Karrikins discovered in smoke trigger Arabidopsis seed germination by a mechanism requiring gibberellic acid synthesis and light

Discovery of the primary seed germination stimulant in smoke, 3-methyl-2 H -furo[2,3-c ]pyran-2-one (KAR 1 ), has resulted in identification of a family of structurally-related plant growth regulators, karrikins. KAR 1 acts as a key germination trigger for many species from fire-prone, mediterranean climates, but a molecular mechanism for this response remains unknown. We demonstrate that Arabidopsis thaliana , an ephemeral of the temperate Northern hemisphere which has never been reported to be responsive to fire or smoke, rapidly and sensitively perceives karrikins. Thus these signaling molecules may have greater significance among angiosperms than previously realized. Karrikins can trigger germination of primary dormant Arabidopsis seed far more effectively than known phytohormones or the structurally-related strigolactone GR-24. Natural variation and depth of seed dormancy affect the degree of KAR 1 stimulation. Analysis of phytohormone mutant germination reveals suppression of KAR 1 responses by abscisic acid and a requirement for gibberellin (GA) synthesis. The reduced germination of sly1 mutants is partially recovered by KAR 1 , which suggests germination enhancement by karrikin is only partly DELLA-dependent. While KAR 1 has little effect on sensitivity to exogenous GA, it enhances expression of the GA biosynthetic genes GA3ox1 and GA3ox2 during seed imbibition. Neither ABA nor GA levels in seed are appreciably affected by KAR 1 treatment prior to radicle emergence, despite marked differences in germination outcome. KAR 1 stimulation of Arabidopsis germination is light-dependent and reversible by far-red exposure, although limited induction of GA3ox1 still occurs in the dark. The observed requirements for light and GA biosynthesis provide the first insights into the karrikin mode of action. ABA germination rates inhibited, however aba3-2 seeds still showed no response to KAR 1 . Similar results obtained with aba2-1 and aba3-1 alleles, and afterripened (AR) L er and aba1-3 germination was not further KAR 1 in the of ABA These results could indicate that KAR 1 acts primarily by reducing ABA synthesis during imbibition, or that high levels of ABA block the karrikin response. triggering Orobanche germination. These results indicate that karrikins and strigolactones are not interchangeable and may act via different mechanisms. The level of GA3ox induction corresponded with the efficacy of these germination stimulants (KAR 2 > KAR 1 > KAR 3 > GR-24). However, it cannot be assessed from GA3ox and CP1 expression alone whether karrikin and strigolactone signals are perceived and transduced via common pathways, as both stimulants may ultimately enhance GA synthesis as part of the germination process. column (SGE 30 m x 0.25 mm, 0.25 µm) using helium as the carrier gas (1 mL/min) and the inlet temperature was 280°C. The initial oven temperature was set at 50°C and held for 1 min before increasing to 200°C at 15°C/min. The temperature was increased at 3°C/min up to 270°C followed by 15°C/ min to 320°C and held for 5 mins. The transfer line was set at 280°C, the ion source was 200°C and the ionization potential was 70 eV. The analyses were performed in SIM mode, monitoring for the following sets of ions 190 and 194 for ABA/d 6 ABA, 284 and 286 for GA 4 /d 2 GA 4 , 506 and 508 for GA 1 /d 2 GA 1 . Amounts were calculated based on the ratio of these ions with corrections made to account for contributions to the area of the deuterated peak from endogenous ions. Deuterated standards were calibrated with authentic samples to provide an accurate measurement of the endogenous hormone levels.

Germination is a critical event in the plant life cycle, as the timing of emergence from the protective seed coat is crucial for survival and reproductive success. A variety of abiotic stimuli, including light, temperature, and nitrates provide information about the external environment which affect germination. Seed dormancy gates responses to these factors.
Upon maturation, physiologically dormant seeds are in a primary dormant (PD) state which is lost during afterripening. The transition between a PD and nondormant state is both gradual and reversible, and results in relaxation of the set of environmental conditions under which a seed will germinate (Baskin and Baskin, 2004;Finch-Savage and Leubner-Metzger, 2006).
Despite decades of research, seed dormancy remains a complex physiological state that is not well understood. The plant hormones abscisic acid (ABA) and gibberellin (GA) are mutually antagonistic central players in the germination decision (Finch-Savage and Leubner-Metzger, 2006;Finkelstein et al., 2008). The role of dormancy establishment and maintenance has been attributed to ABA, while GA has been implicated in the initiation and completion of germination. The ratio of ABA:GA signaling, rather than absolute amounts of the hormones, appears to be critical to dormancy breaking (Finch-Savage and Leubner-Metzger, 2006). Environmental stimuli and phytohormones influence the ABA:GA balance, although the mechanisms of signal integration and hormone crosstalk are still largely unknown.
In many biodiverse regions, fire events provide an irregular but important opportunity for seedling establishment by freeing up key resources such as light, space, and nutrients (Van Staden et al., 2000;Dixon et al., 2008). A clear example of this is seen in the flush of new growth in the immediate post-fire environment, indicating potent activation of the soil seed bank. Heat is not required for the germination response, as cold smoke application induced up to 48 fold increase in the number of germinating seedlings, and ~3 fold enrichment in species abundance in field trials (Roche et al., 1997;Rokich et al., 2002). It has now been well established that smoke is a broadly effective stimulant which enhances germination of ~1200 species in more than 80 genera worldwide (Dixon et al., 2008). Attempts to study smoke effects on plant physiology have been confounded by the complex mixture of components within smoke, some of which confer toxicity at high concentrations. Bioassay-guided fractionation of smoke water culminated in the discovery and synthesis of the primary germination stimulant, 3-methyl-2H-furo[2,3c]pyran-2-one (Flematti et al., 2004)..With the recent identification of three analogous active compounds in smoke water fractions (Fig. 1A, G Flematti, unpublished data), this family of butenolide molecules have been designated karrikins, after 'karrik' the first recorded Aboriginal Nyungar word for smoke (Dixon et al., 2008).
The parent molecule, KAR 1, is a potent stimulant which enhances germination in some species at subnanomolar concentrations (Flematti et al., 2004;Stevens et al., 2007). In field trials KAR 1 is effective at <5 g/ha compared to 10 tonnes/ha of smoke water and thus may have practical value in agriculture, conservation, and restoration (Stevens et al., 2007). Smoke water fractions containing KAR 1 have been reported to enhance seedling vigor of several weed and crop species, indicating potential use for KAR 1 as a seed priming agent to improve germination and seedling establishment (Jain et al., 2006;Jain and Van Staden, 2006;Kulkarni et al., 2006;van Staden et al., 2006;Daws et al., 2007).
Since its discovery, a widespread capacity for KAR 1 germination response among angiosperms has been demonstrated (Flematti et al., 2004;van Staden et al., 2004;Merritt et al., 2006;van Staden et al., 2006;Daws et al., 2007;Stevens et al., 2007). Thus karrikins may be considered a novel class of plant growth regulators with broad impact.
To gain a better understanding of the mechanism by which karrikins trigger seed germination and explore their interaction with ABA and GA, we examined KAR 1 responses in Arabidopsis thaliana.
typically caused an early, limited enhancement of seed germination rates, suggesting that a subset of the seed population had been pushed over a dormancy threshold within an imbibition time window (Fig. S1). Notably, the control levels of germination and responses to KNO 3 were also variable across ecotypes. These results may be attributed to natural variation either in the capacity for KAR 1 perception and response, or in seed dormancy depth. To address the latter hypothesis, we examined the germination of Ler seeds with different depths of dormancy. Although after 7 d of imbibition the germination enhancement by KAR 1 was apparent and equivalent to 6 weeks of afterripening (Fig. S2), KAR 1 was unable to fully stimulate germination of PD seed within a 4 d period (Fig. 2F).
With afterripening, KAR 1 enhanced germination within this time frame at a rate outpacing the rise in control germinability. However, as dormancy was further lost there was a corresponding reduction in the apparent effects of KAR 1 on germination relative to the control. Thus in either highly dormant or nondormant states seed may not show an obvious response to KAR 1 treatment, particularly when assaying germination at a single time point. We found that KAR 1 had a positive effect on seed germination rates even after removal of PD Ler seed dormancy by cold stratification or extended afterripening ( Fig. 2G). Karrikins therefore display characteristics of dormancy breaking and germination stimulating compounds.
GA biosynthesis is required for KAR 1 promotion of germination. To investigate the interaction of karrikins with ABA and GA pathways we tested the germination responses of several phytohormone mutants (Table I). We first examined mutants with reduced ABA biosynthetic capacity. aba3-2 had a small response to KAR 1 after 48 h but otherwise exhibited no difference in germination relative to the control (Fig. 3A). We reasoned that if karrikins act through reducing ABA levels or sensitivity, the lack of ABA in this mutant may prevent detection of a KAR 1 effect. In the presence of exogenous Alternatively, as it has been established that exogenous ABA application does not induce the same transcriptional state in seeds as natural dormancy (Carrera et al., 2008), it may be that ABA is required during seed maturation to form a seed with KAR 1 -responsive dormancy.
ABA catabolism occurs primarily through an oxidation pathway resulting in phaseic acid via an 8' hydroxy ABA intermediate (Nambara and Marion-Poll, 2005). The CYP707A family of cytochrome P450s have been characterized as ABA 8'-hydroxylases. Mutations in these genes lead to increased ABA levels in mature seeds, and a reduction in germinability (Okamoto et al., 2006). PD cyp707a2 cyp707a3 seed was responsive to KAR 1 , while the highly reduced germination of cyp707a1 cyp707a3 and cyp707a1 cyp707a2 showed no enhancement (Fig. S3E). With afterripening, however, germination of all three double mutant lines was mildly enhanced by KAR 1 (Fig. S3F). KAR 1 was also unable to overcome the enhanced dormancy of the ABA hypersensitive era1-2 mutant (Cutler et al., 1996), although KNO 3 could eventually induce germination (Fig.   S3G). Thus high endogenous levels of ABA signaling can block induction of germination by KAR 1 .
GA is typically required for germination and can overcome dormancy or promote germination in restrictive conditions. Alleles of GA3ox1 (ga4-1) and GA20ox1 (ga5-1) have reduced GA biosynthetic capacity, but were both strongly responsive to KAR 1 (Fig.   S4A,B). Germination is blocked in the GA-deficient mutant ga1-3, but this can be overcome by other hormones such as brassinosteroid and ethylene, or by reduced ABA synthesis during seed maturation (Karssen et al., 1989;Debeaujon and Koornneef, 2000;Steber and McCourt, 2001). In our hands, very limited ga1-3 germination was achieved with either ACC or EBR, even with the inclusion of nitrates in the media and a stratification treatment (Fig. 3B). However the ga1-3 mutant had no germination in the presence of KAR 1 . Similarly, while a ga3ox2 allele was KAR 1 -responsive, the highly reduced germination of a ga3ox1 ga3ox2 double mutant was not recovered by KAR 1 (Fig. S4D,E). If KAR 1 acts primarily through enhancement of GA biosynthesis and not sensitivity to GA, a lack of germination promotion in GA-supplemented ga1-3 seed would be expected. Indeed, KAR 1 produced little to no change in the germination response of ga1-3 to different concentrations of GA 3 (Fig. 3C).
GA signaling is mediated by SLEEPY (SLY1), an F-box protein that targets the DELLA repressors proteins for degradation in a GA-dependent manner (Dill et al., 2004;Finkelstein et al., 2008). Interestingly, KAR 1 treatment partially restored germination of the GA signaling deficient sly1-2 and sly1-10 alleles, while KNO 3 was less effective (Fig.   3D). This suggests that nitrate and karrikin have distinct modes of action, and that KAR 1 may partially influence germination in a DELLA-independent manner.

Transcription of GA3-oxidases and CP1 is induced by active karrikins.
To gain further insight into KAR 1 effects on hormone metabolism during PD Ler seed imbibition, we performed qRT-PCR analysis for a set of genes involved in the synthesis, catabolism, or response of ABA and GA. Transcriptional changes were assessed during the first 48 h of imbibition, before KAR 1 -treated seeds begin to germinate ( Figure 2B), in order to identify early regulatory events which may result in a germination decision.
No transcript differences were observed for the major ABA biosynthetic enzymes ABA1, ABA2, ABA3, NCED6, or AAO3 in response to KAR 1 (Fig. S5A). AAO4, which can have a role in ABA biosynthesis in the absence of the major isozyme AAO3 (Seo et al., 2004), was upregulated after 24 h by KAR 1 . In a comparison of dormant and nondormant Arabidopsis seed, expression of the ABA 8'-hydroxylase CYP707A2 was fourfold upregulated in nondormant seed at 6 h of imbibition (Millar et al., 2006). However, CYP707A2 and CYP707A3 showed no change in expression in response to KAR 1 treatment during imbibition (Fig. S5B). ABA can also be inactivated through formation of an ABA-glucose ester conjugate by the glucosyltransferase UGT71B6 (Priest et al., 2005). UGT71B6 transcript abundance was unaffected by KAR 1 (Fig. S5B).
ABI genes were identified as components of ABA signal transduction pathways through genetic screens for ABA-insensitive mutants (Finkelstein et al., 2002). Of the five ABI genes tested, only ABI4 transcripts were affected by KAR 1 (Fig. S5C). At 48 h, ABI4 expression was upregulated by KAR 1 . ABI4 encodes an AP2 domain transcription factor (Finkelstein et al., 1998) and confers sensitivity to sugar and salt stress (Arenas-Huertero et al., 2000;Huijser et al., 2000;Laby et al., 2000;Quesada et al., 2000). The implications of ABI4 induction by KAR 1 are currently unclear, but may reflect preparation for seedling emergence. FRY1/SAL1 encodes an IP 3 phosphatase and acts as a negative regulator of ABA signaling (Xiong et al., 2001). We observed upregulation of FRY1 in KAR 1 -treated seeds after 48 h of imbibition (Fig. S5C).
Among the five DELLA proteins, RGL2 is considered the main repressor in seed germination (Lee et al., 2002;Tyler et al., 2004;Cao et al., 2005). We detected no KAR 1induced changes in transcript abundance during imbibition (Fig. S6C). The putative Cys proteinase CP1 is a useful marker for GA signaling as it is upregulated by GA 4 in a concentration-dependent manner and also induced by GA-sensitizing treatments such as stratification (Yamauchi et al., 2004). CP1 exhibited a very similar pattern of expression as GA3ox2, and was upregulated by KAR 1 after 24 h (Fig. S6C). This suggested a rise in GA levels consistent with GA3ox expression was occurring in KAR 1 treated seeds.
The relatively few changes in gene expression we observed implied that KAR 1 enhances GA synthesis via GA3ox1 and GA3ox2 but does not directly affect ABA pathways. To assess these predictions, we quantitated changes in the levels of ABA and GAs during imbibition of PD seed in response to KAR 1 (Fig. 4B). ABA levels declined substantially during the first 48 h of imbibition, but were unaffected by KAR 1 . Interestingly, we did not detect a dramatic rise in GA 4 levels predicted by GA3ox and CP1 expression levels at 48 h. At 24 h, GA 4 levels were the same in control and KAR 1 -treated seed, and at 48 h there was a statistically significant (Student's paired t-test, p < 0.02), although small (10%) increase in GA 4 induced by KAR 1 .
We examined the transcript levels of GA3ox1, GA3ox2, and CP1 in PD seed imbibed for 24 h to compare the expression changes observed with KAR 1 to other germination stimulants. KAR 1 , KAR 2 , and KAR 3 , but not KAR 4 , induced expression of GA3ox1, GA3ox2, and CP1. The degree of upregulation of these genes corresponded to the effectiveness of each treatment on stimulating germination (Fig. 4C). GR-24 produced a slight enhancement of GA3ox expression, but did not result in CP1 upregulation, at least at this time point. Exogenous GA 4 treatments produced the expected transcriptional effects: GA3ox1, which is feedback inhibited, was downregulated while GA3ox2 was unaffected, and GA-responsive CP1 was strongly induced. KNO 3 was particularly effective at inducing GA3ox and CP1 transcripts, which may explain its broad effectiveness in Arabidopsis as a dormancy breaking treatment.
KAR 1 requires light to enhance Arabidopsis germination. Moisture and light are minimal requirements for normal Arabidopsis seed germination. We found that karrikin could not replace the light requirement (Fig. 5A). Under continuous light KAR 1 induced nearly complete germination of PD Ler seed within 7 d ( Fig. 2A), but caused minimal germination (2%) of PD seeds incubated in the dark after a 2 h initial white light exposure. AR seed was much more responsive than PD seed to the early light exposure, but did not achieve maximal germination without KAR 1 . When the early light treatment was reversed by a far-red (FR) light pulse prior to dark incubation, no germination was observed for KAR 1 -treated seeds regardless of seed dormancy state. Light induces GA synthesis in seeds, and exogenous GA is sufficient to induce germination of Arabidopsis seeds in the dark. To determine whether KAR 1 action requires light because GA synthesis is not triggered, we tested the germination of GA-treated seeds in the presence and absence of KAR 1 during dark incubation. GA supplements were not sufficient to restore KAR 1 promotive effects, and KAR 1 had little or no effect on seed sensitivity to exogenous GA (Fig. 5A). It is also interesting to note that PD and AR seed had nearly equivalent responses to GA after FR reversal of the early light exposure. Thus light, but not dormancy loss through afterripening, enhances seed sensitivity to GA.
To further examine the relationship of light to KAR 1 effects on germination, we tested the expression of the KAR 1 -responsive genes GA3ox1 and CP1 under the same conditions. Interestingly, KAR 1 stimulated GA3ox1 expression in AR seed in the dark even after FR exposure (Fig. 5B). However, the levels of GA3ox1 expression were dramatically enhanced by the combination of light and KAR 1 in AR seeds. CP1 expression showed a similar trend (data not shown). Notably, the strong induction of GA3ox1 occurred only in the treatment producing maximum dark germination. Thus while KAR 1 can upregulate GA3ox1 expression independently of light, its effects are insufficient to induce germination in the absence of light.

Discussion
Karrikins are a novel family of plant growth regulators that impact key processes for a broad range of angiosperms. However, very little is known concerning the potential mechanisms of karrikin action in germination or seedling establishment. We provide the first demonstration that three karrikins, KAR 1 , KAR 2 , and KAR 3 , promote germination of Arabidopsis seed. At high concentrations, the structurally related synthetic strigolactone GR-24 enhanced germination of two karrikin-responsive species. However, KAR 1 was completely inactive on the smoke water and strigolactone-responsive parasitic weed triggering Orobanche germination. These results indicate that karrikins and strigolactones are not interchangeable and may act via different mechanisms. The level of GA3ox induction corresponded with the efficacy of these germination stimulants (KAR 2 > KAR 1 > KAR 3 > GR-24). However, it cannot be assessed from GA3ox and CP1 expression alone whether karrikin and strigolactone signals are perceived and transduced via common pathways, as both stimulants may ultimately enhance GA synthesis as part of the germination process.
KAR 1 was not equally effective across all tested Arabidopsis ecotypes. Different depths of primary seed dormancy in these lines may provide at least a partial explanation for this observation. A progressive receptiveness to germination promoting factors (nitrates, stratification) has been observed during afterripening of Arabidopsis Cvi seed, indicating dynamic capacities for seed response to stimuli (Finch-Savage et al., 2007). Similarly, KAR 1 may be an ineffective stimulant under certain dormancy states. As an example, Brassica tournefortii germination is particularly sensitive to KAR 1, but some seed collections require several months of afterripening to become responsive (Stevens et al., 2007). As seeds in the soil seed bank cycle between dormancy states in response to afterripening or environmental stimuli, limited windows of opportunity for karrikin action may be created. In support of this, smoke treatments to enhance seed bank germination have marked differences in effectiveness at specific times of the year (Rokich and Dixon, 2007). Natural variation provides another likely explanation for variable KAR 1 responses. Here we do not simply refer to variation in the depth of established seed dormancy, but in the mechanisms by which dormancy is broken. Light, cold, and nitrate commonly promote germination across Arabidopsis ecotypes, but the contribution of each signal toward germination commitment can vary. For instance, polymorphisms in the photoreceptor phytochrome B have been identified as a source of natural variation in light responses (Filiault et al., 2008). In consideration of the range of KAR 1 effectiveness even within a single species, it would not be surprising to discover that karrikins activate germination under specific conditions for many more taxa than are currently known to be responsive. Uncovering crosstalk between karrikins and phytohormones is important for determining a mechanism for karrikin promotion of germination. KAR 1 was ineffective at overcoming inhibition of germination by exogenous ABA or high endogenous ABA signaling. While high concentrations of GA 3 are often effective at stimulating germination of KAR 1responsive species, there has been no direct evidence for GA-mediated KAR 1 signaling (Merritt et al., 2006;Daws et al., 2007;Stevens et al., 2007). We demonstrate that KAR 1 requires GA synthesis to induce germination, as both ga1-3 and ga3ox1 ga3ox2 mutants were unresponsive. KAR 1 did not affect seed sensitivity to GA, either in ga1-3 or in darkincubated wildtype.
In support of the germination phenotypes, KAR 1 did not affect transcript abundance of the majority of genes involved in ABA and GA biosynthesis and catabolism, but did induce GA3ox1 and GA3ox2. It is difficult to assess whether GA3ox induction by KAR 1 is a cause or a result of the seed's commitment to germination. As GA3ox1 expression is influenced by cold, light, and GA levels, it may serve as a signal integration point with a direct effect on germination. While KAR 1 enhancement of germination requires both light and GA biosynthetic capacity, it cannot be concluded that KAR 1 acts solely or directly through enhancement of light-induced GA3ox transcription.
The abundance of ABA during imbibition was unaffected by KAR 1 , while GA 4 levels were only slightly increased. As dormancy loss has been reported to result in a faster decline of ABA levels during seed imbibition (Ali-Rachedi et al., 2004;Millar et al., 2006), and afterripening leads to enhanced GA sensitivity (Karssen et al., 1989), KAR 1 does not overcome seed dormancy in a similar manner. Although we had anticipated changes in hormone levels prior to initiation of germination, these results correspond with a similar experiment in which strong induction of GA3ox transcripts preceded germination, but a rise in GA 4 levels was concomitant only with radicle emergence (Ogawa et al., 2003). Therefore a substantial rise in total seed GA 4 levels may be a result of a seed's commitment to germination, rather than a cause. As there is evidence for spatial separation of transcripts for early and late phases of the GA biosynthetic pathway in embryonic tissues (Yamaguchi et al., 2001), it would be interesting if production of active GAs is restricted to a small 'decision' sector of the seed prior to initiation of germination and thus initially contributes little to the overall seed GA 4 abundance.
Alternatively, the initial suppression of GA20ox1 by KAR 1 may restrict the GA precursor supply during the first 24 h of imbibition.
The broad conservation of karrikin perception, even among taxa not obviously subject to selective pressures from a 'fire-prone' environment suggests several intriguing hypotheses. First, karrikins may be generated via other mechanisms than fire. A biotic source such as bacteria or fungi, or the slow chemical breakdown of organic matter at the soil surface could provide alternative sources of karrikin. Second, there may be a strong selective advantage for species that have maintained a karrikin signaling system even in ecosytems with rare fire events. Third, karrikins may be endogenous plant hormones that await identification. Our demonstration of a karrikin response in Arabidopsis seed germination opens the door for a genetic approach to addressing these possibilities.

Plant growth and germination assays
Arabidopsis thaliana were grown in soil under continuous white light, 22 C, 60% RH conditions. Harvested plants were dried for 4-7 d under ambient conditions in paper bags.
Seeds pooled from multiple parent plants (>10), were then cleaned and either stored at -80 C to preserve primary dormancy. Cryostorage had no obvious effect on seed viability or germination. Afterripened seed was maintained in the dark at room temperature. For germination assays, seeds were surface sterilized for 5' with inversion in 70% EtOH, 0.05% Triton X-100, rinsed with 70% EtOH, rinsed again with 95% EtOH, and rapidly dried on filter papers in a sterile laminar flow cabinet. Sterilization treatment had no effect on germination. Sterilized seeds were sprinkled onto 0. (Sigma, MeOH), and ACC (Sigma, water). Karrikins were synthesized as described previously (Flematti et al., 2007). Immediately after plating, plates were sealed with gaspermeable Leukopor tape and transferred to continuous light (~100 μE), 20 C conditions, and 25 seed sectors were outlined prior to germination. Germination, defined as emergence of the radicle, was assessed for three replicates each of 100 or 150 seeds (300 or 450 seeds total/treatment/time point) from one large seed batch per genotype. Average germination and standard error across the three replicates is shown. Similar trends from multiple independent seed batches were consistently observed for Ler and ga1-3. Far-red LEDs (L735-04AU, Epitex) were used to supply FR treatment where indicated. FR LED light intensity (6 μE) was determined by Warsash Scientific EPP2000 spectrometer with irradiance calibration. The ga1-3 mutant required 2-3 spray treatments of 10 μM GA 3 to recover fertility, but all other mutants were grown without additional treatments.
Orobanche minor seeds (collected from King's Park, Perth, Western Australia) were surface sterilized and preconditioned on filter paper dampened with 0.5 mL of water for 2 weeks at 20 C in the dark. A second filter paper was then added to cover the seeds and dampened with 0.5 mL of 2X aqueous germination stimulant. Germination was scored for three replicates each of 75 seeds after 7 d of dark imbibition at 20 C.
Brassica tournefortii (collected from Meckering, Western Australia) was sown on 0.8% agar supplemented with 1000X methanolic stocks of KAR 1 or GR-24. Germination for 3 replicates each of 50 seeds was assayed after 7 d imbibition in the dark at 20 C.

qRT-PCR analysis
Imbibed seed samples (50 or 80 mg pre-imbibition weight) were frozen in liquid nitrogen and stored at -80 until processing. RNA was isolated using the RNAqueous Kit with Plant RNA Isolation Aid (Ambion) and LiCl precipitation to aid in removal of contaminating polysaccharides. RNA integrity was assessed by BioAnalyzer (Agilent).
RNA was treated with Turbo DNA-free (Ambion) and subsequently converted to cDNA using iScript cDNA Synthesis Kit (BIORAD). qRT-PCR was performed on a Roche LC480 using LightCycler 480 SYBR Green I Master (Roche). Cycle conditions were 95 www.plantphysiol.org on August 19, 2017 -Published by Downloaded from Copyright © 2008 American Society of Plant Biologists. All rights reserved. C for 10'; 45 cycles of 95 C for 20", 60 C for 20", and 72 for 20"; followed by melt curve analysis. When possible, primer pairs were designed across introns. Most primer pairs were designed by AtRTPrimer (Han and Kim, 2006), except NCED6 (Millar et al., 2006).
Primer sequences are listed in Table S1. Melt curve analysis of real-time PCR products was always performed to ensure clean amplification. Cp values were calculated under high confidence. Two independently prepared biological replicates per treatment/time point were examined in all studies, with at least two technical replicates of each real-time PCR reaction. The average Cp value of two technical replicates was used to calculate expression relative to an internal reference gene adjusted by primer efficiencies. We tested several potential reference genes identified by Czechowski et al. (Czechowski et al., 2005), and for our analyses chose At5g46630, a clathrin adaptor complex subunit which we term CACS. The average relative expression and standard error for the two biological replicates are shown. Levels of relative expression in dry seed or the water control were typically adjusted to 1, and all other values scaled accordingly. For the PD Ler imbibition time course, two of the three independent seed batches used for ABA/GA quantitation were used. All three seed batches had germination rates similar to that shown in Figure 2B, and no radicle emergence occurred by 48 h imbibition.

Quantitation of ABA and GA
Per time point, three replicates of 300 mg of seeds (pre-imbibition weight) from independent PD Ler seed batches were pulverized in liquid nitrogen using a ball mill.
Powder was extracted with 5 mL 80% (v/v) of methanol / water (with 0.1% acetic acid (AA)) with the following deuterated internal standards added: 10 ng/g of [17,  dissolved in 15% methanol/0.1 % AA water (10 mL) and was applied directly to a preconditioned C 18 Sep-Pak (Waters, 1g) cartridge. The retained material was rinsed with 15% methanol/0.1 % AA water (10 mL) and the GA's and ABA were eluted with 80% methanol/0.1 % AA water (10 mL). The 80% methanol fraction was evaporated to dryness under reduced pressure at 35°C and the sample was dissolved in methanol (2 mL) and methylated with excess ethereal diazomethane. The sample was dried and dissolved in 20% methanol/0.1 % AA water (1 mL) and separated by HPLC (Hewlett-Packard 1050 HPLC). The sample was injected (1 mL) onto a C 18 reversed-phase column     dry, 24 hour, and 48 hour +/-1 μM KAR 1 imbibed seed. y-axis indicates ng/g of preimbibition seed weight. C) Expression of GA3ox1, GA3ox2, and CP1 in PD Ler seed after 24 h imbibition in the light at 20 C on water, 1 μM KAR 1 , KAR 2 , KAR 3 , or KAR 4 , 10 μM GR-24, 10 μM GA 4 , or 10 mM KNO 3 . Relative expression values for each gene in seeds imbibed in water was set to 1, and other expression values were scaled accordingly.  The ecotype for each mutant is indicated. Special conditions (Treatment) are noted, i.e. in the presence (ABA/GA) or absence (-) of exogenous hormone, or dormancy state (PD/AR) of the seed. Where not noted, all seed was of the primary dormancy level within a few days of harvest. KAR 1 response in germination is classified as '-' (0-10%), '+' (10-40%), '++' (>40%).

Allele
Ecotype Treatment KAR 1 Response Figure   ABA