A Raf-like kinase is required for smoke-induced seed dormancy in Arabidopsis thaliana

Significance

Seed dormancy is an important developmental trait for plants, which ensures seed germination under more favorable spatiotemporal environments. A smoke compound, 3,4,5-trimethylfuran-2(5H)-one, which is called TMB for short, has been identified to inhibit seed germination in a still elusive way. Here, we presented several lines of evidence that TMB inhibits seed germination through the induction of physiological dormancy. Furthermore, by isolating TMB-resistant Arabidopsis mutants, we demonstrated that TMB RESISTANT1, encoding a Raf-like kinase, is critically required for the TMB-induced seed dormancy. These results suggest the presence of a smoke sensory pathway that takes part in the multiple layers of the dormancy regulatory network in higher plants.

Abstract

Plants sense and integrate diverse stimuli to determine the timing for germination. A smoke compound, 3,4,5-trimethylfuran-2(5H)-one (trimethylbutenolide, TMB), has been identified to inhibit the seed germination of higher plants. To understand the mode of action, we examined various physiological and molecular aspects of the TMB-dependent inhibition of seed germination in Arabidopsis thaliana. The results indicated that the effect of TMB is due to the enhanced physiological dormancy, which is modulated by other dormancy regulatory cues such as after-ripening, stratification, and ABA/GA signaling. In addition, gene expression profiling showed that TMB caused genome-wide transcriptional changes, altering the expression of a series of dormancy-related genes. Based on the TMB-responsive physiological contexts in Arabidopsis, we performed mutant screening to isolate genetic components that underpin the TMB-induced seed dormancy. As a result, the TMB-RESISTANT1 (TES1) gene in Arabidopsis, encoding a B2 group Raf-like kinase, was identified. Phenotypic analysis of the tes1 mutant implicated that TES1 has a critical role in the TMB-responsive gene expression and the inhibition of seed germination. Taken together, we propose that plants have been equipped with a TMB sensory pathway through which the TMB induces the seed dormancy in a TES1-dependent way.

Seed dormancy is one of the most important developmental traits in higher plants, which contributes to adaptive survival under fluctuating ambient environments. A sophisticated and complex regulatory network of seed dormancy has begun to be revealed using molecular genetic analysis in model plants (1–3). When developing seeds enter into the dormant state during the later stage of seed development, the parental environments such as low temperature, light quality, and nutrients impact the degree of seed dormancy, which is the so-called primary dormancy (1, 4, 5). The dormancy of the seeds can be alleviated with the drying time after harvest, so-called after-ripening (4). Upon imbibition, the dormancy can also be relieved by several environmental cues, which include low temperature, nitrate, karrikin (KAR), and red (R) light in Arabidopsis seeds (6, 7). On the contrary, several, which are often unfavorable, environments such as far-red (FR) light, high temperature, salt stress, and low water content can suppress seed germination through the enhancement of the dormancy of imbibed Arabidopsis seeds, which is called the secondary dormancy (8–10). Besides, the natural chemical cues derived from allelopathic plants, bacteria, fungi, and smoke have been also inferred to impact secondary dormancy (11–14). As such, plants sense and integrate diverse endogenous and exogenous stimuli to determine the right time to germinate.

Plant-derived smoke contains several germination regulating factors (13). The best-characterized smoke compounds are a group of butenolides, KARs. KARs impact multiple aspects of physiology and development, including the promotion of seed germination in many plant species (15, 16). Several components involved in KAR signaling have been identified in Arabidopsis, which include a KAR receptor, KARRIKIN INSENSITIVE2/HYPOSENSITIVE TO LIGHT (hereafter referred to as KAI2); an F-box protein, MORE AXILLARY GROWTH2 (MAX2); and putative transcriptional regulators, SUPPRESSOR OF MAX2 1 (SMAX1)/SMAX1-LIKE2 (17).

Besides germination promoting compounds, smoke also contains several germination-inhibiting compounds (18, 19). As a representative smoke inhibitor, 3,4,5-trimethylfuran-2(5H)-one (trimethylbutenolide, TMB), was identified by its antagonizing effect on the KAR-induced germination (18, 20). Initially, it was hypothesized that the TMB may interfere with the KAR signaling based on its structural similarity to the KAR. Physiological and molecular analyses implicated that the TMB exerts its germination inhibitory effect independently of the KAR signaling (21). However, the mode of action of the TMB remains to be characterized.

To understand how the TMB exerts its inhibitory effect on seed germination, we assessed the TMB-dependent response of Arabidopsis seeds under various physiological contexts in this study. Our experimental data showed that the TMB induces physiological dormancy independently of the KAR–KAI2 signaling, which is accompanied by the genome-wide transcriptional changes which include a series of germination/dormancy regulatory genes. In accordance, we found that several dormancy regulatory cues such as ABA, GA, salt, mannitol, cold temperature, and after-ripening modulated the TMB-dependent germination behavior. Furthermore, by screening TMB-resistant mutants of Arabidopsis, we identified a regulatory gene, designated as TMB RESISTANT1 (TES1), which encodes a B2 group Raf-like kinase. Our physiological and molecular analysis of the tes1 mutants revealed that TES1 plays an essential role in the TMB-induced seed dormancy through its kinase activity.

Results

TMB-Dependent Inhibition of Seed Germination in Arabidopsis.

TMB (Fig. 1A) was originally identified from plant-derived smoke water as an active compound that inhibits the seed germination of Lactuca sativa (18). However, it has not been elucidated how TMB inhibits germination. To understand the mode of action of TMB, we examined the TMB-responsive seed behavior of Arabidopsis, a model plant of molecular genetic analysis. When we tested the inhibitory effect of TMB on seed germination, the effect of TMB was inversely proportional to the storage period (Fig. 1B). Notably, the most inhibitory effect was found in the freshly matured seeds. With the freshly matured seeds, the TMB nearly completely suppressed the germination (Fig. 1C). The TMB inhibited the germination of the freshly matured seeds in a concentration-dependent manner (IC₅₀, ∼5 μM) (Fig. 1D). Besides the prolonged seed storage (after-ripening), cold treatment during imbibition, so-called stratification, relieved the germination-inhibiting effect of the TMB (Fig. 1E). While the after-ripened Arabidopsis seeds showed a reduced or marginal response to TMB under continuous white light, the PhyB-dependent germination by a pulse of R light was inhibited by the TMB treatment (SI Appendix, Fig. S1). Thus, the TMB-dependent inhibition of germination likely comes from the enhanced physiological dormancy rather than toxicity which affects the cellular physiology or growth of the seeds. In accordance with the hypothesis, the nongerminating Arabidopsis seeds, which were kept for 7 d in the presence of the TMB, could resume their germination and postembryonic growth when transferred to the TMB-free media (SI Appendix, Fig. S2).

Fig. 1.

The TMB inhibits the seed germination of Arabidopsis. (A) Chemical structures of KAR₁ and TMB. (B) The effect of the after-ripening on the TMB-dependent inhibition of germination. The dry-stored wild-type (Col-0) seeds for the indicated time (weeks) after harvest were sown on the aqueous media with or without 50 μM TMB. The germination was counted based on the cotyledon emergence at 5 d after incubation under continuous white light. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (C) The TMB-dependent inhibition of the germination of freshly matured Arabidopsis seeds. The freshly matured wild-type (Col-0) seeds were imbibed on the aqueous media either containing mock or 50 μM TMB. The germination was counted based on the cotyledon emergence. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (D) The dose response of the TMB-dependent inhibition of the germination. The freshly matured wild-type (Col-0) seeds were sterilized and imbibed on the aqueous media containing various concentrations of TMB as indicated. The germination rate was counted based on the cotyledon emergence at 4 d after sowing. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (E) The effect of stratification on the germination inhibitory effect of the TMB. The sterilized freshly matured wild-type (Col-0) seeds were water imbibed and kept at 4 °C for the indicated days, and then the seeds were sown on the aqueous media either with or without 50 μM TMB. The germination was counted based on the cotyledon emergence at 5 d after sowing. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (F) The KAI2-independent action of the TMB on the inhibition of the germination. The freshly matured wild-type Landsberg erecta (Ler) and kai2-2 seeds were sterilized and imbibed on the aqueous media without (Mock) or with 50 μM TMB (TMB), 10 μM KAR₁ (KAR), or both 50 μM TMB and 10 μM KAR₁ (TMB+KAR) under the continuous light condition. The germination was counted based on the cotyledon emergence at 4 d after sowing. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate.

KAI2-Independent TMB Response.

Even though KAR and TMB share similar chemical structures (Fig. 1A), TMB did not act as a competitor of KAR in the regulation of seed germination in Lactuca sativa (21). To further investigate the mode of action of TMB relating to KAR, we examined the TMB-dependent germination behavior of kai2-2 mutant, a null allele of a KAR receptor gene, KAI2. While the kai2-2 mutant did not respond to 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikin 1 [KAR₁]), the germination of both the wild-type and kai2-2 seeds was suppressed by the TMB (Fig. 1F). The results imply that TMB can inhibit seed germination independently of KAR–KAI2 signaling. Furthermore, the mutants of KAR-signaling components, including SMAX1 and MAX2/ORE9, responded to the TMB treatment as wild type did, displaying TMB-dependent inhibition of germination (SI Appendix, Fig. S3). These results support the hypothesis that KAR and TMB have distinct modes of action to affect seed germination.

TMB-Responsive Gene Expression Profiling.

It is well established that the modulation of seed dormancy is accompanied by transcriptional reprogramming in Arabidopsis. To investigate the effect of TMB on transcriptional expression, we performed messenger RNA sequencing (mRNA-seq) experiments. Upon imbibition for 12 h, TMB caused genome-wide transcriptional changes, up-regulating 769 genes and down-regulating 1,371 genes with over twofold changes compared to the mock treatment (false discovery rate [FDR]-adjusted P ≤ 0.05) (Dataset S2). When we examined expression profiles of a series of dormancy/germination-related genes in response to TMB, compared to the mock treatment (Fig. 2 A and B), in line with the reduced germination by the TMB, the expression of EMBRYOGENESIS ABUNDANT1 (EM1) and EMBRYOGENESIS ABUNDANT6 (EM6), dormancy-associated genes, was increased, while the transcript level of CYSTEINE PROTEINASE1 (CP1), a germination-associated gene, was reduced by the TMB. Consistent with the expected role of TMB as a dormancy-inducing factor, the transcript level of several dormancy-inducing regulatory genes, including DELAY OF GERMINATION 1 (DOG1), MOTHER OF FT AND TFL1 (MFT), and FUSCA3 (FUS3), were increased by the TMB treatment, compared to the mock treatment. Next, we assessed the expression of several ABA/GA-related genes, which are often associated with seed dormancy. The expression of ABA DEFICIENT 1 (ABA1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 5 (NCED5), encoding rate-limiting ABA biosynthetic enzymes, were increased by the TMB, whereas the transcript level of Gibberellin 3-beta-dioxygenase 1 (GA3ox1), encoding a GA biosynthetic enzyme, was decreased. The effect of the TMB on the ABA-catabolic gene expression was differential. While the transcript level of CYP707A3 was decreased, CYP707A2 transcript level was elevated by the TMB, compared to the mock treatment. The TMB treatment also affected the expression of ABA-signaling genes such as HONSU (HON), ABA INSENSITIVE4 (ABI4), and ABA INSENSITIVE5 (ABI5). Compared to the mock treatment, the TMB treatment increased the expression of HON and ABI5, but it decreased the ABI4 expression. In the case of ABA INSENSITIVE3 (ABI3), there was a marginal effect of the TMB on the expression. Similar expression profiles were found by qRT-PCR analysis with independent seed batches in other seasons (SI Appendix, Fig. S4).

Fig. 2.

The TMB-responsive gene expression profiling. For the analysis of the TMB-responsive gene expression, the freshly matured wild-type (Col-0) seeds were sown on the aqueous media, imbibed for 12 h either without (Mock) or with 50 μM TMB (TMB). Total RNA from three biologically independent samples for each treatment (Mock or TMB) was subject to mRNA-seq analysis or qRT-PCR analysis. (A) Heat map of a selected set of dormancy-associated genes based on fragments per kilobase of transcript per million mapped reads values of mRNA-seq. FC, fold change. **, FDR-adjusted P value < 0.05. (B) Validation of mRNA-seq by qRT-PCR analysis. Relative expression to At1g13320 was normalized to that of the mock treated one, which was set to 1. Values are the mean ± SEM from three biologically independent samples. P values are shown; two-tailed Student’s t test. (C) GO enrichment analysis of differentially expressed genes using the Database for Annotation, Visualization, and Integrated Discovery with default options. The top 10 GO enrichment terms of both up- and down-regulated were shown compared to mock treatment.

Gene ontology (GO) analysis with the TMB-responsive genes showed that genes involved in the various processes, including ABA response and abiotic stress adaptation, were significantly enriched in the TMB-up-regulated genes (TMB-UP) (Fig. 2C). In contrast, genes with functions related to the germination-promoting chemical/hormones KAR and GA were enriched in the TMB-down-regulated genes (TMB-DOWN). Notably, genes of cell wall remodeling or cell growth/division were also enriched in the TMB-DOWN. When we compared the TMB-responsive transcriptome with the reported transcriptome in response to the R light, another dormancy regulatory cue in Arabidopsis seeds (22), the results showed that a large fraction (83%) of TMB-UP overlapped with the list of the R light repressible genes (SI Appendix, Fig. S5). Similarly, a large fraction (75%) of TMB-DOWN were also found in the list of the R light inducible genes. The results imply that the overlapping, if not all, fraction of TMB-responsive genes are associated with the dormancy regulatory network that integrates multiple environmental stimuli. Collectively, the results of gene expression profiling suggested that TMB-dependent inhibition of seed germination entails genome-wide transcriptional changes, including altered expression of a series of dormancy regulatory genes.

ABA/GA Signaling Is Required for TMB-Responsive Dormancy.

Given the prominent effect of TMB on the expression of the ABA/GA-related genes (Fig. 2), we assessed the impacts of ABA/GA signaling on the TMB-induced germination inhibition. Norflurazon (NF), an inhibitor of ABA biosynthesis, suppressed the effect of TMB on the seed germination (Fig. 3A), and the effect of TMB was reduced in the ABA receptor mutants, pyr1pyl1pyl2pyl4, if not completely abolished (Fig. 3B). The results implied that ABA biosynthesis/signaling can modulate the TMB-induced germination inhibition. Exogenously applied GA₃ relieved moderately the inhibitory effects of TMB on the seed germination (Fig. 3C). In line with the results, a della pentuple mutant (della-p, gai/rga1/rgl1/rgl2/rgl3), in which GA signaling is constitutively activated, was shown to be partially resistant to the TMB (Fig. 3D). These results suggest that the TMB-dependent inhibition of germination is partially conditional to ABA/GA homeostasis and signaling.

Fig. 3.

ABA/GA biosynthesis/signaling modulates the TMB inhibition of germination. (A) The effect of NF on the TMB-induced inhibition of the germination. The freshly matured wild-type (Col-0) seeds were sown on the aqueous media without (Mock) or with 50 μM TMB, 100 μM NF, or both 50 μM TMB and 100 μM NF (NF+TMB) under continuous light. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (B) The TMB-dependent germination of the ABA receptor mutant. The freshly matured seeds of wild type (Col-0) and the ABA receptor mutant, pyr1/pyl1/pyl2/pyl4 quadruple (pyrQ), were sown on the aqueous media either containing 50 μM TMB or Mock. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (C) The effect of the GA on the TMB-induced inhibition of the germination. The freshly matured wild-type (Col-0) seeds were sown on the aqueous media without (Mock) or with 50 μM TMB, 100 μM GA, or both 100 μM GA and 50 μM TMB (GA+TMB). Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (D) The TMB-dependent germination of the della mutant. The freshly matured seeds of wild type (Col-0) and the della mutant, rga/rgl1/rgl2/rgl3/gai (dellaP), were sown on the aqueous media either containing 50 μM TMB or Mock. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. The germination was measured based on the cotyledon emergence at the indicated day after sowing.

We also examined the effect of ABA or paclobutrazol (PAC), a GA biosynthetic inhibitor, on the TMB-dependent inhibition of germination. In the presence of a low concentration of ABA (0.5 μM) or PAC (0.1 μM), the TMB completely inhibited the germination of the after-ripened seeds whereas the TMB alone, ABA (0.5 μM) or PAC (0.1 μM) did not inhibit the germination (SI Appendix, Fig. S6 A and B). Thus, the applied low concentration of ABA or PAC appears to sensitize the after-ripened seeds for the response to the TMB. Similarly, the germination of after-ripened seeds was completely inhibited by the TMB in the presence of a moderate concentration of Mannitol or NaCl, other dormancy-inducing cues in Arabidopsis (23–25) (SI Appendix, Fig. S6 C and D). These results implicate that the TMB-mediated signaling pathway may interact with other dormancy-inducing cues integrated into the dormancy regulatory network.

Isolation of tes1 Mutants.

To identify the molecular components mediating TMB response, we adopted a forward genetic approach by making use of the genetic resources in Arabidopsis. A two-round screening scheme was set up to isolate mutants that impaired the TMB sensitivity. With the pools of publicly available transfer DNA (T-DNA) insertional lines (CS76502) from ABRC (Arabidopsis Biological Resource Center), we screened ∼150,000 seeds of 10,000 T-DNA insertional lines to select putative mutants that could germinate on the aqueous media containing both 50 μM TMB and 0.5 μM ABA. The progeny seeds of 137 candidate lines were retested for their germination phenotypes. Among them, we selected three lines that exhibited a strong germination phenotype (over 50% of germination) in the presence of both 50 μM TMB and 0.5 μM ABA. With the three mutant lines, we performed a second round of screening, testing for their sensitivity to TMB or ABA, respectively. Among them, two mutants were resistant to both compounds, which might be potentially ABA-signaling mutants. We found that a mutant was resistant only to the TMB but responded normally to ABA (Figs. 4C and 5). Further genetic analysis was performed with the mutant of TMB-specific resistance, hereafter designated as TMB resistant1-1 (tes1-1). Genetic analysis showed that the F₁ seeds derived from crossing wild type (Columbia [Col-0]) with tes1-1 were normally sensitive to TMB, and the F₂ seeds from self-pollinated F₁ plants displayed Mendelian segregation ratio regarding germination phenotype (nongerminating: germinating in the presence of 50 μM TMB and 0.5 μM ABA, 194: 63, χ² =0.033). The results implied that the tes1-1 is a single recessive mutation.

Fig. 4.

The identification of the TES1 gene. (A) A schematic diagram of the genomic structure of the At4g23050 and the T-DNA insertion sites of tes1-1 and tes1-2. The protein structure of At4g23050 showed typical domains of B2 group Raf-like kinase, predicted by SMART (smart.embl-heidelberg.de/). (B) The TMB-resistant phenotype of the tes1-1 and the tes1-2 mutants. The freshly matured seeds of wild type (Col-0) and the tes1 mutants were sown on the aqueous media with or without 50 μM TMB. The germination was counted at 5 d after imbibition based on the emergence of the green cotyledon. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (C) The TMB-resistant phenotype of the tes1-1 and the tes1-2 mutants during the PhyB-dependent germination. The after-ripened seeds of wild-type (Col-0) and tes1 mutants were sown on the aqueous media without or with 50 μM TMB. The seeds were irradiated with FR light for 15 min and then kept in darkness with or without following a single pulse of R light for 5 min for 4 d. The germination was counted at 4 d after the dark incubation based on the seedling establishment. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (D) The TMB-resistant phenotype of the after-ripened tes1-1 and tes1-2 mutant seeds. The after-ripened seeds of wild-type (Col-0) and tes1 mutants were sown on the aqueous media containing mock solution, 50 μM TMB alone (TMB), 50 μM TMB plus 0.5 μM PAC (TMB + PAC), or 50 μM TMB plus 0.5 μM ABA (TMB + ABA). The germination was scored based on the radicle emergence. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. (E) The effect of the tes1 mutation on the TMB-dependent gene expression. For the analysis of the TMB-responsive gene expression, the freshly matured wild-type (Col-0) and tes1-1 seeds were imbibed for 12 h either without (Mock) or with 50 μM TMB. The total RNA was extracted, and the relative expression level was examined by qRT-PCR analysis. The expression of the At1g13320 gene was analyzed as a control. Values presented are normalized to that of the mock-treated wild type (Col-0), which was set as 1. Values are the mean ± SEM from three biologically independent samples. P values are shown; two-tailed Student’s t test. Similar trends were found with independent seed batches in other seasons (SI Appendix, Fig. S8). (F) The transgenic complementation analysis. The germination analysis was performed with the freshly matured wild-type (Col-0), tes1-1, and the independent transformants seeds of TES1-GFP overexpressor (ox) or TES1^D609E-GFPox. The germination was scored at 4 d after sowing, based on the emergence of green cotyledons. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate.

Fig. 5.

The germination phenotype of the tes1-1 in response to PAC, ABA, or NaCl. (A) PAC dose–response analysis. (B) ABA dose–response analysis. (C) NaCl dose–response analysis. The after-ripened wild-type (Col-0) and tes1-1 mutant seeds were sown on the aqueous media containing various concentrations of PAC, ABA, or NaCl. The germination was counted as the emergence of the green cotyledon at 5 d after sowing. Mean values (± SD) of germination rates were determined from three experimental replicates with at least 50 seeds per plate. For the PAC- or the ABA-sensitivity test, the rgl2 or abi5-7 mutant seeds were included as a control. Representative plate images were shown on the right.

Molecular Cloning of TES1.

Genetic cosegregation analysis with the tes1-1 mutant, which was identified from the T-DNA insertional pooled lines, implied a tight linkage between the T-DNA and the tes1 mutant phenotype. To find the molecular lesion of the tes1-1 mutation, we isolated the genomic DNA flanking the T-DNA of the tes1-1 mutant using a modified thermal asymmetric interlaced PCR (TAIL-PCR) analysis. The sequencing analysis revealed that the T-DNA is inserted into the fourth exon of the At4g23050, encoding a Raf-like kinase which was annotated as MAP3Kδ4 or Raf12 (Fig. 4A). The genotyping PCR analysis confirmed the insertion of the T-DNA in the At4g23050 (SI Appendix, Fig. S7). We obtained an additional T-DNA insertion allele of the At4g23050 (SAIL_122_D01), designated as tes1-2 (Fig. 4A). The RT-PCR analysis confirmed that the tes1-1 and tes1-2 represent the null alleles of the At4g23050 (SI Appendix, Fig. S7). When we examined the TMB-dependent germination, both the tes1-1 and tes1-2 mutants were insensitive to the TMB under the various physiological contexts, which included the primary dormant seeds under white light and a pulse of R light–dependent germination and the germination in the presence of a low concentration of ABA or PAC of the after-ripened seeds (Fig. 4 B–D). Using the TMB-responsive genes as molecular markers (Fig. 2), we examined the TMB-responsiveness of the tes1-1 mutant in regards to gene expression. Compared to the wild type, the tes1-1 mutation impaired severely, if not completely, the TMB-responsive expression of all of the genes tested, including EM1, EM6, CP1, ABA1, ABI4, ABI5, FUS3, and MFT (Fig. 4E). These results implied that TES1 is also required for the TMB-responsive gene expression. Interestingly, in contrast to the severe TMB insensitivity, the tes1 mutants showed typical germination response to other dormancy-inducing cues, including PAC, ABA, and NaCl (Fig. 5). Thus, taken together, we concluded that the loss of function of the At4g23050 gene is responsible for the TMB insensitivity of the tes1 mutants.

The in silico analysis based on expression data (https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) indicated that the TES1 is predominantly expressed in the seeds, which was verified by the RT-PCR analysis (SI Appendix, Fig. S9). Furthermore, we found that the TES1 was inducible by the TMB treatment in the imbibed seeds. Microscopic analysis with the transgenic lines that overexpress the TES1-GFP showed that the TES1-GFP signal was localized in the cytosol (SI Appendix, Fig. S10). In further support of the functionality of the TES1-GFP fusion protein, the transgenic expression of TES1-GFP could rescue the TMB-resistant germination of the tes1-1 mutant (Fig. 4F). In contrast, when we introduced the mutant form of TES1 (TES1^D609E) in which 609th aspartic acid (D), a conserved amino acid in the active site of kinases, was changed to glutamic acid (E), the mutant TES1^D609E-GFP did not rescue the TMB-resistant germination of the tes1-1 mutant (Fig. 4F).

In Arabidopsis, TES1 with five homologous genes, including Raf7, Raf8, Raf9, Raf10, and Raf11, consist of the B2 group of Raf-like kinases, conserved among terrestrial plant species (SI Appendix, Figs. S11 and S12). We asked whether the B2 group Raf genes in Arabidopsis other than TES1 would be involved in the TMB-dependent response during germination. When we examined the germination phenotype of the after-ripened seeds of raf7, raf8, raf9, raf10, raf11, raf10raf11, tes1-1, and tes1-2 mutants in the presence of TMB and ABA, none of the knock-out mutants of the B2 group Raf-like kinase genes showed a strong TMB-resistant phenotype, as the single tes1 mutants did (SI Appendix, Fig. S11). The results indicated the specified role of TES1 for the TMB-dependent responses, suggestive of functional specialization among B2 group Raf-like kinases in Arabidopsis.

Discussion

TMB Induces Physiological Seed Dormancy in Arabidopsis.

Coping with the sessile nature, plants have evolved to sense and respond to diverse chemical and physical cues, increasing their adaptive fitness under fluctuating ambient environments (26). Smoke compounds have long been known to contain several phytoactive compounds, including KARs, of which mode of action in plants has been extensively characterized for the past decade (17, 27). In this study, we investigated how Arabidopsis seeds respond to TMB, another smoke-derived small molecule. Our findings that KAR-signaling mutants such as kai2, smax1, and max2/ore9 were still responsive to exogenously applied TMB supported the hypothesis that KAR and TMB have distinct modes of action to modulate seed germination (Fig. 1 and SI Appendix, Fig. S3).

Several lines of genetic and physiological evidence point to the role of TMB, as a dormancy-inducing cue in Arabidopsis. Firstly, the paused, nongerminating seeds in the presence of the TMB could resume their germination and postembryonic growth when they were transferred to the TMB-free Murashige and Skoog–sucrose media (SI Appendix, Fig. S2). Thus, it is likely that the TMB inhibition of germination is due to physiological dormancy of the imbibed seeds rather than a toxic effect on the cells to inhibit cellular metabolism or growth. Secondly, we found that exogenously applied TMB caused transcriptional changes of over 1,000 genes, including the GA/ABA biosynthetic/signaling genes (GA3ox1, ABA1, NCED5, and ABI5) and several dormancy regulatory genes (DOG1, MFT, and FUS3) (Fig. 2). Taken together with the known roles of the TMB-responsive genes in the dormancy regulation, it is plausible that the TMB-dependent inhibition of germination entails transcriptional regulation of dormancy-associated genes. In support, GO term analysis of the TMB-responsive genes showed that genes in the germination regulatory hormones such as ABA, GA, and KAR are under control of TMB, suggestive of potential cross-talk between TMB and these hormones (see below). It is noteworthy that genes related to abiotic stress adaptation were also enriched in the TMB-UP, in line with the finding that abiotic stress adaptation is a key process in the DOG1-dependent seed dormancy (28). Intriguingly, the TMB caused the down-regulation of ABI4, a positive regulator of ABA signaling, and the up-regulation of CYP707A2, encoding an ABA-catabolic enzyme, which might counteract the effect of TMB for the seed dormancy. It may reflect a system-wide negative feedback regulation in the dormancy regulatory network. Thus, although the gene expression profiling provided a molecular signature of TMB-dependent responses, the biological relevance of the TMB-responsive genes remains to be determined by functional studies combined with in-depth bioinformatics analyses. Finally, the effect of the TMB on the seed germination was evident within certain physiological contexts, which is suggestive of cross-talks between TMB and other dormancy regulatory pathways. The conditional effect of TMB depending on the physiological contexts may reflect multiple layers of genetic and physiological regulation that act together with TMB to modulate seed dormancy (SI Appendix, Fig. S13). As such, the dormancy-inducing effect of the TMB was relieved by the dormancy-releasing conditions including after-ripening and stratification (Fig. 1 B and E). On the other hand, the TMB-dependent inhibition of germination was hypersensitized by several germination-inhibiting cues, including modest salt, osmotic stress, and a low concentration of ABA or PAC (SI Appendix, Fig. S6). In line with the findings, it is notable that the different seed batches in other seasons exhibited a variable degree of TMB responsiveness in regards to the transcriptional gene expression (Figs. 2 and 4E and SI Appendix, Figs. S4 and S7). It is well known that ever-fluctuating parental environments such as light, temperature, and nutrition can affect several physiological features of the seed including dormancy level (29). Thus, assumingly, the seasonal variance in the physiological status of seed may account for the differential extent of TMB response in the gene expression among seed batches. It will be interesting to understand how the TMB response system interacts with other exogenous and endogenous cues, including several plant hormones at the molecular and biochemical level in future studies.

Collectively, our results of physiological and molecular analyses together support the hypothesis that TMB acts as a dormancy-inducing cue, implicating the presence of TMB sensory system in Arabidopsis.

Role of TES1 during TMB-Induced Seed Dormancy.

With the physiological contexts in which TMB can effectively inhibit the seed germination of Arabidopsis (Fig. 1 and SI Appendix, Figs. S1 and S6), our pilot study of genetic screening identified the TMB-RESISTANT1 (TES1) gene, encoding a Raf-like kinase (also called Raf12 or MAP3Kδ4) that plays a critical role in the TMB-induced seed dormancy (Fig. 4). It is worthy to note that the tes1 mutants showed strong, if not complete, TMB insensitivity in regards to the germination and gene expression (Fig. 4 B–E), while exhibiting marginally impaired, if not normal, responses to several dormancy-inducing cues, including FR light, ABA, PAC, and NaCl (Figs. 4C and 5). Thus, the results suggest that the TES1 has specified roles for the TMB-induced seed dormancy. Though the visible manifestation of the altered seed behavior in tes1 mutants was not observed in the absence of the TMB (Fig. 4), we cannot rule out the possibility that TES1 may be involved in the endogenous, as yet unidentified, dormancy regulatory signaling, besides TMB-dependent signaling. Since the wild type (Col) has relatively low primary dormancy among ecotypes and defect in the parental temperature-imposed dormancy (30), which might hamper visible manifestation of germination phenotype of the tes1 mutant, further detailed phenotypic analysis using tes1 mutants in the deep dormant ecotype (e.g., Cvi) would help to elucidate the TMB-independent function of TES1.

Despite the identification of the TES1 as a key component of the TMB-induced seed dormancy, it is still unclear how the TES1 is involved in the TMB-dependent responses (SI Appendix, Fig. S13). It cannot be ruled out that TES1 is involved indirectly in the TMB responsiveness by affecting the level of TMB sensory components, the TMB transport system, or metabolic activation of the TMB in the plant cell. Considering the critical role of the kinase activity of the TES1 for its function (Fig. 4F), it will be interesting to test whether the TES1 kinase activity is regulated by TMB, altering, in turn, the phosphorylation status of the substrate protein(s), which might be involved in the transcriptional regulation in the nucleus. It is also worthwhile to investigate whether TMB may affect the TES1 activity through other posttranscriptional regulation including translational efficiency, subcellular localization, and protein stability, besides the transcriptional induction of TES1 gene (SI Appendix, Fig. S9B).

Although the loss-of-function analysis in our study suggested that TES1 plays, if any, a marginal role in the ABA- or salt-dependent germination (Fig. 5), previous gain-of-function studies implicated the involvement of TES1 in the ABA or salt stress-dependent responses (31). Besides, recent genome-wide interactome analysis revealed that TES1 can interact with diverse regulatory proteins, including transcriptional factors and several ABA-signaling components (32–34). Thus, it is conceivable that TES1 is involved in the cross-talk with ABA or salt response pathway, if not being a canonical component of ABA or salt responses. Future studies toward the functional characterization of the substrates of the TES1 would help to understand how TES1 is involved in the TMB-sensitivity and its possible cross-talk with ABA or salt response pathway. Interestingly, several B2, B3, and B4 groups of Raf-like kinases are recently characterized to relay ABA or osmotic stress signals into SnRK2s, a canonical component of ABA signaling (35–38). These different groups of Raf-like kinases play differential functions depending on the signaling cues, such as ABA and osmotic stress. For example, Raf10, a member of B2 subgroup Raf-like kinases, phosphorylates C-terminal end of SnRK2.2, OST1/SnRK2.6, and SnRK2.3 (39), while a group of B3 Raf-like kinases phosphorylate S171 of OST1/SnRK2.6 upon activation by ABA and osmotic stress signal (35, 36, 38). By analogy, it is tempting to speculate that one of the downstream signals of TES1 may involve activation of SnRK2s, which would lead to inhibition of seed germination. It will be interesting to determine whether TES1 can phosphorylate SnRK2s and other potential substrate proteins in a TMB-dependent manner.

It is noteworthy that Raf10 and Raf11, two functionally redundant B2 subgroup Raf-like kinases, played a marginal role in the TMB-induced dormancy compared to TES1 (SI Appendix, Fig. S11) while modulating ABA-responsive germination (39, 40). The results imply that the signaling pathways of TMB and ABA are, at least in part, independent, and the members of the B2 subgroup of Raf-like kinases might have specified functions, mediating diverse environmental or endogenous signals in the regulation of seed germination. In line with the hypothesis, the phylogenic analysis showed that TES1 and TES1 homologs in other plant species belong to a distinct clade from that of other B2 group members, including Raf7-11 (SI Appendix, Fig. S12). It would be interesting to investigate any functional redundancy or signaling cross-talk among related B2 subgroup members of Raf-like kinases.

In summary, the findings in this study implicate the presence of a smoke sensory system in higher plants by which seed dormancy is induced. Together with the KAR response system, the TMB sensory system may underpin the variable responses toward smoke compounds among plant species (41), contributing to diverse adaptation strategies under the postfire environment. Further genetic identification of additional TES loci, combined with biochemical analyses of TES1 protein and TMB-binding protein(s), will shed light on the TMB sensory system by which plants sense and respond to a smoke-derived chemical cue like TMB, providing further insights into how plants modulate seed dormancy and germination, an agronomically important trait.

Materials and Methods

Plant Materials and Growth Conditions.

Most of the Arabidopsis mutants used are in the Col-0 background unless otherwise stated. The ABA-, GA-, or KAR-related mutants, including della-pentuple (N16298), pyr1pyl1pyl2pyl4 (CS72388), abi5-7, rgl2-ko (SALK_024532C), kai2-2 (N100282), smax1-3, and ore9-1 have been previously described (42–45). T-DNA insertional mutants of raf7-1 (SALK_000887), raf8-1 (SALK_120720), raf9-1 (SALK_022298), and tes1-2 (SAIL_122_D01) were obtained from the ABRC or The Nottingham Arabidopsis Stock Centre (NASC). The raf10-1 (ais143), raf11-1 (SALK_047070), and raf10-1raf11-1 double mutant were previously described (38).

Chemicals.

For hormone-sensitivity tests or the chemical sensitivity tests, an appropriate concentration of KAR₁ (Toronto Research Chemicals), ABA (Sigma-Aldrich), PAC (Duchefa), GA₃ (Duchefa), NF (Sigma-Aldrich), or TMB was added before pouring the aqueous medium. TMB was prepared using organic synthesis, following the procedure as described by Surmont et al. (46).

Germination Assay.

Germination tests were performed with freshly matured seeds that had been dried within 2 wk after harvest or after-ripened seeds that had been dry stored for at least 2 mo, essentially as described in Lee et al. (45). For the PhyB-dependent germination assays, the seeds were surface sterilized and sown on an aqueous medium containing 0.8% agar. Seeds were irradiated with FR light (1.325 μW ⋅ cm⁻²) for 15 min and then kept in darkness with or without a single pulse of R light (5.72 μW ⋅ cm⁻²) for 5 min. After 4 d, the germination rate was determined based on the seedling establishment. The monochromatic light was provided by light-emitting diode light (Joeninsang, Co.). For the hormone-responsive and chemical-responsive germination, the seeds were surface sterilized and sown on an aqueous media with a mock solution or media supplemented with the indicated concentration of plant hormone or chemical. After incubation under continuous white light for 2 to 7 d, the germination rate was scored based on the radicle emergence or the cotyledon greening, as indicated. For germination assay, we used seed batches from independently grown plants (5 to 10 plants per batch) that were grown under the same experimental conditions. A total of ∼50 to 100 seeds per batch were used in the germination assay for each genotype/experiment. Three seed batches were tested for each germination assay to assess biological variations. Similar results were obtained at least twice with independent seed batches from plants grown in other seasons.

Gene Expression Profiling.

For the gene expression profiling, qRT-PCR analyses were performed basically as described (45). Briefly, total RNA was extracted from 12 himbibed seeds. After the DNase I treatment, the total RNA was subjected to reverse transcription for the first-strand complementary DNA (cDNA) synthesis. The cDNA diluted 10-fold was used for the template of real-time PCR analysis. The comparative 2^–ΔΔCt method was used to evaluate the relative quantities of each amplified product in the samples (47). The relative expression levels were normalized based on the value of At1g13320 (PP2A), a stably expressed gene in the seeds under various dormancy-regulating environments (48). The used primer sequences are listed in Dataset S1.

mRNA-Seq Analysis.

For mRNA-seq analysis, freshly matured wild-type (Col-0) seeds were sown onto aqueous media with or without 50 μM TMB. After incubation for 12 h under white light, the seed samples were taken for extraction of total RNA. The total RNA was extracted using a Spectrum Plant Total RNA kit (Sigma) following manufacturer’s instructions. The RNA purity and integrity were validated using Bioanalyzer 2100 (Agilent Technologies Inc.), and mRNA sequencing libraries were prepared using Illumina Truseq stranded mRNA library prep kit, according to the manufacturer’s instructions. The mRNA was purified and fragmented from total RNA (1 μg) using poly-T oligo-attached magnetic beads and two rounds of purification. Cleaved RNA fragments were reverse transcribed into first-strand cDNA using random primers, and dUTP in place of dTTP. A single adenine base was added to these cDNA fragments, followed by adapter ligation. The products were purified and amplified with PCR to create the final-strand–specific cDNA library. The libraries were subjected to 100 bp paired-end sequencing using an Illumina HiSEq. 2500 platform and yielded >55 million reads per library. The low-quality reads and adapter sequences were removed, and then the clean reads were mapped to the TAIR10 Arabidopsis genome reference using HISAT2 with default settings. For analysis of differential gene expression, EdgeR was used (49). Genes with at least a twofold change in expression (FDR ≤ 0.05) with fragments per kilobase of transcript per million mapped reads value >1 were considered to be differentially expressed and subject to GO analysis at the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov/) with default settings.

Isolation of the TES1 Mutants.

The tes1-1 mutant was isolated from mutant screening with a pool of T-DNA insertion lines (CS76502), obtained from the ABRC (https://abrc.osu.edu/). Because the effect of TMB is weak or marginal on the germination of after-ripened seeds including the T-DNA pools obtained from ABRC, we adopted two rounds of mutant screening toward the TMB-resistant mutants. In the first-round screening, the mutagenized pools of seeds (∼150,000 seeds derived from 100 pools of 100 T-DNA lines) were sown on the aqueous media that contained both 0.5 μM ABA and 50 μM TMB. The germinated seedlings were selected at 2 wk after incubation under white light. The putative 137 TMB-resistant mutants were transferred to the soil and grown to set seeds. Using the after-ripened seeds, we repeated the germination assay to confirm the mutant phenotype, germination in the presence of both 0.5 μM ABA and 50 μM TMB. Among the putative candidates, only three mutants exhibited a high germination rate (over 50%) that is feasible for genetic analysis, which were chosen for further analysis. With the three confirmed mutants, the second-round screening was performed. With the assumption that not only TMB specifically resistant mutants but also mutants with impaired ABA signaling can germinate in the presence of both 0.5 μM ABA and 50 μM TMB, we tried to screen out ABA-insensitive mutants that exhibited a high germination rate (over 50%) in the presence of 1 μM ABA and found that two of the three mutants were insensitive to ABA. These newly isolated ABA-insensitive mutants will be described elsewhere. As a result, the remaining one mutant, designated as the tes1-1, with very weak, if not normal, ABA insensitivity was selected for further analysis. At least two rounds of genetic backcrossing of the tes1-1 mutant to the wild type (Col-0) was performed before further physiological analyses.

Molecular Cloning of the TES1.

As the tes1-1 mutation was derived from the T-DNA insertional pools and was found to cosegregate with the T-DNA, we tried to isolate the genomic DNA flanking the T-DNA. Using a modified TAIL-PCR method, the FPNI-PCR (50), we obtained a genomic fragment flanking the T-DNA. The Basic Local Alignment Search Tool analysis at The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/) against the Arabidopsis genome sequence indicated that the T-DNA was inserted into the fourth exon of the At4g23050 gene. To verify that the At4g23050 is the responsible gene, we searched a second allele in Columbia background, designated as tes1-2 (SAIL_122_D01), at T-DNA insertional database (http://signal.salk.edu/cgi-bin/tdnaexpress) and obtained the mutant seeds from TAIR. The genotype-confirmed tes1-2 seeds were used for further physiological analyses.

Transgenic Complementation and Subcellular Localization of the TES1-GFP.

To generate the TES1-overexpressing transgenic plants, the full-length cDNA of the At4g23050 was obtained by the RT-PCR. The cDNA amplicon was cloned into the pENTR-d-Topo (Invitrogen). The sequence-verified clone was subject to an attL and attR recombination with the gCsVMV-eGFP-N1300, a gateway destination vector for the GFP fusion. For the cloning of the TES1^D609E, the mutant TES1 cDNA was obtained by the overlapping PCR strategy. The resulting gCsVMV-eGFP-N1300/ TES1 or TES1^D609E was introduced into the Agrobacterium tumefaciens, GV3101 for in planta transformation of Arabidopsis. The tes1-1 mutant plants were used for the in planta transformation using a floral dipping method (51). The transgenic Arabidopsis plants were selected based on the hygromycin resistance and further screened at the next T2 generation to select the transgenic lines with a single copy of T-DNA. At the T3 and T4 generation, multiple homozygous lines were verified for their transgenic overexpression and used for further physiological and microscopic analysis. The GFP fusion protein expression was observed in the root tip of the 24 h-imbibed embryo using Leica TCS-SP5 confocal microscopy (Leica microsystems).

Data Availability

The mRNA-seq sequence read data reported in this paper have been deposited in the National Center for Biotechnology Information Sequence Read Archive under BioProject ID PRJNA692487. All other study data are included in the article and/or supporting information.