Michael Bunsick

Zhenhua Xu

Gianni Pescetto

George Ly

Jenna Hountalas

François- Didier Boyer

Christopher S P Mcerlean

Julie D Scholes

Shelley Lumba
email: shelley.lumba@utoronto.ca

HTL/KAI2 signalling substitutes for light to control plant germination

Deciphering signalling pathways is essential to understanding how organisms respond to environmental cues but elucidating how these signalling pathways evolve in new environments is less clear. 1,2 Most plants, for example, monitor multiple environmental cues to optimize the time and place to germinate. Some root parasitic plants, however, germinate in response to small molecules like strigolactones (SLs) emanating from host roots 3,4 whilst a number of ephemeral weeds germinate in response to chemicals called karrikins (KARs) released after a forest fire. 5,6 Although these species represent distinct clades, they use the same HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) signalling pathway to perceive strigolactones or karrikins, which suggests convergent evolution. 3,5 Because specialist lifestyles are derived traits, it is not clear if HTL/KAI2 signalling in these species evolved from a specific germination-signalling pathway or whether this pathway had other functions that were co-opted for specialist germination circumstances. Here, we show HTL/KAI2 signalling in Arabidopsis bypasses the light requirement for germination. In part, this is because the HTL/KAI2 downstream component, SMAX1 impinges on PHYTOCHROME INTERACTING FACTOR 1/PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIF1/PIL5)-regulated hormone response pathways conducive to germination. We identified Arabidopsis accessions that can germinate in the dark, which had altered expression of HTL/KAI2 signalling components, suggesting that divergence in this signalling pathway occurs in nature. Moreover, Arabidopsis HTL/KAI2-regulated gene signatures were observed in germinating Striga seed. The ability of HTL/KAI2 signalling to substitute for light advances an explanation for how some specialist plants evolved their underground germination behaviour in response to specific environments. (248 words) RESULT AND DISCUSSION Parasitic plants like Striga hermonthica (Striga) and some pyroendemic plants like Brassica tournefortii germinate underground and do not require a light stimulus. 5,7,8 In the lab, Striga and B. tournefortii seeds are typically germinated in the dark by adding SLs or KARs, respectively. 7,8

Although SLs and KARs are distinct small molecules, they appear to signal through a class of related 𝛼/𝛽 hydrolase receptors called HTL/KAI2 in these species. 5,9,10 In Arabidopsis thaliana (Arabidopsis), mutations in the HTL/KAI2 (AtKAI2) receptor were originally identified through

3 genetic screens as positive regulators of hypocotyl light responses and KARs enhance the effect of light on germination and hypocotyl growth. [START_REF] Qiu | Identification of candidate virulence loci in Striga hermonthica, a devastating parasite of African cereal crops[END_REF][START_REF] Nelson | Karrikins discovered in smoke trigger Arabidopsis seed germination by a mechanism requiring gibberellic acid synthesis and light[END_REF][START_REF] Nelson | Karrikins enhance light responses during germination and seedling development in Arabidopsis thaliana[END_REF] These results suggested that limiting light conditions might uncover new roles for this receptor with respect to germination. Generally, Arabidopsis germination assays involve sterilizing seeds in the light and plating them onto nutrient rich agar. To create conditions more reflective of underground germination, we plated dry, unsterilized, and after-ripened seeds of the common laboratory wild type, Col-0, onto water agar plates and placed them directly in the dark. Imbibed Col-0 seeds did not germinate under these conditions but did show a slow and steady increase in germination over time under low light exposure (0.3 µE) (Figure 1A). A loss-of function AtKAI2 allele (htl-3) also did not germinate in the dark but low light exposure resulted in minimal germination suggesting AtKAI2 signalling contributes to light-dependent germination (Figure 1A). Three additional lines of evidence supported this premise. First, a previously characterized Arabidopsis line expressing the Striga ShHTL7 receptor (ShHTL7OX), which strongly activates the AtKAI2 signalling pathway, [START_REF] Bunsick | SMAX1-dependent seed germination bypasses GA signalling in Arabidopsis and Striga[END_REF] germinated in the dark on nanomolar levels of a racemic mixture of the artificial SL, rac-GR24 (Figures 1B and1C). Second, genetic inactivation of two downstream regulators in KAI2 signalling, [START_REF] Stanga | SUPPRESSOR OF MORE AXILLARY GROWTH2 1 controls seed germination and seedling development in Arabidopsis[END_REF] SUPPRESSOR OF MAX2 1 (SMAX1) and SUPPRESSOR OF MAX2 LIKE 2 (SMXL2), resulted in constitutive dark germination (Figure 1B). Finally, htl-3 Col-0 lines misexpressing KAI2 (AtKAI2OX) germinated in the absence of light at effective KAR2 concentrations (EC50) in the low nanomolar range (Figures 1B and1D). Consistent with this, expression of genes (DLK2, KUF1, BBOX20) known to be induced by activation of AtKAI2 [START_REF] Nelson | F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana[END_REF] increased in KAR2-treated AtKAI2OX seed and these genes were constitutively expressed in smax1-2 single and smax1-2 smxl2-1 double mutant (Figure 1E).

The germination of AtKAI2OX seed at low nanomolar KAR2 concentrations in the dark is somewhat unexpected because adding KAR2 to AtKAI2OX seed in the light only partially rescued seed germination in seeds depleted for the germination stimulant, gibberellin (GA). [START_REF] Bunsick | SMAX1-dependent seed germination bypasses GA signalling in Arabidopsis and Striga[END_REF] It is thought this is because KAR2 does not fully activate the AtKAI2 signalling pathway. Possibly KAR2 is metabolized to a more potent ligand in the dark or light increases its degradation. It is also possible that dark is a less complex environment that sensitizes Arabidopsis germination to AtKAI2 signalling. Surprisingly, AtKAI2OX seed did not germinate in response to rac-GR24 in the dark although the 2'S enantiomer of this racemic mixture preferentially activates AtKAI2 in other assays [START_REF] Flematti | Stereospecificity in strigolactone biosynthesis and perception[END_REF] (Figure S1A). When each enantiomer was tested separately, however, the 2'S-GR24 germinated AtKAI2OX seed in the dark whilst 2'R isomer had no effect (Figure S1B). This suggested that the 2'R isomer was not only inactive but also inhibited 2'S-GR24 action in dark germination assays. Because rac-GR24 is active in many SL assays in the light, the clarity of enantiomeric specificity in dark germination may again reflect the simplicity of these conditions with respect to germination cues. Perhaps light establishes metabolic conditions that modify 2'R isomers thereby removing its inhibitory effects.

The transition from dark-to light-driven development (photomorphogenesis) requires light activated photoreceptors to inhibit specific negative regulators called PHYTOCHROME INTERACTING FACTORS (PIFs). 18 PIF1 (also known as PIL5 and BHLH105) is the major repressor of photomorphogenic germination and loss-of-function pif1-1 mutant seed germinate in the dark. [START_REF] Oh | PIL5, a phytochromeinteracting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana[END_REF] This pif1 constitutive dark germination phenotype was epistatic to the decreased light sensitivity of htl-3, indicating that PIF1 acts genetically at or downstream of AtKAI2 (Figure 2A).

Because of its repressive role in light responses, a simple model is PIF1 function requires SMAX1/SMXL2 action. Consistent with this, transcripts of direct targets of PIF1 repression, HECATE 2 (HEC2) and LONG HYPOCOTYL IN FAR RED 1 (HFR1), 20,21 were highly induced upon activation of a KAI2OX line as were expression levels of photosynthetic genes (LHC, RBC, PS1 and PSII) (Figure 2A).

PIF1 also represses transcription of a seed specific regulator, SOMNUS (SOM), which influences gene expression of abscisic acid (ABA) and GA synthesis [START_REF] Kim | SOMNUS, a CCCH-type zinc finger protein in Arabidopsis, negatively regulates light-dependent seed germination downstream of PIL5[END_REF] . To functionally test the roles of these hormones on AtKAI2 signalling, we fully activated the pathway by exposing ShHTL7OX seed to rac-GR24 and the addition of either ABA or paclobutazol (PAC), a GA biosynthetic inhibitor to decrease GA levels. Both conditions decreased the ability of ShHTL7OX seed to germinate (Figure 2B,C). Whilst PAC completely suppressed germination of ShHTL7OX seed on rac-GR24, the addition of ABA did not inhibit seed coat rupture but did inhibit further development (Figure 2B,C). This is consistent with observations that ABA alone cannot increase a DELLA repressor of GA signalling, REPRESSOR OF GA LIKE 2 (RGL2), to levels required to inhibit seed coat rupture. [START_REF] Piskurewicz | The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity[END_REF] Consistent with the positive role of GA and negative role of ABA in germination, we found GA-and ABA-responsive genes increased and decreased respectively in KAR2-treated AtKAI2OX seed (Figures 2B,C). Moreover, we found AtKAI2 signalling affected expression of SOM-regulated target genes involved in GA production (GA3ox1, GA3ox2, GA2ox2) 5 and SOM-regulated genes involved in reducing ABA levels (ABA1, NCED6, NCED9) (Figures 2B,2C).

Finally, when we introduced a mutation that confers ethylene insensitivity, ein2-1 (ETHYLENE INSENSITIVE 2) into an ShHTL7OX line, it suppressed the dark germination phenotype (Figure 2D). Moreover, activation of AtKAI2 increased transcript levels of genes involved in ethylene synthesis (ACS7, ACO1, ACO2) and affected expression of genes involved in ethylene action (Figure 2D). Interestingly, ethylene partially facilitates dark to light seedling transition by working in concert with PIF1 to induce genes involved in chlorophyll synthesis. [START_REF] Zhong | EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings[END_REF] Consistently, both PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA) and PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B (PORB) transcripts are induced by AtKAI2 signalling (Figure 2D). In this context, ethylene is a positive regulator of germination in a variety of species that is thought to inhibit ABA action. [START_REF] Corbineau | Ethylene, a key factor in the regulation of seed dormancy[END_REF] Ethylene also circumvents the requirement of SL for Striga germination. [START_REF] Sugimoto | Germination strategy of Striga hermonthica involves regulation of ethylene biosynthesis[END_REF][START_REF] Tsuchiya | PARASITIC PLANTS. Probing strigolactone receptors in Striga hermonthica with fluorescence[END_REF] Similarly, activation of the KAI2 pathway leads to the induction of ethylene biosynthesis in Lotus roots. [START_REF] Carbonnel | The karrikin signaling regulator SMAX1 controls Lotus japonicus root and root hair development by suppressing ethylene biosynthesis[END_REF] In summary, it appeared that activating AtKAI2 signalling increased GA and ethylene signalling and decreased ABA signalling. This combination of hormone action, which was necessary for germination in the dark, is also commonly seen during the germination of most plants in the light. [START_REF] See | Interaction of light and hormone signals in germinating seeds[END_REF] This conserved pattern suggested that activation of KAI2/HTL signalling can substitute for light to dial up the correct germination code.

Arabidopsis thrives over a wide number of geographical ranges [START_REF] Mitchell-Olds | Genetic mechanisms and evolutionary significance of natural variation in Arabidopsis[END_REF] , so locally adapted variation in AtKAI2 signaling may contribute to the germination behaviour of some Arabidopsis populations. Because natural variation influences the Arabidopsis germination response to KARs in the light [START_REF] Nelson | Karrikins discovered in smoke trigger Arabidopsis seed germination by a mechanism requiring gibberellic acid synthesis and light[END_REF] , we decided to screen the ability of Arabidopsis seed collections from different geographical areas (accessions) to germinate in the dark. Thirty-seven accessions showed varying degrees of dark germination indicating natural divergence of this trait (Figure 3A). Of the three top accessions (Do-0, Copac-1, and Ara-1), Ara-1 showed elevated AtKAI2 expression compared to the Col-0 accession (Figure 3B and3C). To this point, Ara-1 seed mimicked smax1-2 by germinating under levels of soil that inhibited Col-0 germination (Figure 3D). Moreover, transcript profiles of dark germinating Ara-1 seeds and AtKAI2OX Col-0 seed treated with KAR2 were highly correlated (r 2 =0.86), suggesting that these genotypes used similar genetic programs to germinate in the dark (Figure 3E). The increased expression of AtKAI2 in Ara-1 seeds, however, only partially explains its dark germination phenotype, as this accession does not require KARs 6 for dark germination. It has been hypothesized that Arabidopsis makes an endogenous ligand dubbed AtKAI2 ligand (KL), that activates AtKAI2. [START_REF] Conn | Evidence that KARRIKIN-INSENSITIVE2 (KAI2) Receptors may perceive an unknown signal that is not karrikin or strigolactone[END_REF] Natural variation in KL levels in Arabidopsis accessions could contribute to a spectrum of dark germination behaviours but this variation would have to be coupled with increased AtKAI2 expression.

Interestingly, although dark-germinated Ara-1 and activated AtKAI2OX lines had very similar transcript profiles, expression of the AtKAI2 homolog, DWARF14-LIKE2 (DLK2) was reduced in Ara-1 but was highly induced in KAR2-treated AtKAI2OX seed (Figure S2A). The function of DLK2 is unknown but it does metabolize 2'S-GR24 [START_REF] Végh | Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs[END_REF] suggesting it may have roles in KAI2 signalling homeostasis. DLK2 loss-of-function seeds did not germinate in the dark but they were sensitized to germinate under low light compared to wild type seed (Figure S2B). Furthermore, kai2 mutants were epistatic to dlk2 indicating DLK2 functions genetically at or upstream of the receptor (Figure S2B). If DLK2 were involved in metabolizing KL, then loss of DLK2 function may result in higher levels of KL. Whatever the case, altered expression of both AtKAI2 and DLK2 in Ara-1 seeds suggested that this dark germinating accession, Ara-1 has aberrant AtKAI2 signalling compared to Col-0.

An Arabidopsis KAI2-dependent transcriptional signature for germination in the dark provides a window for looking at the germination code of experimentally intractable specialist plants like Striga. The ability of AtKAI2 signalling to bypass light in Arabidopsis germination predicted that germinating Striga seed may show similar profiles. Indeed, many Striga gene homologs of Arabidopsis light-, GA-, ABA-and ethylene-responsive genes showed similar patterns of gene expression in germinating Striga seeds (Figure 4). Like KAR2-treated AtKAI2OX seed, SL stimulated expression of light-induced genes like LHCP, Rubisco, and components of Photosystems I and II in dark germinated Striga seed (Figure 4). In addition, the ability of SL to repress expression of ABA-induced genes is consistent with the observation that Striga seeds are capable of germinating on high concentrations of ABA. [START_REF] Fujioka | Aberrant protein phosphatase 2C leads to abscisic acid insensitivity and high transpiration in parasitic Striga[END_REF] Interestingly, the expression of Arabidopsis and Striga genes involved in GA and ABA synthesis did not strongly correlate although we know levels of these hormones change in response to rac-GR24-treated Striga seed (Figure 4). [START_REF] Toh | Thermoinhibition uncovers a role for strigolactones in Arabidopsis seed germination[END_REF] This suggested that SL regulates GA and ABA levels differently in Striga versus Arabidopsis. By contrast, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 7 (ACS7), a key enzyme in ethylene synthesis that is AtKAI2inducible [START_REF] Carbonnel | The karrikin signaling regulator SMAX1 controls Lotus japonicus root and root hair development by suppressing ethylene biosynthesis[END_REF] is upregulated in both species (Figure 4A). In summary, HTL/KAI2 signalling dials up a correct code of light, GA, ABA, and ethylene responses that favour germination. This germination code appears to be conserved between a generalist plant like Arabidopsis which normally uses light as a germination cue and a specialist species like Striga which uses a small molecule cue.

Our analysis of HTL/KAI2 signalling pathway gives some insight into the evolution of germination signalling pathways in Striga. Adapting a signalling pathway for a particular process broadly follows scenarios where either the pathway is established for its function by natural selection (historical genesis) or the pathway, which has other functions, is co-opted for new uses (current utility). [START_REF] Gould | Exaptation-a Missing Term in the Science of Form[END_REF] Although HTL/KAI2 signalling has been implicated in a variety of developmental responses, our findings suggest its role in Striga germination is the product historical genesis rather than current utility. In generalist plants like Arabidopsis, SMAX1/SMXL2 inhibits germination in the absence of light similar to the repressive role of PIF1. Unlike PIF1, however, which is modulated by a ubiquitous environmental cue, light, SMAX1/SMXL2 levels are dependent on specific small molecules generated by distinct environmental conditions. 3,5 Because SMAX1/SMXL2 function is necessary to repress dark germination, any selective pressures affecting their levels will dominate germination behaviours. We know substituting ShHTL receptors for AtKAI2 changes the small molecules that activate signalling and the magnitude of signal through the pathway. 10 We also know only a few mutations are required to change a KAR receptor into an SL receptor. [START_REF] Arellano-Saab | Detection of Parasitic Plant Suicide Germination Compounds Using a High-Throughput Arabidopsis HTL/KAI2 Strigolactone Perception System[END_REF] It is therefore easy to envision how selective pressures on HTL/KAI2 receptors contributed to the evolution of a conserved germinationsignalling pathway. Our results emphasize that defining signalling pathways by their ligands and receptors can be confounding when these signals influence conserved downstream effectors that are also modulated by other signalling pathways. We suggest when looking at fundamental processes such as germination it is more informative to reference pathways by their bottom effectors like SMAX1. S1. Boxplot analysis represents three sets of biological replicates. Heatmap represents select ethyleneregulated genes in response to rac-GR24. The scale bar represents expression values on rac-GR24 relative to the control that have been log2-transformed. The heatmaps depicts fold-change at 24h.

See Table S2 for gene lists and values.

Figure 3. An A. thaliana accession, Ara-1 germinates in the dark and misexpresses AtKAI2.

(A) The germination frequency of a collection of Arabidopsis accessions under dark conditions.

The Ara-1 accession is highlighted in blue. The list of accessions is provided in Table S3. S2 for gene lists and values.

Materials and Methods

Plant Materials and Growth Conditions 14

All Arabidopsis strains, except the accession collection, are in the Col-0 background. We have previously described the generation of KAI2OX, ShHTL7OX, and htl-3 dlk2-1 strains. [START_REF] Pires-Dasilva | The evolution of signalling pathways in animal development[END_REF]2 The smax1-2 mutant and smax1-2 smxl2-1 double mutant were gifts from Dr. David Nelson. 3,4 The Arabidopsis accessions from the 1001 genomes project were a gift from Dr. David Guttman. The pif1-1 and ein2-1 mutants were obtained from the Arabidopsis Biological Resource Center. Striga hermonthica seeds used in this study were collected from infested maize from the Kibos region in Kenya. All genotypes used in this study are described in the Table S3. All plants were grown under 24h, continuous white light at room temperature. Seeds were allowed to ripen for at least six months before use.

Germination Assays

Our germination assays were performed on 0.8% agar plates supplemented with 10 µM benomyl.

Stocks of GR24, KAR2, and GA3, were prepared in DMSO; paclobutrazol (PAC) was prepared in EtOH; and ABA was prepared in MeOH. For each experiment, we prepared a working stock 1,000 times the experimental concentration. Each stock was diluted 1:1,000 in 0.8% agar to a final solvent percentage of 0.1 (v/v). For all germination assays, the seeds were at least 6 months old.

We emphasize that seeds ripened for less than 6 months have elevated background germination.

For dark germination experiments, the seeds were sprinkled onto plates within 1 minute and then immediately wrapped in 3 layers of aluminum foil. For low light experiments, seeds were sprinkled onto plates and exposed to (0.3 µE) for specified durations, then wrapped in three layers of aluminum foil. Seeds were then stratified for 4 d at 4 °C and then placed in a dark cabinet at room temperature. After 5 days, germination was scored using radicle emergence. Dose response curves and effective concentration (EC50) values were generated with SigmaPlot 11.0.

Accession Screen and Soil Germination Experiment

For germination experiments on agar, 104 accessions from the 1001 genome project were individually plated on 0.8% agar water medium supplemented with 10 µM benomyl. The plates were wrapped in three layers of aluminum foil within 1 minute of plating. After the seeds were plated, they were stratified for 4 d at 4°C and then placed in a dark cabinet at room temperature.

After 5 days, germination was scored using radicle emergence.

For germination at different levels of soil, 0.6 g of damp soil was added to a 90 mm Petri dish.

Approximately 10 mg of dry seeds per genotype were sprinkled on top. Damp soil was then sprinkled on top of the seeds to various levels. Plates were wrapped in three layers of aluminum foil and stratified at 4°C in the dark for 4 days. After this, the foil was removed from the top the plates and each plate was placed at room temperature in the light (15 µE) for 5 days.

Double mutant construction

Putative pif1 htl-3 double mutants were identified from F2 offspring of the cross between pif1 and

Quantitative Gene Expression Analysis by RT-qPCR

Tissue Preparation and RNA Extraction

For DLK2, KUF1, and BBX20 expression, seeds were sprinkled on 0.8% agar plates supplemented with vehicle or 0.5 µM KAR2 and immediately wrapped in aluminum foil. For KAI2 expression, seeds were plated on 0.8% agar plates and immediately wrapped in foil. The seeds were stratified for 4 d at 4 °C and then placed in a dark cabinet at 25-26 °C. After 24 h, the seeds were flashfrozen in liquid N2. Seeds were then ground to a fine powder form using a frozen mortar and pestle.

From this powder, total RNA was extracted in hot phenol (80 °C) using the phenol-chloroform extraction protocol, followed by treatment with DNase I. 5 The samples were then purified with the Monarch® RNA Cleanup Kit (New England Biolabs).

Reverse transcription and qPCR

One μg of RNA was reverse transcribed using the LunaScript RT SuperMix Kit (New England Biolabs) according to the manual. One μl of 10-fold diluted cDNA was used in the qPCR. Primers (Supplementary Dataset) were designed using Beacon Designer 8 (PREMIER Biosoft International). Real-time PCR was performed using Luna Universal qPCR Master Mix (New England Biolabs) and quantified on a Bio-rad CFX96 Real-time Detection System (Bio-rad). Each reaction was performed in triplicate for each biological replicate. Expression levels were normalized against the expression of endogenous control gene, AtACTIN8, and were calculated using 2 -ΔΔCt method. 6 All the expression data are presented as fold change value.

Transcriptome studies

Tissue preparation and RNA extraction from Arabidopsis thaliana

AtKAI2OX seeds were plated on 0.8% agar plates supplemented with either 0.5 µM KAR2 or the vehicle; Ara-1 seeds were plated on a 0.8% agar plate supplemented with the vehicle. Each condition was prepared in triplicate. The plates were then immediately wrapped in aluminum foil and stratified for 4 d at 4 °C and then placed in a dark cabinet at room temperature. After 24h, the seeds were flash-frozen in liquid N2. Seeds were then ground to a fine powder form using a frozen mortar and pestle. From this powder, total RNA was extracted in hot phenol (80 °C) using the phenol-chloroform extraction protocol.

Transcript profiling and data analysis from Arabidopsis

The triplicate total RNA samples used for single-end 1X75bp RNA-Seq on a NextSeq500 sequencing platform (Illumina). After examining the data quality using FastQC (v0.11.5), the fastq data containing the raw reads for each sample was trimmed and filtered using htStream (v1. Seeds were washed from the plates onto clean GF/A filter paper. They were then placed in Eppendorf tubes and frozen in liquid nitrogen.

Total RNA was extracted using a RNeasy plant mini kit (Qiagen). Striga seeds were initially ground to a powder by placing two sterile 5 mm tungsten ball bearings into the Eppendorf tubes and shaking at 25 hz for 45s followed by an additional 60s, using a tissue lyser (Qiagen). Samples were eluted in 30 µl of dH2O. RNA was then extracted following the RNeasy plant mini kit protocol. RNA was eluted in 30 µl dH2O and the concentration determined using a Nanodrop-1000. RNA samples (3 µg) were treated with dsDNase (Thermocientific) to remove genomic DNA

38 12 karrikin

 12 receptor to a strigolactone receptor. Proc. Natl. Acad. Sci. U S A. 118, e2103175118. doi: 10.1073/pnas.2103175118. 38. Bunsick, M., McCullough, R., McCourt, P., and Lumba, S. (2021). Plant hormone signaling: Is upside down right side up? Curr. Opin. Plant Biol. 63, 102070. doi: 10.1016/j.pbi.2021.102070.

Figure 1 .

 1 Figure 1. Dark germination in Arabidopsis (A) Germination of Col-0 and an AtKAI2 loss-offunction mutant (htl-3) seed in the dark and after discrete low light exposures (0.3 µE). Each point represents three independent biological replicates. Bar=SD (B) Dark germination of Arabidopsis genotypes placed on minimal media (mock or DMSO), 0.5 µM strigolactone (rac-GR24) or 0.5 µM karrikin (KAR2) containing media under dark conditions. (C) Effective concentrations (EC 50 ) of rac-GR24 required to germinate 50% of Col-0, htl-3 and an ShHTL7OX line. Each point represents three independent biological replicates. Bar=SD. (D) Effective concentrations (EC 50 ) of KAR2 required to germinate 50% of Col-0, htl-3, ShHTL7OX and two KAI2 misexpression lines (KAI2OX-A, KAI2OX-B). Each point represents three independent biological replicates. Bar=SD. (E) Expression (RT-qPCR analysis) of KAR-induced genes (DLK2, KUF1, BBOX20) in darkgerminated Arabidopsis seeds on 0.5 µM KAR2. An 18S rRNA gene was used as an internal control. Letters indicate significant differences (P < 0.05, ANOVA with post-hoc Tukey HSD Test). P values, sample sizes and box plot elements are provided in TableS1.

Figure 2 .

 2 Figure 2. Dark germination of activating AtKAI2 signalling lines perturbed in light or hormone signalling. (A) Left panel, representative pictures of pif1-1, htl-3 and pif1-1 htl-3 seed germinating in the dark. Right panel, heatmap of light-regulated genes expression in response to rac-GR24 addition in an ShHTL7OX line. (B) Germination of an ShHTL7OX line on paclobutrazol (PAC). Boxplot analysis represents three sets of biological replicates. Asterisk indicates a significant difference from the mock treatment (P < 0.05, ANOVA with one-sided Fisher's LSD Test). Heatmap represents select GA-regulated genes in response to rac-GR24. (C) Germination of ShHTL7OX seed on ABA. Boxplot analysis represents three sets of biological replicates. Heatmap represents select ABA-regulated genes in response to rac-GR24. (D) Germination of ShHTL7OX seed containing a wild type EIN2 or the loss-of-function ein2-1 allele. Asterisk

  (B) Germination frequency of Col-0 and the top three dark germination lines (Ara-1, Do-1 and Copac-1) in the next generation. Each box represents three sets of biological replicates. (C) AtKAI2 receptor expression of Col-0, Ara-1, Do-1, and Copac-1. RT-qPCR analysis of AtKAI2 expression level relative to its expression in Col-0 seeds imbibed in the dark. An 18S rRNA gene was used as an internal control. Letters indicate significant differences (P < 0.05, ANOVA with post-hoc Tukey HSD Test). P values, sample sizes and box plot elements are provided in Table S1. (D) Representative picture of the germination of Col-0, smax1-2 and Ara-1 seed buried at different depths of soil (1.5 to 4.5 cm). (E) Whole transcriptome comparison of 24h dark-germinating Ara-1 versus KAR2-treated KAI2OX seed. Log2-transformed values for probe responses for Ara-1 and KAI2OX + KAR2 are plotted on the y-and x-axis, respectively. The Pearson correlation coefficient (r 2 = 0.86) is shown (degrees of freedom = 1, P-Value = 0, 99% confidence interval = 1.597281-1.647912). Three sets of independent biological replicates were used for transcriptome studies.

Figure 4 .

 4 Figure 4. Expression of light and hormone regulated genes in germinating KAR2-treated KAI2OX, Ara-1, and Striga seeds. Heatmaps depict Log2 fold-change in gene expression during germination (24h) of KAR2 -treated KAI2OX (left box), Ara-1 (middle box), and Striga (right box) seeds. Partial biosynthetic pathways are shown for each hormone. Hormone-responsive genes are represented in panels. SeeTable S2 for gene lists and values.

htl- 3 ;

 3 ShHTL7OX by first screening for the pif1 dark germination phenotype. Plants were later screened for the htl-3 round rosette leaves and long hypocotyl phenotype. Potential pif1 htl-3 homozygous double mutants were confirmed by genotyping of individual plants by PCR (see below). a Schematic illustration of WT and pif1 T-DNA 436 insertion allele of AtPIF1 gene, as well as the primers 437 for pif1 allele genotyping. 438 b Confirmation of htl-3 allele in the individual pif1 439 htl-3 double mutants. 440 c Confirmation of pif1 allele in the individual pif1 htl-441 3 double mutants. 442 d Confirmation of pif1 allele in the individual pif1 htl-

  Figure 1

  3.0)(UC, Davis) to generate clean reads. For each read, trimming and filtering processes include removing the last base, removing the low-quality fragments, removing 'N's and returning the longest fragments, and discarding reads that are less than 25 bp. Clean reads from each sample were subjected to HISAT2 7 (v2.1.0) for genome mapping using Arabidopsis thaliana TAIR10 genome as reference. Samtools (v1.10) was used to remove the unmapped and multi-loci mapped reads and covert the uniquely mapped reads into a BAM file. Transcript assembly for each sample was performed using StringTie 8 (v2.1.3) against Arabidopsis thaliana Araport11 annotation 9 and transcriptome assemblies from each sample were merged using the merge function in StringTie to generate a combined transcriptome across all sequencing samples to serve as a new reference MSTRG'-tagged gene_id with the TAIR10 gene locus name to facilitate the comparisons of genes and transcripts between different samples. A StringTie provided python script, prepDE.py, was used to extract the read counts matrix from each transcriptome assemble and this counts matrix served as the input data for DESeq2 (v1.18.1).

	transcriptome.	A	customized	python	script,	stringtieGeneIdReplace2.py
	(https://github.com/maplexuci/stringtie_gene_id_replacement) was written to replace the
	StringTie default '

10

The data are found and described in NCBI GEO under GSE161704 entitled, Natural variation in activation of KAI2/HTL pathway promotes germination in the dark in Arabidopsis.

Tissue preparation and RNA extraction from Striga hermonthica

Forty mg aliquots of S. hermonthica seeds (collected from plants parasitizing maize in Kibos, Kenya in 2013) were surfaced sterilised with 10 % bleach for 7 min. Seeds were washed thoroughly with distilled water, placed on moistened filter paper (GF/A, Whatman TM , Buckinghamshire) in Petri dishes and incubated in the dark at 30 o C for 12 days. Seeds were collected at 12 days (prior to the addition of rac GR24) and 3 and 16 h after application of GR24.

Acknowledgements

We thank Dr. David Nelson for the gift of smax1-2 and smax1-2; smxl2-1. We acknowledge the contribution of Dr. Peter McCourt for discussion and feedback on our manuscript. We acknowledge resource centers like the Arabidopsis Biological Resource Center (ABRC) for 20 providing Arabidopsis mutants and accessions as well as the Parasitic Plant Genome Project for publicly available Striga transcriptome datasets. This work was supported by the New Frontiers in Research Fund Exploration program (2018-00118) and the Natural Sciences and Engineering Research Council of Canada (NSERC) in the form of a Discovery Grant (06752), an Accelerator Supplement (507992) and a Research Tools and Instruments grant awarded to S. Lumba.

contamination following the manufacturer's instructions. Samples were then purified and concentrated using a Monarch RNA Cleanup Kit (NEB). RNA samples were quantified and sent to BGI (China) for processing and sequencing.

Transcript profiling and data analysis from Striga hermonthica

Sequencing was performed on a BGISEQ-500 (BGI). Data quality was examined using FastQC (v0.11.5). Clean reads were obtained by trimming adaptors and filtering for quality score. After filtering, reads were mapped to S. hermonthica genome provided by J. Scholes 11 using HISAT2.

Gene expression levels were calculated using RSEM. Gene expression levels were calculated in Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Sequencing data were deposited in NCBI under PRJNA802803, https://dataview.ncbi.nlm.nih.gov/object/PRJNA802803?reviewer=h9qigloj2glj899mome2m5mp la Corresponding A. thaliana genes were identified in S. hermonthica by performing a BLAST analysis against the S. hermonthica genome provided by J. Scholes. 11 S. hermonthica hits were confirmed by reciprocal BLAST to A. thaliana datasets. An e-value cut-off of 1e-20 was applied.

Author Contributions