Background It has been known for centuries that cats respond euphorically to Nepeta cataria (catnip). Recently, we have shown that Lonicera tatarica (Tatarian honeysuckle), Actinidia polygama (silver vine), and Valeriana officinalis (valerian) can also elicit this "catnip response". The aim of this study was to learn if the behavior seen in response to these plants is similar to the response to catnip. Furthermore, we studied if these responses are fixed or if there are differences between cats. While nepetalactone was identified decades ago as the molecule responsible for the "catnip response", we know that this volatile is found almost exclusively in catnip. Therefore, we also aimed to identify other compounds in these alternative plants that can elicit the blissful behavior in cats. Bioassays with 6 cats were performed in a low-stress environment, where 5 plants and 13 single compounds were each tested for at least 100 and 17 h, respectively. All responses were video recorded and BORIS software was used to analyze the cats' behavior. Results Both response duration and behavior differed significantly between the cats. While individual cats had preferences for particular plants, the behavior of individual cats was consistent among all plants. About half a dozen lactones similar in structure to nepetalactone were able to elicit the "catnip response", as were the structurally more distinct molecules actinidine and dihydroactinidiolide. Most cats did not respond to actinidine, whereas those who did, responded longer to this volatile than any of the other secondary plant metabolites, and different behavior was observed. Interestingly, dihydroactinidiolide was also found in excretions and secretions of the red fox, making this the first report of a compound produced by a mammal that can elicit the "catnip response". A range of different cat-attracting compounds was detected by chemical analysis of plant materials but differences in cat behavior could not be directly related to differences in chemical composition of the plants. Together with results of, among others, habituation / dishabituation experiments, this indicates that additional cat-attracting compounds may be present in the plant materials that remain to be discovered. Conclusions Collectively, these findings suggest that both the personality of the cat and genetic variation in the genes encoding olfactory receptors may play a role in how cats respond to cat-attracting plants. Furthermore, the data suggest a potential distinct mechanism of action for actinidine.

It has been known for centuries that cats respond euphorically tocatnip. We showed that among others Tatarian honeysuckle and silver vine can also elicit this “catnip response”. The behavior seen during the responses was different between cats. While individual cats had preferences for particular plants, the behavior of individual cats was consistent among all plants. About half a dozen lactones similar in structure to nepetalactone were able to elicit the “catnip response”, as were the structurally more distinct molecules actinidine and dihydroactinidiolide. Most cats did not respond to actinidine, whereas those who did, responded longer to this volatile than any of the other secondary plant metabolites, and different behavior was observed. A range of different cat-attracting compounds was detected by chemical analysis of plant materials but differences in cat behavior could not be directly related to differences in chemical composition of the plants. Collectively, these findings suggest that both the personality of the cat and genetic variation in the genes encoding olfactory receptors may play a role in how cats respond to cat-attracting plants. Furthermore, the data suggest a potential distinct mechanism of action for actinidine.

Cross-kingdom mimicry of female insect sex pheromones by sexually deceptive orchids has evolved multiple times.1 Fungus gnats (Diptera) are predicted to be pollinators of hundreds of sexually deceptive orchids,2-4 yet unlike orchids that sexually attract bees and wasps (Hymenoptera),5-11 the chemistry of fungus gnat-pollinated orchids remains unknown. Furthermore, despite the importance of fungus gnats as pollinators, pests, and decomposers of organic material, and evidence for sex pheromones since 1971,12-17 no structure of any fungus gnat sex pheromone has to date been confirmed. In this study, we found a mixture of five hydrocarbons shared between Pterostylis orbiculata orchids and female Mycomya sp. (Mycetophilidae) fungus gnats, which included three alkanes, a C23 diene, and a C23 triene. The triene was an undescribed natural product, which we synthesized and confirmed to be (6Z,9Z)-1,6,9-tricosatriene. Field bioassays with a synthetic blend of the five hydrocarbons elicited attraction and sexual behavior from male gnats. The triene alone elicited attraction and low levels of sexual behavior, but the blend without it was unattractive, suggesting that this compound is a key component of orchid pollinator attraction and the female fungus gnat sex pheromone. In two closely related Pterostylis species, we found related C23 trienes, but not (6Z,9Z)-1,6,9-tricosatriene. These results suggest that unusual long-chain unsaturated hydrocarbons hold the key to sexual deception in Pterostylis orchids, and are an important step toward deciphering female fungus gnat sex pheromones.

Summary Strigolactones and karrikins are butenolide molecules that regulate plant growth. They are perceived by the α/β‐hydrolase DWARF14 (D14) and its homologue KARRIKIN INSENSITIVE2 (KAI2), respectively. Plant‐derived strigolactones have a butenolide ring with a methyl group that is essential for bioactivity. By contrast, karrikins are abiotic in origin, and the butenolide methyl group is nonessential. KAI2 is probably a receptor for an endogenous butenolide, but the identity of this compound remains unknown. Here we characterise the specificity of KAI2 towards differing butenolide ligands using genetic and biochemical approaches. We find that KAI2 proteins from multiple species are most sensitive to desmethyl butenolides that lack a methyl group. Desmethyl‐GR24 and desmethyl‐CN‐debranone are active by KAI2 but not D14. They are more potent KAI2 agonists compared with their methyl‐substituted reference compounds both in vitro and in plants. The preference of KAI2 for desmethyl butenolides is conserved in Selaginella moellendorffii and Marchantia polymorpha, suggesting that it is an ancient trait in land plant evolution. Our findings provide insight into the mechanistic basis for differential ligand perception by KAI2 and D14, and support the view that the endogenous substrates for KAI2 and D14 have distinct chemical structures and biosynthetic origins.

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii , a noxious weed of Mediterranean climates, is strongly stimulated by KAR 1 , the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR 2 . The α/β-hydrolase KARRIKIN INSENSITIVE 2 (KAI2) is the putative karrikin receptor identified in Arabidopsis . Here we show that B. tournefortii expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR 1 relative to KAR 2 when expressed in Arabidopsis . We identify two amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer differential responses to chemical cues produced by environmental disturbance, including fire.

Bioactive natural products underpin the intriguing pollination strategy used by sexually deceptive orchids. These compounds, which mimic the sex pheromones of the female insect, are emitted in particular blends to lure male insect pollinators of specific species. By combining methods from field biology, analytical chemistry, electrophysiology, crystallography, and organic synthesis, we report that an undescribed β-hydroxylactone, in combination with two specific hydroxymethylpyrazines, act as pollinator attractants in the rare hammer orchid Drakaea micrantha. This discovery represents an unusual case of chemically unrelated compounds being used together as a sexual attractant. Furthermore, this is the first example of the identification of pollinator attractants in an endangered orchid, enabling the use of chemistry in orchid conservation. Our synthetic blend is now available to be used in pollinator surveys to locate suitable sites for plant conservation translocations.

Synthetic auxin herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), are widely used for selective control of broadleaf weeds in cereals and transgenic crops. Although the troublesome weed wild radish ( Raphanus raphanistrum) has developed resistance to 2,4-D, no populations have yet displayed an enhanced capacity for metabolic detoxification of the herbicide, with both susceptible and resistant wild radish plants readily metabolizing 2,4-D. Using mass spectrometry and nuclear magnetic resonance, the major 2,4-D metabolite was identified as the glucose ester, and its structure was confirmed by synthesis. As expected, both the endogenous and synthetic compounds retained auxin activity in a bioassay. The lack of detectable 2,4-D hydroxylation in wild radish and the lability of the glucose ester suggest that metabolic 2,4-D resistance is unlikely to develop in this species.

Sexually deceptive orchids attract specific pollinators by mimicking insect sex pheromones. Normally this mimicry is very specific and identical compounds have been identified from orchids and matching females of the pollinators. In this study, we conduct a detailed structure-activity investigation on isomers of the semiochemicals involved in the sexual attraction of the male pollinator of the spider orchid Caladenia plicata. This orchid employs an unusual blend of two biosynthetically unrelated compounds, (S)-β-citronellol and 2-hydroxy-6-methylacetophenone, to lure its Zeleboria sp. thynnine wasp pollinator. We show that the blend is barely attractive when (S)-β-citronellol is substituted with its enantiomer, (R)-β-citronellol. Furthermore, none of the nine-possible alternative hydroxy-methylacetophenone regioisomers of the natural semiochemical are active when substituted for the natural 2-hydroxy-6-methylacetophenone. Our results were surprising given the structural similarity between the active compound and some of the analogues tested, and results from previous studies in other sexually deceptive orchid/wasp systems where substitution with analogues was possible. Interestingly, high-level ab initio and density functional theory calculations of the hydroxy-methylacetophenones revealed that the active natural isomer, 2-hydroxy-6-methylacetophenone, has the strongest intramolecular hydrogen bond of all regioisomers, which at least in part may explain the specific activity.

Karrikins are butenolide compounds present in post-fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE 2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2-dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)-mutagenized populations of A. thaliana for mutants with kai2-like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2-10 encoded a D184N protein variant that was stable in planta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2-active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.

Sexually deceptive orchids lure their specific male pollinators using volatile semiochemicals that mimic female sex pheromones. To date, the semiochemicals known to be involved consist of blends of chemically and biosynthetically related compounds. In contrast, we report that (S)-beta-citronellol and 2-hydroxy-6-methylacetophenone, two biosynthetically distinct compounds, are the active semiochemicals in Caladenia plicata, which is pollinated by male Zeleboria sp. thynnine wasps. They are also sex pheromone components of the female Zeleboria. A 1: 4 blend elicits a high rate of attempted copulation (similar to 70%) in bioassays, equivalent to rates observed at orchid flowers. Whereas beta-citronellol is well known, 2-hydroxy-6-methylacetophenone appears to be previously unknown as a floral volatile. Production of the two compounds is restricted to glandular sepal tips; thus, differential expression analysis of contrasting floral tissue transcriptomes was employed to illuminate the biosynthesis. As expected, production of (S)-beta-citronellol commences with the terpene synthase GES1 catalyzing the irreversible conversion of geranyl diphosphate (GPP) to geraniol. Contrary to prediction, biosynthesis subsequently proceeds in three steps, commencing with the oxidation of geraniol to geranial by alcohol dehydrogenase ADH3, followed by the enantioselective reduction of a double bond in geranial by geranial reductase GER1 to give (S)-beta-citronellal. Finally, ADH3-catalyzed reduction of (S)-beta-citronellal results in (S)-beta-citronellol. In line with previous work on insects showing that 2-hydroxy-6-methylacetophenone is derived from a polyketide pathway, we report a differentially expressed polyketide synthase (PKS) gene candidate. Thus, in this unique example of sexual deception, pollination is achieved by co-opting and regulating two independent biosynthetic pathways of floral volatile compounds.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants.

Plants produce strigolactones with different structures and different stereospecificities which provides the potential for diversity and flexibility of function.

Acalypha indica is a herb that grows throughout the tropical regions of the world. As well as being exploited for medicinal use, the roots of this plant are known to elicit a drug-like effect in cats. Recent research into feral cat control on Christmas Island has investigated whether a preparation of the roots of A. indica might be effective in traps to attract feral cats. However, the volatile nature of the attractants made it unviable for use in traps for more than a few days. In this study, we investigated the volatile components emitted by the plant roots and identified two iridoid compounds, (4R,4aR,7S,7aR)-isodihydronepetalactone and (4R,4aS,7S,7aR)-isoiridomyrmecin, which are known to affect behavioural activity in cats. Synthesis of standards confirmed the stereochemistry of both compounds emitted by the plant. Potential application of these compounds in feral cat control is discussed.

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCF(D3) that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process.

In Arabidopsis thaliana, the α/β-fold hydrolase KARRIKIN INSENSITIVE2 (KAI2) is essential for normal seed germination, seedling development, and leaf morphogenesis, as well as for responses to karrikins. KAI2 is a paralog of DWARF14 (D14), the proposed strigolactone receptor, but the evolutionary timing of functional divergence between the KAI2 and D14 clades has not been established. By swapping gene promoters, we show that Arabidopsis KAI2 and D14 proteins are functionally distinct. We show that the catalytic serine of KAI2 is essential for function in plants and for biochemical activity in vitro. We identified two KAI2 homologs from Selaginella moellendorffii and two from Marchantia polymorpha. One from each species could hydrolyze the strigolactone analog GR24 in vitro, but when tested for their ability to complement Arabidopsis d14 and kai2 mutants, neither of these homologs was effective. However, the second KAI2 homolog from S. moellendorffii was able to complement the seedling and leaf development phenotypes of Arabidopsis kai2. This homolog could not transduce signals from exogenous karrikins, strigolactone analogs, or carlactone, but its activity did depend on the conserved catalytic serine. We conclude that KAI2, and most likely the endogenous signal to which it responds, has been conserved since the divergence of lycophytes and angiosperm lineages, despite their major developmental and morphogenic differences.

Derived from burnt vegetation, karrikins (KAR) are butenolide chemicals that stimulate seed germination and enhance seedling responses to light. Genetic and biochemical studies have identified KARRIKIN INSENSITIVE2 (KAI2) as a putative karrikin receptor protein (reviewed by Waters et al., 2014Waters M.T. Scaffidi A. Sun Y.K. Flematti G.R. Smith S.M. The karrikin response system of Arabidopsis.Plant J. 2014; 79: 623-631Crossref PubMed Scopus (91) Google Scholar). KAI2 is an α/β-fold hydrolase and a paralog of DWARF14 (D14; AtD14 in Arabidopsis), and both possess the conserved catalytic triad of Ser, His, and Asp residues typical of this class of hydrolytic enzymes. D14 proteins are likely receptors for strigolactones (SL), a group of butenolide plant hormones involved in the regulation of shoot architecture, and an intact catalytic triad is essential for D14-mediated SL responses (Hamiaux et al., 2012Hamiaux C. Drummond R.S. Janssen B.J. Ledger S.E. Cooney J.M. Newcomb R.D. Snowden K.C. DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone.Curr. Biol. 2012; 22: 2032-2036Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). It has been proposed that the catalytic serine of KAI2 and AtD14 initiates nucleophilic attack on the butenolide moiety of KAR and SLs, respectively (Scaffidi et al., 2012Scaffidi A. Waters M.T. Bond C.S. Dixon K.W. Smith S.M. Ghisalberti E.L. Flematti G.R. Exploring the molecular mechanism of karrikins and strigolactones.Bioorg. Med. Chem. Lett. 2012; 22: 3743-3746Crossref PubMed Scopus (66) Google Scholar, Zhao et al., 2013Zhao L.-H. Zhou X.E. Wu Z.S. Yi W. Xu Y. Li S. Xu T.H. Liu Y. Chen R.Z. Kovach A. et al.Crystal structures of two phytohormone signal-transducing α/β hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14.Cell Res. 2013; 23: 436-439Crossref PubMed Scopus (181) Google Scholar), and that these proteins have both enzyme-like and receptor-like qualities (Waters et al., 2014Waters M.T. Scaffidi A. Sun Y.K. Flematti G.R. Smith S.M. The karrikin response system of Arabidopsis.Plant J. 2014; 79: 623-631Crossref PubMed Scopus (91) Google Scholar). Recently, it was reported that AtD14 was degraded as a result of the SL perception process (Chevalier et al., 2014Chevalier F. Nieminen K. Sánchez-Ferrero J.C. Rodríguez M.L. Chagoyen M. Hardtke C.S. Cubas P. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis.Plant Cell. 2014; 26: 1134-1150Crossref PubMed Scopus (154) Google Scholar). Crucially, this event was dependent on the F-box protein MORE AXILLARY GROWTH2 (MAX2), which is required for seedling responses to both SL and KAR in Arabidopsis (Nelson et al., 2011Nelson D.C. Scaffidi A. Dun E.A. Waters M.T. Flematti G.R. Dixon K.W. Beveridge C.A. Ghisalberti E.L. Smith S.M. F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA. 2011; 108: 8897-8902Crossref PubMed Scopus (313) Google Scholar). Since the proteasome inhibitor MG132 also blocked degradation of AtD14, it was concluded that AtD14 was likely ubiquitinated in a MAX2-dependent process and thus targeted for degradation by the ubiquitin–proteasome system (Chevalier et al., 2014Chevalier F. Nieminen K. Sánchez-Ferrero J.C. Rodríguez M.L. Chagoyen M. Hardtke C.S. Cubas P. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis.Plant Cell. 2014; 26: 1134-1150Crossref PubMed Scopus (154) Google Scholar). However, it was not clear if this degradation event was a requirement for or a consequence of SL perception. In addition, it is not known if KAI2 undergoes a similar degradation process as part of karrikin signaling. We investigated KAI2 protein levels in Arabidopsis seedlings exposed to KAI2 substrates. First, we assessed the relative effect of KAR1 and KAR2 upon KAI2 levels in Landsberg erecta (Ler) seedlings grown in liquid culture. We found that both karrikin analogs could trigger the destabilization of KAI2, but surprisingly KAR2 was at least 100-fold more potent than KAR1 (Figure 1A). Notably, this difference is more accentuated than that observed in morphological responses: KAR1 is about 10-fold less potent than KAR2 in terms of inhibiting hypocotyl elongation (Waters et al., 2012Waters M.T. Nelson D.C. Scaffidi A. Flematti G.R. Sun Y.K. Dixon K.W. Smith S.M. Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis.Development. 2012; 139: 1285-1295Crossref PubMed Scopus (348) Google Scholar), suggesting that molecular activity and the degree of developmental outputs are not linearly related. Substantially reduced levels of KAI2 could be detected within 2 h of continuous exposure to 5 μM KAR2, reaching a plateau within 4–8 h (Figure 1B), potentially reflecting a steady-state level in which the rates of protein degradation and biosynthesis are similar. Besides KAR, KAI2 can also mediate seed and seedling responses to the synthetic SL analog GR24 (Waters et al., 2012Waters M.T. Nelson D.C. Scaffidi A. Flematti G.R. Sun Y.K. Dixon K.W. Smith S.M. Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis.Development. 2012; 139: 1285-1295Crossref PubMed Scopus (348) Google Scholar). GR24 as used routinely is a racemic mixture (rac-GR24) of two enantiomers, which have distinct biological activity in Arabidopsis (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar). To examine whether other butenolides that signal via KAI2 might also affect KAI2 stability, we tested these two enantiomers of GR24. We found that, while less potent than KAR2, GR24ent-5DS stimulated a reduction in KAI2 levels, but GR245DS did not (Figure 1C). This differential effect of the two enantiomers is consistent with previously observed KAI2-dependent responses in Arabidopsis in which only GR24ent-5DS is effective (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar). We considered that apparent KAR2-dependent reduction in KAI2 protein levels might reflect reduced transcription, or a change in the epitope recognized by the KAI2 antibody. However, a GFP–KAI2 fusion protein expressed from the constitutive 35S promoter and detected with an anti-GFP antibody showed similar KAR2-dependent destabilization, suggesting that changes in KAI2 levels do not result from a change in KAI2 transcripts or from obfuscation of the native KAI2 epitope (Figure 1D). Thus KAR2, and to a lesser extent KAR1 and GR24ent-5DS, trigger a reduction in KAI2 protein levels by a post-translational mechanism. To establish whether MAX2 is necessary for the response of KAI2 to KAR2, we examined KAI2 responses in three different max2 mutants. Surprisingly, patterns of KAI2 destabilization were indistinguishable between all max2 alleles and their respective wild-types, although the loss of KAI2 was much more pronounced in Ler background than in Col-0 (Figure 1E). Next, to test whether the loss of KAI2 requires a functional catalytic serine, we mutated this to alanine (S95A). We expressed both wild-type and mutant proteins under the control of the native KAI2 promoter in the null kai2-2 background. Whereas KAI2 levels responded in the wild-type (KAI2:KAI2) control similarly to Ler, KAI2 S95A levels showed little to no reduction over the same time period in two independent transgenic lines (Figure 1F). This result suggests that substrate-induced KAI2 instability is a consequence of ligand attack mediated by the catalytic serine. When purified KAI2 and KAI2 S95A proteins were incubated with KAR2 in vitro, no change in protein abundance or size was observed (Supplemental Figure 1A). In addition, KAI2 levels did not change substantially in response to KAR2 in cell-free lysates extracted from Ler seedlings (Supplemental Figure 1B). Together, these findings imply that KAR2 does not affect KAI2 stability directly but that KAI2 breakdown requires cellular integrity. To establish whether KAR2 triggers degradation of KAI2 via the 26S proteasome, we tested the effect of the proteasomal inhibitor MG132. Surprisingly, MG132 could not prevent the loss of KAI2 in the presence of KAR2, but did lead to an increase in abundance of poly-ubiquitinated proteins, suggesting that proteasome activity was indeed blocked (Figure 1G). Furthermore, we could find no evidence that KAI2 protein is ubiquitinated in planta (Figure 1H). We also tested the broad-spectrum serine protease inhibitor AEBSF and the cysteine protease inhibitors ALLN and E-64d. None of these compounds could prevent KAI2 destabilization by KAR2 (Figure 1I and 1J; Supplemental Figure 1C). Overall, these data imply that KAI2-dependent butenolide signaling induces the degradation or loss of KAI2, via a mechanism that follows enzymatic attack upon the substrate. This mechanism does not depend on MAX2 or the 26S proteasome, but does require other unidentified cellular components. Changes in tertiary structure of DAD2/D14 following interaction with GR24 in vitro have been inferred based on a shift in protein melting temperature, and the corresponding serine mutant does not show such a response (Hamiaux et al., 2012Hamiaux C. Drummond R.S. Janssen B.J. Ledger S.E. Cooney J.M. Newcomb R.D. Snowden K.C. DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone.Curr. Biol. 2012; 22: 2032-2036Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Assuming these changes are also true for KAI2 in planta, our results are consistent with a model in which serine-mediated hydrolytic attack upon the ligand triggers a conformational change in protein structure, which in turn leads to protein destabilization. Recent crystallographic data indicating that the pyran ring of KAR1 interacts with phenylalanine residues lining the KAI2 active-site pocket, distal from the catalytic triad itself (Guo et al., 2013Guo Y. Zheng Z. La Clair J.J. Chory J. Noel J.P. Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis.Proc. Natl. Acad. Sci. USA. 2013; 110: 8284-8289Crossref PubMed Scopus (121) Google Scholar), are at odds with the functional requirement for the catalytic serine. It is formally possible that the S95A mutation prevents the reported conformational changes in KAI2 induced by KAR1 binding, without KAR1 actually interacting with the catalytic triad. However, we do not favor this explanation for two reasons. First, crystallography of rice D14 has shown the hydrolyzed butenolide moiety (D ring) from rac-GR24 covalently attached to the active-site serine (Zhao et al., 2013Zhao L.-H. Zhou X.E. Wu Z.S. Yi W. Xu Y. Li S. Xu T.H. Liu Y. Chen R.Z. Kovach A. et al.Crystal structures of two phytohormone signal-transducing α/β hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14.Cell Res. 2013; 23: 436-439Crossref PubMed Scopus (181) Google Scholar), making it more likely that a similar mechanism holds for the action of KAI2 on GR24ent-5DS and karrikins. Second, karrikin analogs with a saturated, non-aromatic pyran ring are still biologically active, albeit at reduced levels (Scaffidi et al., 2012Scaffidi A. Waters M.T. Bond C.S. Dixon K.W. Smith S.M. Ghisalberti E.L. Flematti G.R. Exploring the molecular mechanism of karrikins and strigolactones.Bioorg. Med. Chem. Lett. 2012; 22: 3743-3746Crossref PubMed Scopus (66) Google Scholar). Hence, pi-pi stacking of these saturated karrikin analogs with the phenylalanine residues is unlikely to occur. Instead, to account for the observed ligand–receptor structure (Guo et al., 2013Guo Y. Zheng Z. La Clair J.J. Chory J. Noel J.P. Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis.Proc. Natl. Acad. Sci. USA. 2013; 110: 8284-8289Crossref PubMed Scopus (121) Google Scholar), it is plausible that the karrikin ligand may be in a position that precedes subsequent relocation allowing catalysis, or has moved within the KAI2 pocket after interaction with the catalytic triad. Under the conditions of protein crystallization, KAR1 may remain in this position. The KAI2 degradation mechanism is evidently different from that proposed for rac-GR24-induced destabilization of AtD14 (Chevalier et al., 2014Chevalier F. Nieminen K. Sánchez-Ferrero J.C. Rodríguez M.L. Chagoyen M. Hardtke C.S. Cubas P. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis.Plant Cell. 2014; 26: 1134-1150Crossref PubMed Scopus (154) Google Scholar). KAI2 degradation is independent of MAX2, is not proteasome mediated, and we could not detect ubiquitinated KAI2; pertinently, these three observations are mutually supportive because MAX2 is part of a complex that poly-ubiquitinates substrates such as D53 for proteasomal degradation (Jiang et al., 2013Jiang L. Liu X. Xiong G. Liu H. Chen F. Wang L. Meng X. Liu G. Yu H. Yuan Y. et al.DWARF 53 acts as a repressor of strigolactone signalling in rice.Nature. 2013; 504: 401-405Crossref PubMed Scopus (506) Google Scholar, Zhou et al., 2013Zhou F. Lin Q. Zhu L. Ren Y. Zhou K. Shabek N. Wu F. Mao H. Dong W. Gan L. et al.D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling.Nature. 2013; 504: 406-410Crossref PubMed Scopus (528) Google Scholar). The mechanism for removal of KAI2 protein after signaling is therefore currently unclear. One possibility is lysosomal or vacuolar degradation, a process that would require cellular compartmentalization. Nevertheless, the process is highly specific, being triggered only by ligands that operate through KAI2. Complete absence of the protein (i.e. in kai2-2 mutants) imparts a phenotype that is opposite to that of karrikin signaling. Therefore, loss of KAI2 is likely to be a consequence rather than a cause of the signaling process. Furthermore, that an enzyme be destroyed as a result of its activity is unusual, suggesting that KAI2 does not simply modify a substrate into a further bioactive compound. Rather, KAI2 degradation may be an integral element of its function, allowing removal of the activated or "used" receptor in its post-signaling state. Screening KAI2 mutant proteins for stabilized but constitutively active variants might be instructive in establishing the biological significance of, and mechanistic basis for, KAI2 turnover. No conflict of interest declared. Download .pdf (1.29 MB) Help with pdf files Document S1. Supplemental Figure 1, Supplemental Methods, and Supplemental References

Arabidopsis thaliana provides a powerful means to investigate the mode of action of karrikins, compounds produced during wildfires that stimulate germination of seeds of fire-following taxa. These studies have revealed close parallels between karrikin signalling and strigolactone signalling. The two perception systems employ similar mechanisms involving closely related α/β-fold hydrolases (KAI2 and AtD14) and a common F-box protein (MAX2). However, karrikins and strigolactones may be distinguished from each other and elicit different responses. The karrikin response requires a newly discovered protein (SMAX1), a homologue of rice protein D53 that is required for the strigolactone response. Mutants defective in the response to karrikins have seeds with increased dormancy, altered seedling photomorphogenesis and modified leaf shape. As the karrikin and strigolactone response mechanisms are so similar, it is speculated that the endogenous signalling compound for the KAI2 system may be a specific strigolactone. However, new results show that the proposed endogenous signalling compound is not produced by the known strigolactone biosynthesis pathway via carlactone. Structural studies of KAI2 protein and its interaction with karrikins and strigolactone analogues provide some insight into possible protein-ligand interactions, but are hampered by lack of knowledge of the endogenous ligand. The KAI2 system appears to be present throughout angiosperms, implying a fundamentally important function in plant biology.

Biosynthesis of the strigolactones—important plant hormones—has been solved up to carlactone. Biochemical and genetic evidence now demonstrate that homologous enzymes perform two subsequent oxidations, setting the strigolactone scaffold in place.

Two α/β-fold hydrolases, KARRIKIN INSENSITIVE2 (KAI2) and Arabidopsis thaliana DWARF14 (AtD14), are necessary for responses to karrikins (KARs) and strigolactones (SLs) in Arabidopsis (Arabidopsis thaliana). Although KAI2 mediates responses to KARs and some SL analogs, AtD14 mediates SL but not KAR responses. To further determine the specificity of these proteins, we assessed the ability of naturally occurring deoxystrigolactones to inhibit Arabidopsis hypocotyl elongation, regulate seedling gene expression, suppress outgrowth of secondary inflorescences, and promote seed germination. Neither 5-deoxystrigol nor 4-deoxyorobanchol was active in KAI2-dependent seed germination or hypocotyl elongation, but both were active in AtD14-dependent hypocotyl elongation and secondary shoot growth. However, the nonnatural enantiomer of 5-deoxystrigol was active through KAI2 in growth and gene expression assays. We found that the four stereoisomers of the SL analog GR24 had similar activities to their deoxystrigolactone counterparts. The results suggest that AtD14 and KAI2 exhibit selectivity to the butenolide D ring in the 2'R and 2'S configurations, respectively. However, we found, for nitrile-debranone (CN-debranone, a simple SL analog), that the 2'R configuration is inactive but that the 2'S configuration is active through both AtD14 and KAI2. Our results support the conclusion that KAI2-dependent signaling does not respond to canonical SLs. Furthermore, racemic mixtures of chemically synthesized SLs and their analogs, such as GR24, should be used with caution because they can activate responses that are not specific to naturally occurring SLs. In contrast, the use of specific stereoisomers might provide valuable information about the specific perception systems operating in different plant tissues, parasitic weed seeds, and arbuscular mycorrhizae.