The Tryptophan Conjugates of Jasmonic and Indole-3-Acetic Acids Are Endogenous Auxin Inhibitors1,[W],[OA]

摘要

Most conjugates of plant hormones are inactive, and some function to reduce the active hormone pool. This study characterized the activity of the tryptophan (Trp) conjugate of jasmonic acid (JA-Trp) in Arabidopsis (Arabidopsis thaliana). Unexpectedly, JA-Trp caused agravitropic root growth in seedlings, unlike JA or nine other JA-amino acid conjugates. The response was dose dependent from 1 to100 µM, was independent of the COI1 jasmonate signaling locus, and unlike the jasmonate signal JA-isoleucine, JA-Trp minimally inhibited root growth. The Trp conjugate with indole-3-acetic acid (IAA-Trp) produced a similar response, while Trp alone and conjugates with benzoic and cinnamic acids did not. JA-Trp and IAA-Trp at 25 µM nearly eliminated seedling root inhibition caused by 2 µM IAA. The TIR1 auxin receptor is required for activity because roots of tir1-1 grew only approximately 60% of wild-type length on IAA plus JA-Trp, even though tir1-1 is auxin resistant. However, neither JA-Trp nor IAA-Trp interfered with IAA-dependent interaction between TIR1 and Aux/IAA7 in cell-free assays. Trp conjugates inhibited IAA-stimulated lateral root production and DR5-β-glucuronidase gene expression. JA-deficient mutants were hypersensitive to IAA and a Trp-overaccumulating mutant was less sensitive, suggesting endogenous conjugates affect auxin sensitivity. Conjugates were present at 5.8 pmol g–1 fresh weight or less in roots, seedlings, leaves, and flowers, and the values increased approximately 10-fold in roots incubated in 25 µM Trp and IAA or JA at 2 µM. These results show that JA-Trp and IAA-Trp constitute a previously unrecognized mechanism to regulate auxin action. nnnn--------------------------------------------------------------------------------nThroughout their life, plants use a variety of hormonal signals to adjust growth in response to developmental and external cues. Central among the growth regulating hormones is the auxin indole-3-acetic acid (IAA), which is involved in nearly all aspects of plant development (for review, see Woodward and Bartel, 2005). Optimal growth requires tight control of IAA activity, which is accomplished by diverse mechanisms that include regulating IAA biosynthesis, its transport among tissues, cycling between active and inactive forms of auxin, and hormone degradation through various oxidative pathways (see Ljung et al., 2002; Leyser, 2006; Scheres and Xu, 2006). Regulatory control is also accomplished at the level of IAA interaction with auxin receptors and at numerous steps downstream of this signaling interaction (Mockaitis and Estelle, 2008). nWhile jasmonates are best known for their role in defense against herbivores and certain pathogens, they also control growth (for recent overviews, see Wasternack, 2007; Howe and Jander, 2008; Browse, 2009). Exogenous jasmonic acid (JA) inhibits growth (Yamane et al., 1980; Dathe et al., 1981; Ueda and Kato, 1982; Staswick et al., 1992), and both JA biosynthesis and jasmonate signaling mutants display growth abnormalities, particularly in reproductive organs and in vegetative tissues during defense responses (Mandaokar et al., 2006; Zavala and Baldwin, 2006; Yan et al., 2007; Zhang and Turner, 2008). It makes sense that jasmonate defense signaling would be tied to growth regulation because under stress plants must adjust their development and reallocate resources toward defense (Herms and Mattson, 1992). Although the full mechanism of jasmonate-mediated growth regulation is unclear, it may involve cell cycle transitions, cell division, and IAA-induced cell elongation (Miyamoto et al., 1997; Swiatek et al., 2002, 2004; Zhang and Turner, 2008). nnPlants synthesize conjugated forms of both JA and IAA. Indeed, most IAA in Arabidopsis (Arabidopsis thaliana) is bound through ester or amide linkages to various constituents, including sugars, amino acids, and peptides (Ljung et al., 2002). Conjugates are generally considered inactive metabolites of the hormone. For example, IAA-Asp and IAA-Glu are catabolized, while IAA-Ala and IAA-Leu are stored forms of IAA that can be accessed at a later time by hydrolysis of the amide linkage (Rampey et al., 2004). Early physiological studies suggested that some JA conjugates might have biological activity, but like the IAA conjugates, most were considered inactive or of minor importance compared with JA and its methyl ester (JA-Me; Kramell et al., 1997; Miersch et al., 1999). That perspective changed with the discovery that the Ile conjugate of JA (JA-Ile) is a key jasmonate signal (Staswick and Tiryaki, 2004). nnJA-Ile accumulation is strongly but transiently induced in plant defense responses in both Arabidopsis and tobacco (Nicotiana tabacum), and this accumulation is tied to productive defense reactions (Kang et al., 2006; Wang et al., 2007; Suza and Staswick, 2008). Furthermore, JA-Ile promotes interaction between the COI1 F-box protein, the presumed jasmonate receptor, and its ubiquitination targets, the JAZ transcriptional repressors (Chini et al., 2007; Thines et al., 2007; Katsir et al., 2008a; Staswick, 2008). In contrast, JA and JA-Me have essentially no activity in this interaction. Instead, they are precursors that become effective signals when added exogenously to plants because they are converted to JA-Ile by the JAR1 conjugating enzyme (Staswick and Tiryaki, 2004; Tamogami et al., 2008). nnJA-Ile signaling closely parallels the mechanism previously established for auxin, although in the latter case, free rather than conjugated IAA is the active signal. IAA binds in a pocket of the TIR1 auxin receptor, which is an F-box component of the E3 ubiquitin ligase complex called SCFTIR1. Auxin binding strengthens the interaction between TIR1 and the Aux/IAA protein targets for ubiquitination (Dharmasiri et al., 2005a; Kepinski and Leyser, 2005; Tan et al., 2007). Degradation of the Aux/IAA transcriptional repressors by the 26 s proteasome then leads to auxin-dependent gene transcription and auxin response. Five TIR1-related proteins, AFB1 through AFB5, have been identified, and they have partially overlapping functions with TIR1 (Dharmasiri et al., 2005b; Mockaitis and Estelle, 2008). In contrast, COI1 is the primary, if not the sole, F-box protein in jasmonate signaling. nnCritical tools that have shaped our understanding of auxin activity are auxin response inhibitors of two general classes: those that alter auxin transport and those that perturb auxin signaling. Although a few endogenous auxin inhibitors have been reported, little is known about their in vivo roles. Most studies have employed synthetic inhibitors, such as 1-naphthylphthalamic acid (NPA), p-chlorophenoxyisobutyric acid (PCIB), 4,4,4-trifluoro-3-indole-3-butyric acid, tri-iodobenzoic acid, and 1-naphthoxyacetic acid (MacRae and Bonner, 1953; Jönsson, 1961; Katayama et al., 1995; Tomic et al., 1998; Parry et al., 2001; Rahman et al., 2002). The TIR1 auxin receptor was discovered as a mutant (tir1) resistant to the NPA transport inhibitor, and new antagonists and agonists have been identified in recent chemical genetic screens (Ruegger et al., 1997; Armstrong et al., 2004; Surpin et al., 2005; Yamazoe et al., 2005; Hayashi et al., 2008). nnWhile synthetic auxin inhibitors have been immensely informative, there are potential drawbacks to using these unnatural compounds. They may work in ways that differ from endogenous inhibitors, and potential xenobiotic responses to the foreign compounds can complicate analysis of their mechanism of action (Armstrong et al., 2004). Naturally occurring auxin inhibitors, on the other hand, may refine our understanding of how auxin activity is controlled in planta, and they may illuminate new components of the auxin regulatory pathway. This study was initiated to evaluate possible jasmonate activity for a series of JA-amino acid conjugates. Surprisingly, the Trp conjugates of both JA and IAA are naturally occurring auxin antagonists that interfere with a broad range of IAA-mediated processes. Trp conjugates require TIR1 for full activity, and determining how they work may shed new light on the control of auxin signaling in plants.