F-box protein MAX2 has dual roles in karrikin and signaling in Arabidopsis thaliana

David C. Nelsona, Adrian Scaffidib, Elizabeth A. Dunc, Mark T. Watersa, Gavin R. Flemattib, Kingsley W. Dixond,e, Christine A. Beveridgec, Emilio L. Ghisalbertib, and Steven M. Smitha,b,1

aAustralian Research Council Centre of Excellence in Plant Energy Biology, bSchool of Biomedical, Biomolecular and Chemical Sciences, and dSchool of Plant Biology, University of Western Australia, Crawley WA 6009, Australia; and cSchool of Biological Sciences, University of Queensland, St. Lucia QLD 4072, Australia; and eKings Park and Botanic Garden, West Perth WA 6005, Australia

Edited* by Peter H. Quail, University of California at Berkeley, Albany, CA, and approved April 19, 2011 (received for review January 18, 2011) Smoke is an important abiotic cue for plant regeneration in Karrikins share partial structural similarity with postfire landscapes. Karrikins are a class of compounds discovered (Fig. 1A), a family of phytohormones present in root exudates that in smoke that promote seed germination and influence early de- stimulate germination of parasitic weeds (Orobanche and Striga velopment of many plants by an unknown mechanism. A genetic spp.) (12, 13) and enhance hyphal branching in arbuscular mycor- screen for karrikin-insensitive mutants in Arabidopsis thaliana rhizal fungi (14). Additionally, strigolactones or a strigolactone- revealed that karrikin signaling requires the F-box protein MAX2, derived signal repress shoot branching. For the remainder of this which also mediates responses to the structurally-related strigolac- article, we will simply refer to the active signal as strigolactone. As tone family of phytohormones. Karrikins and the synthetic strigo- demonstrated by an array of mutants in pea, rice, petunia, and lactone GR24 trigger similar effects on seed germination, seedling A. thaliana, strigolactone deficiency or insensitivity gives rise to photomorphogenesis, and expression of a small set of genes dur- increased shoot branching (reviewed in refs. 15, 16). In Arabi- ing these developmental stages. Karrikins also repress MAX4 and dopsis, several more axillary growth (max) mutants have been IAA1 transcripts, which show negative feedback regulation by stri- characterized. Two carotenoid cleavage dioxygenases, MAX3/ golactone. We demonstrate that all of these common responses CCD7 and MAX4/CCD8, and a cytochrome P450 enzyme, are abolished in max2 mutants. Unlike strigolactones, however, MAX1, are implicated in the formation of strigolactone (17–23). karrikins do not inhibit shoot branching in Arabidopsis or pea, in- Multiple strigolactones are produced in Arabidopsis, including dicating that plants can distinguish between these signals. These orobanchol and orobanchyl acetate (24, 25). The ccd7 and ccd8 results suggest that a MAX2-dependent signal transduction mech- mutants are strigolactone-deficient, and application of the syn- anism was adapted to mediate responses to two chemical cues thetic strigolactone GR24 restores inhibition of axillary shoot with distinct roles in plant ecology and development. outgrowth to these mutants in rice, pea, and Arabidopsis (18, 19). Reciprocal grafting experiments demonstrated that the F-box eed germination is a crucial developmental transition in the protein MAX2 acts downstream of MAX1/MAX3/MAX4 (26). Splant life cycle. As such it is highly regulated in many plant Consistent with this result, shoot branching in max2 is insensitive species by physiological seed dormancy, which prevents germi- to GR24 (18, 19). Recently, max1 and max4 mutants in Arabi- nation except under specific environmental conditions (1). Smoke dopsis, but not max2, have been shown to have significantly re- elicits a significant ecological impact on postfire environments, as duced levels of orobanchol (25), providing further evidence that it is known to stimulate germination of over a thousand plant the increased branching of max2 is not caused by a defect in species (2, 3). A key bioactive signal in smoke was discovered to strigolactone production. be the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one (4, 5), There are distinct as well as common responses to these two plant-growth regulators. As is the case with karrikins, GR24 has which is now known as karrikinolide or KAR1 (3). Five additional analogous butenolide compounds have since been identified in been shown to stimulate seed germination and inhibit hypocotyl smoke and comprise a unique family known as karrikins (6). elongation in Arabidopsis (10, 11, 27). However, GR24 is typically Karrikins trigger germination and promote seedling vigor for required at much higher concentrations than karrikins to promote a broad range of plant species at concentrations as low as 1 nM seed germination of Arabidopsis and Brassica tournefortii, and (7–9). KAR1 is unable to stimulate germination of at least four GR24- Karrikins can strongly enhance germination of primary dor- responsive parasitic weed species (7, 10). KAR1 is also inactive in mant Arabidopsis thaliana seed, although this response depends assays for induction of hyphal branching in arbuscular mycor- upon the depth of seed dormancy and requires both light and rhizal fungi, although strigolactones are extremely effective (14, synthesis of the phytohormone (10). KAR and KAR 28). Karrikin molecules have a butenolide moiety in common with 1 2 the D-ring of strigolactones (Fig. 1A, highlighted in red). The (Fig. 1A) were found to be the most active karrikins in Arabidopsis fi (10). A microarray-based investigation of molecular responses to butenolide D-ring of strigolactones is necessary, but not suf - cient, to stimulate germination of parasitic weeds and hyphal KAR1 preceding germination revealed a predominance of light- regulated transcripts among karrikin-responsive genes (11). We branching of arbuscular mycorrhizal fungi (28, 29). As several also discovered that germination of nondormant Arabidopsis seed variants of this butenolide ring were also ineffective as Arabi- dopsis seed-germination stimulants (Fig. S1), the similar respon- is triggered at lower fluences of red light when treated with KAR1. Cotyledon expansion and inhibition of hypocotyl elongation are enhanced in the presence of germination-active karrikins in Author contributions: D.C.N., K.W.D., C.A.B., E.L.G., and S.M.S. designed research; D.C.N., a light-dependent manner, providing further evidence of karrikin A.S., E.A.D., and M.T.W. performed research; A.S. and G.R.F. contributed new reagents/ crosstalk with light signaling pathways. Indeed, the transcription analytic tools; D.C.N., A.S., E.A.D., and M.T.W. analyzed data; and D.C.N. and S.M.S. wrote factor HY5, which is a key player in light signal transduction, is the paper. up-regulated by karrikins and has a role in postgerminative kar- The authors declare no conflict of interest. rikin responses. We found, however, that HY5 is not required for *This Direct Submission article had a prearranged editor. the induction of early KAR-responsive transcripts in seeds (11). 1To whom Correspondence should be addressed. E-mail: [email protected].

Therefore, central components of the karrikin response mecha- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PLANT BIOLOGY nism remain unknown. 1073/pnas.1100987108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1100987108 PNAS | May 24, 2011 | vol. 108 | no. 21 | 8897–8902 Downloaded by guest on October 1, 2021 O O Two kai mutants, kai1-1 and kai1-2, shared multiple pheno- A O O O types, including increased axillary shoot branching, reduced in- florescence height, delayed senescence, leaf curling, and elongated KAR1 O O B–D kai1 GR24 hypocotyls (Fig. 1 ). The seed had enhanced primary O O O O dormancy, which could be partially overcome by 10 mM KNO3 treatment, but was completely unresponsive to promotion of KAR 2 germination by KAR1,KAR2, or GR24 (Fig. 1E). We noted that kai1-1 and kai1-2 exhibited several phenotypes B C in common with the Arabidopsis mutant more axillary growth 2 kai1-1 (max2), which has also been identified in genetic screens as oresara9 (ore9) (30), a delayed senescence mutant, and pleiotro- pic photosignaling (pps) (31), a light hyposensitive mutant. Se- quencing of the MAX2 gene in kai1-1 and kai1-2 mutants revealed two unique alleles: a 4-bp net insertion (now called max2-7) and a 4-bp deletion (now called max2-8), resulting in D frame-shifts of the 693 aa-encoding MAX2 ORF after 248 and 66 codons, respectively (Fig. 2A).

Germination and Photomorphogenesis Responses to Karrikins and Ler kai1-1 Ler kai1-1 kai1-2 GR24 Are Abolished in max2. To further test the link between kai1 phenotypes and the MAX2 locus, we examined two additional E H 2O fi 100 KAR1 alleles, max2-1 and max2-2, which were previously identi ed in KAR2 the Columbia (Col) ecotype (22). Supporting our results with 80 GR24 max2-7 and max2-8, these max2 alleles also exhibited highly re- KNO3 60 duced germination (Fig. 2B). After 92 h in continuous white light conditions, max2-1 and max2-2 seeds attained ∼10% to 20% 40

germination (%) germination 20 kai1-1 (max2-7) 0 A TTTTT Ler kai1-1 kai1-2 kai1-2 (max2-8) TCAATCTCAC CTA----AGT Fig. 1. Identification of two karrikin-insensitive mutants. (A) Structure of the two most highly active karrikins on A. thaliana, KAR1 and KAR2, and the MAX2 synthetic strigolactone GR24. The common butenolide ring is highlighted in red. (B) Reduced stature, increased axillary branching, and delayed senes- cence in adult kai1-1 compared with wild-type. (C) Peripheral leaf curling in 1000 2000 kai1-1.(D) Eight-day-old seedlings grown on 0.5× MS + 0.5% (wt/vol) sucrose 2 H O KAR KAR GR24 at 21 °C under continuous white light, 100 μmol·m ·s. (Scale bar, 2 mm.) (E) B 100 2 1 2 Germination after 1 wk in continuous white light, 20 °C, of primary dormant 80 Ler and kai1 seed on water-agar supplemented with 1 μM KAR1,1μM KAR2, 10 μM GR24, or 10 mM KNO3. Data are means ± SE (n = 4 independent seed 60 batches, ≥75 seeds per sample). 40

ses to karrikins and strigolactones in Arabidopsis are not because (%) germination 20

of the butenolide moiety alone. Thus, two hypotheses have 0 emerged: karrikins and strigolactones have completely distinct Col max1 max2-1 max2-2 max3 max4 receptors and signaling mechanisms, or they share a common MS KAR KAR GR24 signal transduction pathway with species/developmental stage- C 12 1 2 specific ligand preferences. In this work we investigate possible 10 mechanistic and genetic links for these two classes of chemical 8 signals in regulation of Arabidopsis development. 6

Results 4

Two max2 Alleles Are Identified from a Genetic Screen for Karrikin- 2 hypocotyl length (mm) length hypocotyl Insensitive Mutants. To identify components of the karrikin re- 0 sponse mechanism, we took advantage of the ability of KAR1 to Col max1 max2-1 max2-2 max3 max4 promote Arabidopsis seed germination strongly (10). A primary dormant M population of γ-irradiated Ler (Landsberg erecta Fig. 2. MAX2 is required for germination and photomorphogenesis 2 responses to karrikins and strigolactone. (A) kai1-1 (now max2-7)andkai1-2 ecotype) seed was screened for failure to germinate on water- (now max2-8) have frame-shift mutations in MAX2. The arrow indicates the agar supplemented with 1 μM KAR1. Nongerminating seed were 2,082-bp MAX2 single exon ORF. Wild-type nucleotides in light gray are transferred to nitrate-containing media to overcome dormancy absent in these alleles. (B) Germination after 92 h in continuous white light, μ μ and induce germination of viable seed. Primary dormant M seed 20 °C, of max mutants on water-agar supplemented with 1 M KAR1,1 M 3 μ ± – from candidate karrikin-insensitive (kai) mutants were rescreened KAR2,or10 M GR24. Data are means SE (n =34 independent seed batches, ≥75 seeds per sample). (C) Hypocotyl elongation responses of max for germination insensitivity to KAR1. Seven of 245 putative kai mutants grown 4 d under continuous red light on 0.5× MS media (white

lines maintained a reduced germination phenotype in the pres- bars) supplemented with 1 μM KAR1 (black), 1 μM KAR2 (dark gray), or 1 μM ence of KAR1. GR24 (light gray). Data are means ± SE (n =3,≥15 seedlings per sample).

8898 | www.pnas.org/cgi/doi/10.1073/pnas.1100987108 Nelson et al. Downloaded by guest on October 1, 2021 germination on water-agar control media, compared with ∼70% H 2O KAR1 KAR2 GR24 80 14 germination for Col wild-type. In addition, germination was not STH7 *** KUF1 max2 *** 12 enhanced by KAR1, KAR2, or GR24 in either allele (Fig. 60 10 2B). It was previously reported that max2 alleles have reduced 8 40 germination following red or far-red light pulses because of light 6 hyposensitivity (31). However, as light exposure was not a limit- 20 4 ing factor in our assays, we propose that the low germination of 2 max2 is also consistent with increased seed dormancy. This max2 20 1.5 *** phenotype may not have been observed by other researchers, as ** KUOX1 HY5 primary dormancy in Arabidopsis is readily alleviated by the 15

relative expression relative 1 common laboratory practices of after-ripening, sowing seed on 10 nitrate-containing media [e.g., Murashige and Skoog (MS)], and 0.5 cold stratification (32), procedures which we did not follow. 5 Seed dormancy state has been demonstrated to restrict the germination response to karrikins or smoke (9, 10). In our hands, Ler max2-7 max2-8 Ler max2-7 max2-8 Col seed has low primary dormancy and its germination is not strongly promoted by karrikin (Fig. 2B and ref. 10). Thus, it Fig. 3. Early transcriptional responses to karrikin and strigolactone are fi abolished in max2 seed. Relative expression (to CACS reference transcript) of could be argued that this assay is not suf ciently amenable to test STH7, KUF1, KUOX1,andHY5 in primary dormant wild-type (Ler) and max2 for karrikin responses in max2 seed. However, seedling photo- seed imbibed 24 h in continuous white light, 20 °C, on water-agar (white)

morphogenesis is consistently enhanced by karrikins in multiple supplemented with 1 μM KAR1 (black), 1 μM KAR2 (dark gray), or 10 μM Arabidopsis ecotypes, including Col, even after alleviation of GR24 (light gray). Expression is scaled to the water control expression level in ± – < seed dormancy (11). One micromolar KAR1, KAR2, and GR24 max2-7. Data are means SD (n =3 4 independent seed batches). *P 0.01, inhibited hypocotyl elongation of wild-type Col under continuous two-tailed t test comparison with control expression in each genotype. red light by ∼25%, 45%, and 65%, respectively, and similar degrees of inhibition were observed in max1, max3, and max4 seedlings (Fig. 2C). In contrast, hypocotyl elongation of max2 markers by either karrikin or GR24 was observed in imbibed was unaffected by treatment with karrikins or GR24 (Fig. 2C). max2-7 or max2-8 seed, demonstrating that this is a MAX2- The max2 hypocotyls were also more elongated compared with dependent response (Fig. 3). In seedlings of max2-7 and max2-8,a wild-type (10.5 mm vs. 8.4 mm, P < 0.001), consistent with prior similar lack of STH7 and KUF1 transcriptional responses to kar- reports (22, 31). We conclude that MAX2 regulates seed dor- rikin or GR24 was observed (Fig. S2). These findings are further mancy, and is also required for early developmental responses to supported by a recent report that STH7 is induced by GR24 ap- both karrikins and strigolactones. plication within 3 h in max1, but not max2, seedlings (33). Unlike max2, the max1, max3, and max4 mutants exhibited Notably, STH7 and KUF1 transcripts were respectively re- normal seed germination and hypocotyl length under control duced by ∼25- and 3.5-fold in untreated max2 seed (Fig. 3) and conditions, and maintained wild-type growth responses to both ∼4- and 1.5-fold in max2 seedlings (Fig. S2), indicating that karrikin and GR24 (Fig. 2 B and C). Thus, other components of MAX2 promotes their expression even in the absence of exoge- the MAX pathway, which are involved in the biosynthesis of nous treatments. In contrast to the strong, early markers of strigolactones, are not required for karrikin activity. This finding karrikin response, HY5 is only mildly induced by karrikin after also demonstrates that although Arabidopsis plants are compe- ∼12 to 24 h of seed imbibition (11). Although HY5 transcript tent to respond to exogenous strigolactone application during abundance was not decreased in max2 seed relative to wild-type, early developmental stages in a MAX2-dependent manner, stri- as it was for the early markers, max2 also lacked the small but golactone deficiency does not appear to affect normal seed significant up-regulation of HY5 by karrikin or GR24 treatment dormancy or hypocotyl elongation. (Fig. 3). As early transcriptional markers as well as devel- In contrast with our results, Tsuchiya et al. (27) found that opmental responses to karrikins and strigolactone are abolished max1-1 seed has reduced germination compared with wild-type in max2, we conclude that MAX2 is an essential part of the signal fi under a speci c light condition. As only a single strigolactone- transduction mechanism for both of these classes of plant- fi de cient allele was reported, and details with regard to seed growth regulators. dormancy, light intensities, and growth media are not described, fi it is dif cult to address whether this observation does, in fact, MAX4 and IAA1 Are Suppressed by Karrikins in a MAX2-Dependent indicate a role for endogenous strigolactones in promoting seed Manner. Negative feedback regulation of transcripts involved in germination in particular circumstances. strigolactone biosynthesis (e.g., MAX3/CCD7 and MAX4/CCD8) has been previously observed in several species (23, 33–38). In MAX2 Is Required for Induction of Early Transcriptional Markers of the Arabidopsis max1, max2, max3, and max4 mutants, MAX3 Karrikin Response. We previously demonstrated that the ga1-3 and and MAX4 as well as -responsive IAA1 transcripts are all hy5-1 mutants maintain transcriptional responses to karrikin de- spite displaying karrikin hyposensitivity in germination or hypo- elevated (34). Complementing this observation, application of cotyl elongation assays, respectively (11). To determine if MAX2 GR24 to max1 seedlings suppressed the overexpression of MAX3 has a central role in karrikin signaling, we examined the effects and MAX4 (33). The feedback inhibition mechanism appears to be MAX2-dependent (33). To test whether karrikins can mimic of KAR1, KAR2, and GR24 on early transcriptional markers of karrikin response in the max2 mutant. The most quickly and strigolactone feedback, we examined the expression of MAX4 strongly karrikin-induced transcripts in primary dormant Ler and IAA1 in treated wild-type and max2 seedlings (in our hands, seed include a double B-box domain transcription factor, STH7 MAX3 expression was too low for reliable detection). KAR1 and (At4g39070), an oxidoreductase, KUOX1 (At5g07480), and an- KAR2 reduced MAX4 and IAA1 expression by approximately other F-box protein, KUF1 (At1g31350) (11). We found that twofold in wild-type seedlings (Fig. 4). These responses were GR24 was as effective as karrikins in up-regulating these three similar to those observed in GR24-treated seedlings, except that marker genes after 24 h of seed imbibition, providing molecular although karrikins and GR24 have the same effects on IAA1

evidence that strigolactones and karrikins may share some sig- expression, karrikins were less effective than GR24 as inhibitors PLANT BIOLOGY naling mechanisms (Fig. 3). However, no induction of these of MAX4 expression. As expected, MAX4 and IAA1 were up-

Nelson et al. PNAS | May 24, 2011 | vol. 108 | no. 21 | 8899 Downloaded by guest on October 1, 2021 MS KAR1 KAR2 GR24 2.5 4 A max4 MAX4 IAA1 2 3 1.5 2 1 * * 0.5 1 relative expression * * * Ler max2-7 max2-8 Ler max2-7 max2-8

Fig. 4. Karrikins suppress MAX4 and IAA1 transcript levels in a MAX2- MS KAR KAR GR24 dependent manner. Relative expression (to CACS reference transcript) of 1 2 MAX4 and IAA1 in wild-type (Ler) and max2 seedlings grown as described for hypocotyl elongation assays after 3 d in continuous red light on 0.5× MS 6 B H2O KAR (white) supplemented with 1 μM KAR1 (black), 1 μM KAR2 (dark gray), or 5 1 1 μM GR24 (light gray). Expression is scaled to the control expression level in KAR2 4 GR24 wild-type Ler. Data are means ± SD (n =3–4 independent pools of several dozen seedlings). *P < 0.01, two-tailed t test comparison with control ex- 3 * pression in each genotype. * 2

rosette axillary shoots 1

regulated in max2 seedlings and unresponsive to either karrikin 0 or GR24 treatment (Fig. 4). Col max3 max4

Karrikins Do Not Suppress Shoot Branching in Arabidopsis or Pea. C 3 The above data suggest that karrikins and strigolactones share a common MAX2-dependent signal transduction mechanism in Arabidopsis. However, as there are multiple examples that in- 2 dicate these signals are not interchangeable (see Introduction), distinct functional roles for karrikins and strigolactones in other 1 H] in aerial tissue (dpm/mg)

aspects of plant development may be expected. To determine 3 [ whether karrikins can compensate for the effect of strigolactone 0 fi 31224 48 96 de ciency in adult plants, we tested the response of max3 and time (h) max4 axillary shoot development to KAR1 and KAR2. Shoot branching in both mutants was clearly suppressed by GR24 de- Fig. 5. Karrikins do not inhibit axillary shoot branching in Arabidopsis.(A) livered hydroponically through roots, but was insensitive to an max4 mutants grown hydroponically under 16-h light:8-h dark photoperiod × μ μ μ equivalent karrikin application (Fig. 5 A and B). The lack of on 0.3 MS media supplemented with 5 M KAR1,5 M KAR2,or5 M GR24. a max4 branching response to karrikins was confirmed in- White arrows denote axillary shoots. (B) Number of rosette axillary shoots for 42-d-old wild-type Col, max3,ormax4 plants grown hydroponically on 0.3× dependently by two of us in a second laboratory (E.A.D. and C. MS (white bars), 1 μM KAR1 (black), 1 μM KAR2 (dark gray), or 5 μM GR24 A.B.) using an alternative semihydroponic application method (light gray). Data are means ± SE (n =16–20 plants). *P < 0.01, two-tailed t (Fig. S3). As expected, the increased shoot branching phenotype test comparison with control media axillary shoot number in each genotype. of max2 mutants was insensitive to any treatments (Fig. S3). (C) Accumulation of [3H] in aerial tissue of wild-type (Ler) plants after ad- 3 ± Thus, strigolactones regulate shoot branching by a MAX2-de- dition of [ H]-KAR1 to hydroponic media. Data are means SE (n =4). pendent mechanism that does not recognize karrikins. To address the possibility that karrikins only appear to be ineffective shoot branching inhibitors in our assays because they Discussion are not taken up by roots and delivered to shoots, we examined As structurally related molecules, it is perhaps not altogether 3 transport of [ H]-KAR1 in hydroponically grown Arabidopsis surprising that karrikins and strigolactones can similarly promote plants. Radioactivity was detectable in the lower stem within 3 h seed germination, inhibit hypocotyl elongation, and up-regulate of addition to hydroponic media, and continued to increase in expression of a common subset of genes in Arabidopsis. Even abundance throughout the plant over the 96-h time course (Fig. more suggestive of a common perception mechanism is the 5C). Although transport was initially observed in stem tissue, feedback effect of karrikins on MAX4 and IAA1 expression. by 96 h the radiolabel was present in all aerial tissues and most highly concentrated in the siliques (Fig. S4). Scintillation fi counting of HPLC-fractionated (6) plant extracts con rmed that 1 µM KAR 3 fi 35 control 2 [ H]-KAR1 was present in each part of the plant. This nding 1 µM KAR1 10 µM KAR2 30 10 µM KAR 1 µM GR24 demonstrates that karrikin is readily taken up by roots and dis- 1 25 tributed throughout the aerial tissue of adult plants. fl 20 We performed an additional test for karrikin in uence on bud 15 outgrowth in pea, which is responsive to GR24 at concentrations 10 as low as 10 nM (18). As previously demonstrated, bud elongation bud growth (mm) 5 * of rms1 (equivalent to Arabidopsis max4) plants was strongly sup- 0 pressed by direct application of 1 μM GR24, but rms4 (equivalent rms1 rms4 to Arabidopsis max2) buds had no significant response to the treatment (Fig. 6). In contrast, even at a 10-μM concentration Fig. 6. Bud outgrowth in pea does not respond to karrikins. Buds at node 2 neither KAR nor KAR reduced bud elongation in the rms1 of 7-d-old rms1-11 and rms4-3 plants grown in 18-h light:6-h dark photo- 1 2 period were treated with control solution (white bars), 1 (black) or 10 μM mutant. Furthermore, rms4 was similarly unresponsive to karri- (dark hatched) KAR1, 1 (dark gray) or 10 μM (light hatched) KAR2,or1μM kins. Thus, karrikins appear to be ineffective inhibitors of axillary GR24 (light gray), and growth was measured 7 d later. Data are means ± SE shoot outgrowth in both pea and Arabidopsis, and cannot com- (n =11–14 plants). *P < 0.01, two-tailed t test comparison with control bud pensate for strigolactone deficiency in this aspect of development. growth in each genotype.

8900 | www.pnas.org/cgi/doi/10.1073/pnas.1100987108 Nelson et al. Downloaded by guest on October 1, 2021 However, these signaling molecules are also quite distinct in plant-growth regulators during different stages of development. terms of their known sources (i.e., in planta vs. smoke), as well as As future studies reveal additional components of the MAX2- the effects they produce on seed from different species, arbus- dependent pathway, it will be interesting to determine how an cular mycorrhizal association, and shoot branching. Thus, the abiotic smoke signal and a phytohormone came to share a com- extent of the relationship between karrikins and strigolactones mon signal integration point. has in the past remained unclear. Through a forward genetic We finally note that the max2 mutants exhibit enhanced seed screen we have now clearly linked the signal transduction for dormancy, elongated hypocotyls, and gene expression (e.g., STH7, these two families of plant-growth regulators and identified KUF1, KUOX1) phenotypes that are opposite to the effects of previously unexplored roles for MAX2 as a key regulator of karrikin or GR24 application on wild-type plants (summarized in karrikin signaling and seed dormancy. Fig. S6). As these phenotypes are not present in the strigolactone- With the revelation that karrikin and strigolactone signaling deficient max1, max3,ormax4 mutants, it may indicate over- must converge at or before MAX2, we wished to address whether accumulation of a constitutive karrikin pathway repressor in max2. these ligands are metabolically linked to produce a common Alternatively, this observation may suggest the existence of an en- signal transduced by MAX2. For example, karrikins may not have dogenous nonstrigolactone-derived signal at these growth stages restored branching suppression in the max3 or max4 mutants to which max2 mutants can no longer respond. If such a signal if they must be converted to a strigolactone-related signal in a were structurally similar to karrikins and strigolactones, it could MAX3/MAX4-dependent manner. However, as max3 and max4 explain the broad conservation of a karrikin-response mechanism were fully responsive to karrikin during germination and seedling fi development, this contradicts a MAX3/MAX4-dependent metab- in plant species from non re-prone environments, such as A. olism model. One possible explanation for this discrepancy is that thaliana, in the absence of an obvious selective pressure. conversion of karrikin to an active strigolactone-related signal Materials and Methods may be specifically lacking in adult tissues. However, we alter- natively propose that karrikins converge with strigolactones at the Plant-Growth Conditions, Germination Tests, Hypocotyl Elongation Assays, and qRT-PCR. Plant-growth conditions, germination tests, hypocotyl elongation point of signal transduction rather than through metabolism. assays, and qRT-PCR were performed as previously described (10, 11). MAX4 MAX2 encodes an F-box protein with 18 leucine-rich repeats and IAA1 primer sequences were the same as in Hayward et al. (34). Karrikin (30). F-box proteins act as components of the SCF (Skp1, Cullin, and GR24 stock (1,000×) solutions were prepared in acetone. In each ex- RBX1, F-box protein) complex, a class of E3 ligases that ubiq- periment, the control contained an equivalent amount of acetone. uitinate specific target proteins. Depending on the degree of fi ubiquitination, modi ed proteins may have altered function or kai Screen. γ-Ray irradiated (Co-60 source, 45 krad) M1 Landsberg erecta seed be targeted for degradation by the 26S proteasome. An F-box (Lehle Seeds) was grown under continuous light conditions. Primary dor-

protein acts as an adapter protein for the SCF complex; the mant M2 seed were after-ripened 2 wk and selected on water-agar sup- μ N-terminal F-box domain associates with Skp1 and the C ter- plemented with 1 M KAR1 for nongermination after 7 to 8 d in continuous minus confers substrate specificity (39). MAX2 was shown to light, 20 °C, and transferred to 0.5× MS media to recover germination of interact with the Arabidopsis homolog of Skp1, ASK1, in yeast viable seed. two-hybrid and in vitro binding assays (30). F-box proteins have been implicated in the direct perception Arabidopsis Shoot Branching Assays. Seedlings were grown in 16-h light:8-h × or early signal transduction of several phytohormones, including dark for 7 d and transferred to perlite substrate in clear, sterile pots with 0.3 auxin (40), (41), and gibberellin (42). For each of MS + Gamborg vitamins (Sigma), 2 mM Mes, pH 5.7 liquid media. Media supplemented with 5 μM KAR ,5μM KAR ,or5μM GR24 was exchanged these hormones, the F-box protein targets negative regulators of 1 2 weekly. All solutions contained 0.05% (vol/vol) acetone. Rosette shoot transcription factors that carry out the hormone responses. In branches ≥ 5 mm were assayed when plants were 42 d old. Semihydroponic the case of auxin, association of the F-box protein TIR1 with an phytatray assays shown in Fig. S3 were performed as previously described Aux/IAA negative regulator is promoted by auxin-binding in a (48), except growth was at 24 °C and rosette branches longer than 5 mm TIR1 pocket, a nonallosteric interaction in which the ligand acts were counted when plants were 35 d old. as “molecular glue” (43). In the current model of gibberellin signaling, however, the receptor GID1 undergoes allosteric ac- Pea Bud Outgrowth Assays. Pea bud outgrowth assays were performed as tivation after gibberellin-binding, which enhances its association previously described (49), except solutions contained 0.1% (vol/vol) acetone. with the DELLA negative regulators and promotes their recog- 3 nition by the F-box protein SLY1 (44). [ H]-KAR1 Transport Assays. For a more detailed description of the experi- As max2 is insensitive to the synthetic strigolactone GR24, it mental setup, please see SI Materials and Methods. In brief, A. thaliana (Ler) has been hypothesized that MAX2 may either be the strigo- plants were grown on a rockwool substrate plug in the lid of 15-mL tubes. Roots were fed liquid 0.5× MS + Gamborg vitamins, 2 mM Mes, pH 5.7 lactone receptor or associate with a receptor complex (18, 39). 3 media. Before addition of [ H]-KAR1, an air gap was made between the Whatever the case may be, we now propose that MAX2 also 3 media and rockwool to prevent direct moistening of the plug. [ H]-KAR1 was manages karrikin signal transduction in a manner similar to that fl for strigolactones. In a dual-role scenario, how might MAX2 added to a 10-ppm concentration to the media of 5-wk-old owering plants, and plant tissue was harvested over a 96-h period. Scintillation activity of distinguish between karrikin and strigolactone signals during tissue in Beckman Coulter Ready-Safe Liquid Scintillation Mixture was de- regulation of shoot branching, but not during seed germination termined over a 10-min interval using a Beckman Coulter LS6500 Multipur- and seedling establishment? The d14/htd2/d88 mutant in rice pose Scintillation Counter. exhibits strigolactone-insensitive, enhanced tillering, as well as feedback up-regulation of strigolactone biosynthesis genes (45– Synthesis of Karrikins, GR24, and Butenolides. KAR (50), [7-3H ]-KAR (51), fi α β 1 1 1 47). D14 is classi ed as an / -fold hydrolase superfamily mem- KAR2 (50), GR24 (52), butenolide B1 [3,4,5-trimethylfuran-2(5H)-one] (53), ber, as is the gibberellin receptor GID1, and has been proposed B2 [3-methylfuran-2(5H)-one 3-methyl] (54), B3 [4-methylfuran-2(5H)-one] to be a potential component of strigolactone signaling (45). In (54), and B4 [3,4-dimethylfuran-2(5H)-one] (55) were prepared as previously a model based upon the gibberellin signaling mechanism, MAX2 described. could target strigolactone or karrikin pathway repressors for proteasomal degradation upon their specific association with a ACKNOWLEDGMENTS. We thank Winslow Briggs for feedback on the manuscript. The European Arabidopsis Stock Centre provided the max respective ligand-activated D14 homolog (Fig. S5). The spatio- mutant seed. Funding was provided by the Australian Research Council

temporal availability of either the receptors or signaling pathway (Grants DP0667197, DP0880484, DP0880627, DP110100851, LP0882775, and PLANT BIOLOGY repressors could then restrict the effects of these two families of FF0457721) and by the Government of Western Australia.

Nelson et al. PNAS | May 24, 2011 | vol. 108 | no. 21 | 8901 Downloaded by guest on October 1, 2021 1. Baskin JM, Baskin CC (2004) A classification system for seed dormancy. Seed Sci Res 14: 29. Mangnus EM, Zwanenburg B (1992) Tentative molecular mechanism for germination 1–16. stimulation of Striga and Orobanche seeds by strigol and its synthetic analogues. 2. van Staden J, Brown NAC, Jäger AK, Johnson TA (2000) Smoke as a germination cue. J Agric Food Chem 40:1066–1070. Plant Species Biol 15:167–178. 30. Woo HR, et al. (2001) ORE9, an F-box protein that regulates leaf senescence in 3. Dixon KW, Merritt DJ, Flematti GR, Ghisalberti EL (2009) Karrikinolide–A Arabidopsis. Plant Cell 13:1779–1790. phytoreactive compound derived from smoke with applications in horticulture, 31. Shen H, Luong P, Huq E (2007) The F-box protein MAX2 functions as a positive ecological restoration, and agriculture. Acta Hortic 813:155–170. regulator of photomorphogenesis in Arabidopsis. Plant Physiol 145:1471–1483. 4. Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD (2004) A compound from smoke 32. Ali-Rachedi S, et al. (2004) Changes in endogenous levels during that promotes seed germination. Science 305:977. dormancy release and maintenance of mature seeds: Studies with the Cape Verde 5. van Staden J, Jager AK, Light ME, Burger BV (2004) Isolation of the major germination Islands ecotype, the dormant model of Arabidopsis thaliana. Planta 219:479–488. cue from plant-derived smoke. S Afr J Bot 70:654–659. 33. Mashiguchi K, et al. (2009) Feedback-regulation of strigolactone biosynthetic genes 6. Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD (2009) Identification of alkyl and strigolactone-regulated genes in Arabidopsis. Biosci Biotechnol Biochem 73: – substituted 2H-furo[2,3-c]pyran-2-ones as germination stimulants present in smoke. 2460 2465. J Agric Food Chem 57:9475–9480. 34. Hayward A, Stirnberg P, Beveridge C, Leyser O (2009) Interactions between auxin and – 7. Chiwocha DS, et al. (2009) Karrikins: A new family of plant growth regulators in strigolactone in shoot branching control. Plant Physiol 151:400 412. smoke. Plant Sci 177:252–256. 35. Foo E, et al. (2005) The branching gene RAMOSUS1 mediates interactions among two – 8. Light ME, Daws MI, Van Staden J (2009) Smoke-derived butenolide: Towards novel signals and auxin in pea. Plant Cell 17:464 474. understanding its biological effects. S Afr J Bot 75:1–7. 36. Drummond RS, et al. (2009) Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 9. Stevens JC, Merritt DJ, Flematti GR, Ghisalberti EL, Dixon KW (2007) Seed germination is involved in the production of negative and positive branching signals in petunia. – of agricultural weeds is promoted by the butenolide 3-methyl-2H-furo[2,3-c]pyran-2- Plant Physiol 151:1867 1877. one under laboratory and field conditions. Plant Soil 298:113–124. 37. Zou J, et al. (2006) The rice HIGH-TILLERING DWARF1 encoding an ortholog of 10. Nelson DC, et al. (2009) Karrikins discovered in smoke trigger Arabidopsis seed Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. Plant J 48:687–698. germination by a mechanism requiring gibberellic acid synthesis and light. Plant 38. Snowden KC, et al. (2005) The Decreased apical dominance1/Petunia hybrida Physiol 149:863–873. CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a 11. Nelson DC, et al. (2010) Karrikins enhance light responses during germination and role in leaf senescence, root growth, and flower development. Plant Cell 17:746–759. seedling development in Arabidopsis thaliana. Proc Natl Acad Sci USA 107:7095–7100. 39. Somers DE, Fujiwara S (2009) Thinking outside the F-box: Novel ligands for novel 12. Cook CE, Whichard LP, Turner B, Wall ME, Egley GH (1966) Germination of witchweed receptors. Trends Plant Sci 14:206–213. (Striga lutea Lour): Isolation and properties of a potent stimulant. Science 154: 40. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin regulates SCF(TIR1)- 1189–1190. dependent degradation of AUX/IAA proteins. Nature 414:271–276. 13. Humphrey AJ, Beale MH (2006) Strigol: Biogenesis and physiological activity. 41. Thines B, et al. (2007) JAZ repressor proteins are targets of the SCF(COI1) complex Phytochemistry 67:636–640. during signalling. Nature 448:661–665. 14. Akiyama K, Matsuzaki K, Hayashi H (2005) Plant sesquiterpenes induce hyphal 42. Dill A, Thomas SG, Hu J, Steber CM, Sun TP (2004) The Arabidopsis F-box protein branching in arbuscular mycorrhizal fungi. Nature 435:824–827. SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation. 15. Beveridge CA, Kyozuka J (2010) New genes in the strigolactone-related shoot Plant Cell 16:1392–1405. branching pathway. Curr Opin Plant Biol 13:34–39. 43. Tan X, et al. (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. 16. Xie X, Yoneyama K, Yoneyama K (2010) The strigolactone story. Annu Rev Nature 446:640–645. Phytopathol 48:93–117. 44. Sun TP (2010) Gibberellin-GID1-DELLA: A pivotal regulatory module for plant growth 17. Dun EA, Brewer PB, Beveridge CA (2009) Strigolactones: Discovery of the elusive shoot and development. Plant Physiol 154:567–570. branching hormone. Trends Plant Sci 14:364–372. 45. Arite T, et al. (2009) d14, a strigolactone-insensitive mutant of rice, shows an 18. Gomez-Roldan V, et al. (2008) Strigolactone inhibition of shoot branching. Nature accelerated outgrowth of tillers. Plant Cell Physiol 50:1416–1424. – 455:189 194. 46. Gao Z, et al. (2009) Dwarf 88, a novel putative esterase gene affecting architecture of 19. Umehara M, et al. (2008) Inhibition of shoot branching by new terpenoid plant rice plant. Plant Mol Biol 71:265–276. – hormones. Nature 455:195 200. 47. Liu W, et al. (2009) Identification and characterization of HTD2: A novel gene fl 20. Lazar G, Goodman HM (2006) MAX1, a regulator of the avonoid pathway, controls negatively regulating tiller bud outgrowth in rice. Planta 230:649–658. vegetative axillary bud outgrowth in Arabidopsis. Proc Natl Acad Sci USA 103: 48. Brewer PB, Dun EA, Ferguson BJ, Rameau C, Beveridge CA (2009) Strigolactone acts – 472 476. downstream of auxin to regulate bud outgrowth in pea and Arabidopsis. Plant 21. Stirnberg P, Furner IJ, Ottoline Leyser HM (2007) MAX2 participates in an SCF complex Physiol 150:482–493. – which acts locally at the node to suppress shoot branching. Plant J 50:80 94. 49. Dun EA, Hanan J, Beveridge CA (2009) Computational modeling and molecular 22. Stirnberg P, van De Sande K, Leyser HM (2002) MAX1 and MAX2 control shoot lateral physiology experiments reveal new insights into shoot branching in pea. Plant Cell 21: – branching in Arabidopsis. Development 129:1131 1141. 3459–3472. 23. Johnson X, et al. (2006) Branching genes are conserved across species. Genes 50. Goddard-Borger ED, Ghisalberti EL, Stick RV (2007) Synthesis of the germination controlling a novel signal in pea are coregulated by other long-distance signals. Plant stimulant 3-methyl-2H-furo[2,3-c]pyran-2-one and analogous compounds from Physiol 142:1014–1026. carbohydrates. Eur J Org Chem 23:3925–3934. 24. Goldwasser Y, Yoneyama K, Xie X, Yoneyama K (2008) Production of strigolactones 51. Scaffidi A, et al. (2011) The synthesis and biological evaluation of labelled by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination. karrikinolides for the elucidation of the mode of action of the seed germination Plant Growth Regul 55:21–28. stimulant. Tetrahedron 67:152–157. 25. Kohlen W, et al. (2011) Strigolactones are transported through the xylem and play 52. Mangnus EM, Dommerholt FJ, De Jong RLP, Zwanenburg B (1992) Improved synthesis a key role in shoot architectural response to phosphate deficiency in non-AM host of strigol analog GR24 and evaluation of the biological activity of its diastereomers. Arabidopsis thaliana. Plant Physiol 155:974–987. J Agric Food Chem 40:1230–1235. 26. Booker J, et al. (2005) MAX1 encodes a cytochrome P450 family member that acts 53. Surmont R, Verniest G, De Kimpe N (2010) Short synthesis of the seed germination downstream of MAX3/4 to produce a carotenoid-derived branch-inhibiting hormone. inhibitor 3,4,5-trimethyl-2(5H)-furanone. J Org Chem 75:5750–5753. Dev Cell 8:443–449. 54. Kayser MM, Breau L, Eliev S, Morand P, Ip HS (1986) Regioselectivity control in metal 27. Tsuchiya Y, et al. (2010) A small-molecule screen identifies new functions for the plant hydride reductions of substituted maleic anhydrides. Can J Chem 64:104–109. hormone strigolactone. Nat Chem Biol 6:741–749. 55. Abe N, Fujisaki F, Sumoto K, Miyano S (1991) Reduction of five-membered 28. Akiyama K, Ogasawara S, Ito S, Hayashi H (2010) Structural requirements of α,β-unsaturated lactones and related compounds with the Ni2+/BH4- System. Chem strigolactones for hyphal branching in AM fungi. Plant Cell Physiol 51:1104–1117. Pharm Bull (Tokyo) 39:1167–1170.

8902 | www.pnas.org/cgi/doi/10.1073/pnas.1100987108 Nelson et al. Downloaded by guest on October 1, 2021