mail: [email protected] or alain. duction of pyridine alkaloids (10, 11). [email protected]. When JAs, such as jasmonic acid (JA) and methyl jasmonate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (MeJA), are applied to plants or produced endogenously from 1073/pnas.1103010108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1103010108 PNAS | April 5, 2011 | vol. 108 | no. 14 | 5891–5896 Downloaded by guest on September 23, 2021 as that of terpenoid indole alkaloids in Catharanthus roseus (28) A and anthocyanins in Arabidopsis (Arabidopsis thaliana) (29), both pathways that are also controlled by JAs in these plants. Antag- onistic as well as synergistic interactions can occur between ABA and JA: in Arabidopsis, for instance, endogenous ABA can have distinct regulatory effects on the JA-responsive insect and path- ogen response (7, 25, 30). Validated by the recent identification of the PYR/PYL/RCAR proteins, a model for ABA action has been established (27, 31–33). The PYR/PYL/RCAR proteins function as direct ABA receptors that recruit group A PP2C proteins after ABA binding and thereby inhibit their function. B 100 80 In the absence of ABA, these PP2C phosphatases are negative 60 40 regulators of ABA signaling, because they inhibit the SnRK2 20 ABA 0 - + - - + + - + - - + + - + - - + + kinases that, in turn, act as the global positive regulators of ABA- PYL4 - - + - + - - - + - + - - - + - + - induced gene expression by phosphorylating downstream tran- NtPYL4 - - - + - + - - - + - + - - - + - + scription factors and/or other regulatory proteins (27). Here we identified NtPYL4, a member of the PYR/PYL/ C RCAR protein family, from tobacco and show that it acts as an ABA receptor in a signaling network conserved between Arabi- dopsis and tobacco. Our results define a connection between the core ABA signaling pathway and the JA responses to monitor elicitor-induced reprogramming of plant metabolism and growth. Results NtPYL4 Is a Functional ABA Receptor and an Ortholog of the Arabidopsis PYL4 Protein. Previously, a gene collection had been Fig. 1. The NtPYL4 protein is a functional ABA receptor. (A) Cladogram established, based on a transcriptome analysis of MeJA-elicited obtained from the ClustalW multisequence alignment. Numbers in the x axis tobacco cells to identify proteins involved in alkaloid biosynthesis indicate the amino acid substitutions (×100). (B) Inhibitory effect of NtPYL4 (10). Several functional screens revealed an AP2/ERF transcrip- and PYL4 proteins on the phosphatase activity of Arabidopsis ABI1, HAB1, and ± tional activator of nicotine biosynthesis (34), a nicotine trans- PP2CA. Data are means SE from three independent experiments. Numbers in porter (35),