Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.


Through millions of years of evolution, plants have gained a sophisticated molecular machinery to sense and respond to a plethora of environmental stimuli. So far, plant responses to biotic and abiotic factors have been extensively studied and characterized at the molecular level. Nonetheless, insights on how plants respond to mechanical cues, like rain, wind, touch, and bending, are just beginning to unfold. Plant responses to mechanical cues are very diverse. Some plants (e.g., Mimosa, Venus flytrap, and sundew) have specialized sensory cells that help them to respond to touch with impressive rapidity (1). In addition, in Arabidopsis, trichomes on the leaf surface have been demonstrated to act as rapid touch sensors, triggering Ca2+ responses in neighboring cells (2). On the other hand, periodic mechanical stimulation to plants results in various morphological changes at a slower rate. Repeated mechanostimulation leads to severe alterations of the plant morphology, like dwarfism, pithiness, altered mechanical properties of stem, delayed flowering, improved anchorage strength of roots, and reduced stomatal aperture, which is collectively termed as thigmomorphogenesis (3). These morphological changes improve the mechanical safety of plants against strong wind, which often causes lodging due to anchorage failure and stem breakage (46). In addition to this, it has been noted that repetitive mechanical stimulation improves plant performance in stressful environments like pathogen attack, drought, salinity, and cold (4, 79). Thus, mechanostimulation has been gaining attention as a potential method for sustainable agriculture practices to improve food security (10). However, the plant response to mechanical stimulation is very complex, as it depends on the intensity of mechanical load and frequency of exposures (10). Understanding the molecular mechanism of plant mechanoperception and thigmomorphogenesis is imperative to apply this method for large-scale farming.
To date, two major classes of molecular players have been identified in plant mechanoperception. Among them, mechanosensitive ion (MS) channels form the most prominent group, converting mechanical cues into chemical signals (11). Increased membrane tension due to mechanical force facilitates the gating of MS channels through conformational changes of the transmembrane domain, which generates an electrical current followed by various biological responses. There are several types of MS channels in the plant, which include mechanosensitive channel of small conductance-like (MSL) and Mid1-complementing activity family (MCA). Patch-clamp electrophysiological analysis has identified stretch-activated gating of Arabidopsis MSL1, MSL8, MSL9, MSL10, and MCA1 (1215). Another group of mechanoreceptor includes receptor-like kinases (RLKs), such as Feronia (FER) (belonging to a Catharanthus roseus RLK1-like subfamily). Arabidopsis loss-of-function feronia mutants show altered root growth responses (like enhanced skewing, reduced penetration ability, and abnormal tracking response) to mechanically challenging environments, e.g., caused by a hard agar surface or an impenetrable glass barrier (16). In addition to this, reduced expression of mechanoresponsive genes after hypo-osmotic shock (which modifies cell wall mechanical tension) and impaired Ca2+ signaling after touching was observed in roots of feronia mutants (16). As in other plant-environment interactions, calcium signaling also plays a central role in plant mechanotransduction. Some MS channels, like MCAs, cause mechanosensitive gating of Ca2+ (11). As a result, a spiking of cytosolic Ca2+ occurs immediately after the perception of mechanical signals. To convert Ca2+ signals into transcriptional responses, calcium-dependent protein kinases and calcium-binding proteins might be involved.
Although substantial progress has been made in unraveling the molecular underpinning of plant mechanoperception using Arabidopsis roots as a model, the molecular mechanism of thigmomorphogenesis of the aboveground part of plants is just beginning to unfold. A couple of studies indicated that thigmomorphogenesis is a more complex developmental process than mere cellular mechanosensing alone. For example, no difference in thigmomorphogenic responses was observed between wind-stimulated mslΔ5 (msl4;msl5;msl6;msl9;msl10) and wild-type Arabidopsis plants (12). Conversely, some mutants impaired in thigmomorphogenesis have been identified recently through reverse genetic approaches. For instance, the Arabidopsis ga2ox7 loss-of-function mutant does not show stunted morphology and delayed flowering in response to touch (17). GA2ox7 is a mechanoresponsive gene that encodes a gibberellin (GA) inactivating enzyme. Arabidopsis mutants defective in jasmonic acid (JA) biosynthesis and signaling also show impaired thigmomorphogenesis (7, 18). Among them, allene oxide synthase (AOS) is involved in JA production, the jasmonate-resistant 1 (JAR1) conjugase converts JA into its bioactive form jasmonate-isoleucine (JA-Ile), coronatine-insensitive 1 (COI1) is a critical component of the JA coreceptor complex, and MYC2/MYC3/MYC4 encode JA-activated basic helix-loop-helix (bHLH) transcription factors. It has also been demonstrated that a reduced concentration of bioactive GA and an increased level of JA is required for touch-mediated growth alteration (7, 17). However, wild-type–like touch-inducible expression of many mechanoresponsive genes (e.g., calmodulin-like TCH2 and TCH3, and cell wall–modifying xyloglucan endotransglycosylase/hydrolase TCH4) was still noted in the JA mutants aos and myc2 myc3 myc4, which indicates the presence of a JA-independent mechanosignaling pathway in plants (7, 18).
In this study, we have compiled a wide selection of mutants with reported or suspected roles in mechanosensitive signaling, based on literature searches and meta-analysis of large-scale datasets. We have screened these mutants for altered molecular responses, in particular gene expression, to touch stimulation by gentle brushing. We identified calmodulin-binding transcriptional activator CAMTA3 as a previously unidentified positive regulator of touch-induced gene expression, required for thigmomorphogenesis and touch-induced delay of flowering. In contrast, we found that Feronia is a strong repressor of JA-dependent touch responses. Moreover, we found that CAMTA3 cooperates with its homologs CAMTA1/2 to directly control touch-induced gene expression of JA-independent pathway genes like TCH2/4, but not JA-dependent genes like JAZ8 and CML39.


Generation of a mutant collection of putative regulators of plant touch responses

To obtain better insight into which genetic factors contribute to touch-induced transcriptional responses, we performed a survey to select promising candidate genes (Table 1). MYC2, MYC3, and MYC4 were previously identified as crucial regulators of the JA-dependent transcriptional responses to water spray and mechanical stimulation (18). Recently, a quadruple myc2 myc3 myc4 myc5 mutant was reported, which also has lost the function of the fourth member of this clade of MYC2-related bHLH transcription factors (19). WRKY40 and its close paralogs WRKY18 and WRKY60 have been implicated in a range of responses, e.g., to abscisic acid (ABA) (20) and retrograde signaling (21), but are also among the most touch responsive genes in Arabidopsis (18, 22, 23). Oxidative signal-inducible (OXI1) kinase has been implicated in mediating oxidative bursts to downstream signaling, making it of interest as touch responses are believed to involve early reactive oxygen species (ROS) signals (24). Touch-responsive phosphoprotein 1 (Treph-1) was picked up in a screen for touch-induced protein phosphorylation targets and was shown to be important for thigmomorphogenesis and regulating specific genes of the transcriptional touch response (25). In addition, Feronia receptor-like kinase, which binds to rapid alkalinization factors (RALFs), has been implicated in a wide range of plant responses, including immune signaling, development, flowering, cell wall integrity, pollen tube growth, and stomatal movement, as recently summarized (26). Moreover, it was suggested to mediate responses to osmotic swelling, which is considered as a type of mechanical stimulation (16). In particular, it was noted to be required for full induction of TCH2 and TCH4 touch marker genes after hypo-osmotic shock. Its homolog Theseus1 (THE1) is a receptor-like kinase that also binds RALF factors and is involved in cell wall integrity signaling (27, 28). The Jumonji domain protein ethylene insensitive 6 (EIN6) was picked up in a screen for ethylene insensitivity (29). It has also been reported to be required for induction of TCH3 by mechanical stimulation (30). The glutamate receptor-like (GLR) proteins are crucial receptors for the neurotransmitter glutamate in animal nervous cells (31). In plants, they are thought to operate as cation channels transporting Ca2+ across the plasma membrane, which may be activated by pH and glutamate released actively or from, e.g., wounded cells (3234). In particular, GLR3.3 and GLR3.6 have been shown to mediate systemic signaling via electric potentials and Ca2+ waves, thereby affecting JA-induced gene expression (33, 34). Given the close similarity between the transcriptomes of wounding and touch signaling (23), and the involvement of JA and Ca2+ in touch responses, the glr3.3 glr3.6 double mutant was included.
Mutant nameGene descriptionFunctionReference
myc2345MYC2 MYC3 MYC4 MYC5JA signaling bHLH transcription
Major et al. (19)
caf1a caf1bCCR4-associated factors 1a and 1bmRNA poly-A tail degradationWalley et al. (40)
oxi1Oxidative signal-inducible 1Protein kinaseRentel et al. (72)
camta3-1 (SALK_001152)Calmodulin-binding transcription
activator 3
Calcium-related transcription factorBenn et al. (36)
camta3-2 (SALK_064889)Calmodulin-binding transcription
activator 3
Calcium-related transcription factorJiang et al. (50)
p35s::CAMTA3- gFPCalmodulin-binding transcription
activator 3
Calcium-related transcription factorJiang et al. (50)
fer-4FeroniaReceptor-like kinaseEscobar-Restrepo et al. (73)
theseus1Theseus1Receptor-like kinaseHematy et al. (28)
wrky18 40 60WRKY18 WRKY40 WRKY60Touch-induced transcription factorsShang et al. (20)
treph1-1Touch-regulated phosphoproteinWEB1/PMI2-related proteinWang et al. (25)
vcs-8VaricosemRNA decappingBasbouss-Serhal et al. (74)
vcs-7VaricosemRNA decappingSorensen et al. (44)
xrn4Exoribonuclease 4/ethylene
insensitive 5
5′ to 3′ exoribonucleases of mRNABasbouss-Serhal et al. (74)
piezo-2 (SAIL_856_B11)PiezoMS channelThis study
piezo-3 (SALK_003004)PiezoMS channelThis study
ein6Ethylene insensitive 6Jumonji/zinc finger proteinRoman et al. (29)
glr3.3 glr3.6Glutamate receptorsSystemic Ca2+ signalingMousavi et al. (34)
camta1/2/3Calmodulin-binding transcription
Calcium-related transcription factorKim et al. (51)
Table 1. List of mutants.
Besides a literature search, we also studied a range of datasets to find clues to previously unidentified players in touch signaling. In one approach, we selected the top 50 most induced Arabidopsis genes 25 min after water spray in Col-0 (table S1) (18). This set of genes was searched for overrepresented transcription factor binding sites using the TF2Network online tool (35). In total, 108 motifs were identified as overrepresented (table S2). At the top of the list, we found three similar motifs that were predicted to be recognized by the calmodulin-binding transcription factor CAMTA3. CAMTA3 has been found to be involved in rapid stress responses, by activating the rapid stress response element in wound-responsive promoters (36). CAMTA3 is furthermore of interest given the role of Ca2+ in early touch signaling (24) and was thus selected for further characterization. Further down, the list was dominated by (putative or experimentally verified) motifs recognized by in total 51 unique WRKY transcription factors including AtWRKY40, supporting our choice to include the wrky18 wrky40 wrky60 mutant in our screen.
Previous studies could not detect changes in touch induced–gene expression in mutants in mechanosensitive channels such as mechanosensitive channel–like (MSL) multiple mutants (18) and Ca2+-permeable mechanosensitive channels mca1 mca2 mutants (23). A different class of MS channels that were initially characterized in animal systems is Piezo-type proteins (37). We searched the Arabidopsis genome and could only identify a single homolog (At2g48060; Piezo; PZO1), which at the start of this study had not been characterized. The Piezo protein is unusually large with 2462 amino acids, resulting in a molecular weight of 282 kDa. To study its function in mechanoperception, we obtained two transferred DNA (T-DNA) mutants in the At2g48060 locus and screened for homozygous mutants. The piezo-2 allele has previously been shown to be a null mutant (38). Quantification of the PIEZO transcript in piezo-3 (SALK_003004) mutants by quantitative real-time polymerase chain reaction (qRT-PCR) also confirmed a clear lack of mRNA (fig. S1). Both mutant alleles grew vigorously and did not show any obvious growth defects when grown on soil. In addition, when grown on standard horizontal MS agar plates, no clear differential phenotypes could be detected, suggesting that Piezo proteins are largely dispensable for normal growth in optimal conditions in plants. We were nevertheless interested in whether molecular phenotypes could be observed in the transcriptome after touch treatment.
One of the most remarkable aspects of the transcriptional response to touching is not only its rapid induction but also its steep decline. The peak expression for most touch-responsive genes is around 22 to 25 min after treatment, yet the expression levels for many genes are down to nearly preinduction levels by 40 min (Fig. 1) (18, 23). This rapid decrease in mRNA levels within 10 to 15 min suggests an active mechanism of mRNA degradation, rather than a general unregulated decay. The half-life of some classic touch-inducible transcripts after inhibition of RNA polymerase by actinomycin D is much longer, e.g., 106 min for AtWRKY40 and 4.5 hours for TCH4 (39). We thus also looked for factors that may regulate this rapid mRNA decay. Two CCR4-associated factors Caf1a and Caf1b are consistently induced by touch treatments (18, 22). CCR4-CAF protein complexes are involved in mRNA destabilization by removing the poly-A tail of mRNAs and were reported to affect deadenylation of wound-induced transcripts (40). Therefore, the caf1a caf1b double mutant was obtained and included in this study.
Fig. 1. qPCR analysis of selected touch-responsive genes in seedlings of Col-0, camta3-1, fer-4, and piezo mutants 0, 22, and 40 min after gentle brushing.
The y axis represents fold inductions relative to 0-min Col-0 value (set to 1). Error bars designate SEM (n = 5). Statistical significance was determined by two-way analysis of variance (ANOVA) followed by post hoc Tukey tests (letters show significant difference between all samples at P < 0.05).
Another mechanism of mRNA degradation is by removal of the protective 5′ 7-methyl guanosine mRNA cap (decapping). The scaffolding protein Varicose (VCS) facilitates mRNA decapping (41, 42), exposing the mRNA to 5′ to 3′ exonuclease activity by, e.g., exoribonuclease XRN4 (43), while the suppressor of VCS (SOV) protein degrades the RNAs in 3′ to 5′ direction (44). A recent study assessed the contribution of mRNA degradation enzymes VCS and SOV after inhibition of transcription with cordycepin (44). Col-0 has a natural null mutation in SOV, so the vcs mutant in Col-0 is effectively a sov vcs double mutant. We analyzed the RNA sequencing (RNA-seq) dataset comparing vcs and sov mutants to Col-0 (technically sov) and Col-0 with a restored SOV gene (“wild type”), specifically focusing on well-known touch-responsive transcripts (fig. S2 and table S3). Notably, the transcripts of, e.g., TCH2-4, JAZ8, WRKY40, and Caf1a were much more stable in the vcs and vcs sov mutants compared to wild type and sov. The mRNA degradation profiles of vcs and vcs sov were overall very similar, indicating that VCS is the main contributor. Thus, at least under controlled conditions and after inhibition of transcription, VCS seems to be important for efficient degradation of many core touch-responsive transcripts. Therefore, the vcs mutant, as well as the xrn4 mutant, were included in the screen.

CAMTA3 and Feronia are regulators of touch response signaling

The transcriptomic responses to touch were assessed in the collection of mutants selected above (Table 1). For touch treatment, plants were gently brushed with a paint brush. Twelve-day-old in vitro grown seedlings were mechanostimulated and samples were collected just before, or 22 or 40 min after touch treatment, and transcripts were measured using qRT-PCR. We collected samples at 22 min after touching, which is around the peak of expression for many touch-responsive genes (18), to assess mutants for their capacity to induce the transcriptional touch responses. We also collected samples at 40 min after touching, when transcript levels had nearly returned to basal levels, to assess whether mutants were affected in the rapid degradation of mRNA (Fig. 1 and figs. S3 to S5). A range of touch-inducible transcripts were selected for qRT-PCR analysis on the basis of previous studies.
The myc2 myc3 myc4 myc5 mutant showed reduced induction of known MYC2 target genes such as bHLH19, ERF109, and CML39 relative to wild type (Col-0) at 22 min, whereas TCH4 transcript levels were induced as in wild type (fig. S3). Overall, the quadruple mutant responded similarly to the triple myc2 myc3 myc4 mutant reported previously (18), indicating that MYC5 likely has a very minor contribution. In contrast, the wrky18 wrky40 wrky60 triple mutant showed a wild-type–like response for all the genes we tested, covering JA-dependent (e.g., bHLH19 and ERF109) and JA-independent transcripts (TCH3 and TCH4). Mutants in the glutamate-like receptors glr3.3 glr3.6 involved in systemic Ca2+ and JA signaling also showed a wild-type–like touch response (fig. S4). The ein6 mutant was previously reported to be deficient in TCH3 induction in response to mechanical stimulation (30); however, in our study and experimental conditions, the mutant actually responded slightly stronger to touch than wild type (Ler-0 in this case) at a transcript level for TCH3, while other tested touch marker genes bHLH19 and TCH2 responded like wild type. Mutants in TREPH1, a protein phosphorylated in response to mechanical stimulation, were previously reported to have impaired touch-induced gene expression of CML38, ERF11, and JAZ7 (25). Using the same treph1-1 T-DNA insertion mutant, we could not see significant alterations in touch-induced gene expression of ERF11, JAZ7, or CML38 in comparison with the touch-treated Col-0 under our growth conditions and using our experimental setup (fig. S5). In addition, treph1-1 plants showed wild-type–like responses for other touch-induced genes like TCH4, Caf1a, bHLH19, JAZ8, TCH2, and OM66.
We also assessed the touch-induced gene expression patterns in mutants of related receptor-like kinases Feronia (FER) and Theseus1 (THE1), and OXI1 kinase. Although the1 and oxi1 mutants displayed wild-type–like touch responses in bHLH19, TCH3, TCH4, and ERF109 expression (fig. S3), clear differences with Col-0 could be observed in fer-4 mutant plants (Fig. 1). Already in untouched control seedlings, we observed significantly higher transcript levels of touch marker genes including bHLH19, CML39, and JAZ8 in fer-4 compared to Col-0. Moreover, a hyperinduction 22 min after brushing treatment could be observed compared to touch-treated Col-0 for bHLH19 and CML39. This elevated expression compared to Col-0 continued 40 min after touching for bHLH19, CML39, JAZ7, JAZ8, and ERF11, while in Col-0 their expression had returned to near untouched control levels. However, the expression levels of TCH2 and TCH4 in fer-4 remained similar to Col-0 in untouched control, as well as after 22 and 40 min of induction (Fig. 1). These results indicate that Feronia kinase is a strong negative regulator of touch-inducible gene expression for several genes that have been associated with JA-responses like JAZ8, while JA-independently regulated genes (e.g., TCH2 and TCH4) do not appear to be affected directly.
Conversely, touch-induced gene expression of bHLH19 was strongly repressed in untouched control and at 22 min after touch treatment in mutants of the Ca2+-signaling related transcription factor CAMTA3 (Fig. 1). While most other touch-induced genes were not significantly affected, TCH2 induction was significantly lower at both 22 and 40 min after touching relative to touched Col-0. This suggests that CAMTA3 is affecting a separate branch of the touch-induced transcriptomic response. bHLH19 touch-induced expression also appeared suppressed at 22 min in the piezo mutant (Fig. 1), although the difference did not meet P value criteria in the two-way analysis of variance (ANOVA) analysis. The induction of CML39 appeared to be significantly repressed in the piezo mutant.
Last, we assessed the rapid reduction of mRNA levels observed at 40 min in Col-0 in the mutants with defects in mRNA decapping (vcs), deadenylation (caf1a caf1b), and mRNA degradation (xrn4). Despite the reported roles of these enzymes in mRNA stability, the mutants had a normal mRNA expression pattern for all tested touch-responsive genes at 40 min. As we had expected the expression of touch-induced genes to remain elevated for longer especially in the vcs-7 mutant based on the previous mRNA stability study (fig. S2) (44), we assessed a second allele vcs-8. In addition, in this mutant, mRNA levels declined as normal at 40 min (fig. S5).

Piezo does not play a major role in touch-responsive gene expression

As the impact of loss of Piezo expression on the overall transcriptome before and after touch is poorly understood, we performed a genome-wide RNA-seq transcriptomics study comparing Col-0 and piezo mutants before (i.e., untouched) and 22 min after touch (brushing) treatment (fig. S6 and table S4). Under untreated conditions, 160 genes were differentially expressed compared to Col-0, of which 82% was less expressed in the piezo (SALK_003004) mutant, including the Piezo gene itself. No significantly overrepresented Gene Ontology (GO) category could be identified and only a relatively small portion of the genes (22) were part of the previously defined 1671 “core touch-responsive” genes that were commonly differentially expressed in a wide range of mechanical stimulation–related whole transcriptomes (fig. S6A) (18). Two related MADS box transcription factors Sepallata1 (AtSEP1) and Sepallata3 (AtSEP3), which redundantly regulate flower development, were approximately five- to sevenfold down-regulated (fig. S6B) (45). In addition, Terminal Flower 1 (TFL1) involved in inflorescence identity determination (46) was approximately 2.5-fold down-regulated. Two ROXY-type glutaredoxins ROXY13 and ROXY14 were down-regulated and cysteine-rich receptor kinase 30 (CRK30). Four protein-encoding genes were more than twofold significantly up-regulated in piezo mutants, including expressed protein At3g01345 (153-fold), a nodulin transporter Umamit25 (6-fold), and a ROX1-like repressor (3-fold).
A total of 2265 genes were differentially expressed 22 min after brushing in Col-0 (padj < 0.05, 1.5× fold change). Of these, 70% were up-regulated and 30% were down-regulated (Fig. 2A). Nearly 50% of the differentially expressed genes (DEGs) after brushing in Col-0 were considered as commonly responsive to mechanical stimulation (core touch-responsive genes) as defined previously (18), demonstrating that a successful mechanical stimulation was achieved. We then looked for genes that showed altered touch-responsive gene expression in piezo mutants compared to Col-0 at 22 min after brushing (fig. S6C) (padj < 0.05). Only nine such genes could be identified, including bHLH19 as observed in the qRT-PCR experiment (Fig. 1) and the (mechanical) stress-activated transcription factor WRKY48, which has been shown to repress plant basal defense (47). Both transcription factor genes were less induced after touching in the piezo mutant compared to in Col-0. In conclusion, our findings indicate that the unique Piezo protein in the Arabidopsis genome does not appear to have a crucial role in regulation of mechanosensitive gene expression in seedlings.
Fig. 2. The fer-4–dependent touch-transcriptome.
(A) DEGs in Col-0 after 22 min of touch treatment. Of 2265 genes differentially expressed in Col-0 after 22 min of touch treatment, 70% of these genes were up-regulated and 48% are core touch-responsive genes (B) Genes affected in fer-4 mutants under untouched conditions. Of 1035 DEGs in fer-4 mutant versus Col-0 (untouched conditions), 56% showed less accumulation and 16% were core touch-responsive genes. (C) Genes with differential touch response in fer-4 mutants. Of 2265 genes differentially expressed in Col-0 after 22 min of touch treatment, Expression of 411 genes showed significant difference in fer-4 22 min versus Col-0 22 min. A total of 50% of those genes were core touch-responsive genes and 55% showed more induction in fer-4 mutants. (D) Heatmap of all 411 fer-4–dependent touch-responsive transcripts. (E) Heatmap of selected fer-4–dependent transcripts related to JA signaling and biosynthesis. Gene expression values were normalized per gene, with 1 indicating the highest expression level. Normalization between 0 and 1 was performed within each dataset (Col-0 versus fer-4). UT, untouched.

The Feronia kinase is a strong negative regulator of touch-induced gene expression

RNA-seq analysis revealed strong effects on gene expression in the fer-4 mutant compared to Col-0 under untreated conditions. In total, 1035 genes were differentially expressed, with 456 genes more highly expressed and 579 genes with reduced expression (Fig. 2B). As observed in the qPCR, 164 of these were core touch-responsive genes such as JAZ8, CML39, lipoxygenase 4 (first step in JA biosynthesis), and ERF11, with around two-thirds showing higher basal expression in fer-4 mutants (table S5). GO signatures of wounding, hypoxic, and biotic responses were up-regulated among these touch-responsive genes, while response to salicylic acid (SA) appeared down-regulated. However, TCH2/3/4 gene expression was not significantly affected. Among the non–core touch-responsive genes that were up-regulated in fer-4, we found an up-regulation of iron starvation and iron response–related genes, suggesting that the lack of Feronia triggers an iron starvation–like response. Among non–core touch-regulated genes, we found that genes involved in cell wall organization, GA response, and gravitropism were down-regulated.
In total, 411 genes had an altered touch response in the fer-4 mutant (Fig. 2, C and D). Most genes showed a stronger touch responsiveness. Among these hypertouch-sensitive genes, we observed a wide range of JA biosynthesis components such as Lipoxygenases LOX3 and LOX4, allene oxide cyclase AOC3, and 12-oxophytodienoate reductase OPR3 (Fig. 2E). In addition, key JA signaling components such as MYC2 and 5 JAZ/TIFY genes were much more induced after touching in fer mutants. In agreement, GO analysis indicated an overrepresentation of JA response, response to fungus, wounding, and (a)biotic stress responses. This indicates that Feronia plays a significant role in touch-responsive gene expression by repressing at least part of the JA-response, rather than by activating touch response signaling as proposed previously for mechanical stimulation by hypo-osmotic swelling (16). In our hands and growth conditions, and using whole seedlings, we did not observe the reduced expression of, e.g., TCH2, CML49, and WRKY33 in fer-4 before or after touching as reported previously for osmotic swelling in root tissue (16).

CAMTA3 is a positive regulator of touch-induced gene expression

The qRT-PCR analysis of selected touch marker genes revealed that the transcription factor CAMTA3 is required for full touch-induced expression of bHLH19 and TCH2 (Fig. 1). The contribution of CAMTA3 in genome-wide transcriptional response to touching was further assessed using RNA-seq analysis (Fig. 3 and table S6). Under untreated conditions, we observed that 516 genes were differentially expressed in camta3-1 mutants compared to Col-0 (padj < 0.05, 2× fold change), and most of which (80%) were lower expressed in camta3-1 than in wild type (Fig. 3A), including CAMTA3 itself. A GO analysis revealed that genes involved in defense to fungi were particularly overrepresented among down-regulated genes in camta3 mutants (fig. S7). Twelve genes were more than 10-fold down-regulated in camta3 indicating a strong positive contribution of CAMTA3 to their expression. Approximately 20% of DEGs in untreated camta3 plants were part of the previously defined 1671 core touch-responsive genes (18). This is much higher than expected by chance (5%), indicating a role for CAMTA3 in the regulation of touch-responsive genes.
Fig. 3. The camta3-1–dependent touch transcriptome.
(A) Genes affected in camta3-1 mutants under untouched conditions. Of 516 DEGs in camta3 under untouched conditions, 80% showed less accumulation and 19% was core touch-responsive genes. (B) Genes with differential touch response in camta3-1 mutants. Expression of 79 genes showed significant difference in camta3-1 mutants, 63% of these genes were core touch-responsive genes and 54% showed less induction in camta3-1 mutants. (C) Heatmap of all 79 camta3-1–dependent touch-responsive transcripts. Gene expression values were normalized per gene, with 1 indicating the highest expression level. Normalization between 0 and 1 was performed within each dataset (Col-0 versus camta3-1).
To assess the contribution of CAMTA3 to touch-responsive gene expression, we searched the 2265 genes that were touch-responsive in Col-0 for genes that were differentially expressed in camta3 at 22 min versus Col-0 at 22 min (padj < 0.05; Fig. 3, B and C). In total, 79 genes showed an altered touch response in camta3-1 mutants (Fig. 3, B and C), with around half of the genes (54%) showing an attenuated touch responsiveness. This was as opposite to what was observed in the fer-4 mutant (Fig. 2), where most genes showed an increased touch responsiveness. A total of 63% of these genes were considered “core” touch-responsive genes, showing that CAMTA3 has a predominantly positive role in gene expression regulation of touch responsive genes. Among these genes, we confirmed bHLH19 as being less induced in camta3-1 mutants as observed previously by qRT-PCR (Fig. 1). Transcript levels of TCH2 were induced 8-fold in Col-0 and only 5.6-fold in camta3-1, although the significant P value of 0.02 fell below the cutoff after the stringent multiple testing correction (padj < 0.05). This is likely explained by the lower number of biological replications used for the RNA-seq (three) versus qRT-PCR (five) for the camta3-1 22-min sample group. In addition, calmodulin-like 23 (CML23), the closest homolog of TCH2/CML24, was significantly less induced in camta3-1 mutants after touching compared to in Col-0 (Fig. 3C). GO analysis further showed that genes involved in defense, cell wall modification, SA response, abscisic acid response, ROS, hypoxia, regulation of flower development, and vegetative to reproductive phase transition of meristem were affected (table S7). To support that CAMTA3 is a regulator of these 79 genes, we used the TF2Network tool to search for transcription factor binding. Consistently, overrepresented sites were found for CAMTA3 and related proteins CAMTA1 and CAMTA2 (table S7).

CAMTA3 and Feronia cooperate with MYC2 MYC3 MYC4 to regulate touch signaling

Now, the best-defined component of touch-responsive transcriptional regulation involves the JA-dependent pathway, which to a large extent is regulated by the MYC2 MYC3 MYC4 transcription factors (18). Therefore, we performed a comparative analysis to evaluate the relative contribution of CAMTA3, MYC2 MYC3 MYC4, and Feronia to touch signaling (Fig. 4). Previously, 357 genes were identified to have a differential touch response in myc2 myc3 myc4 at 25 min after water spray compared to Col-0 (18), which is not an identical but for this context comparable experimental setup. A comparison of fer-4 and myc234 mutants revealed that 50 genes showed an altered touch response in both mutants relative to wild type (Fig. 4, A and B). This list was very strongly overrepresented in genes related to JA biosynthesis (e.g., LOX3/4, OPR3, and AOC3) and signaling (JAZ/TIFY proteins and MYC2 itself). In agreement, the GO categories of JA and wounding responses were overrepresented among these genes (table S8). To get a better overview of how these common genes are regulated in fer-4 and myc234 mutants, the expression values were plotted in heatmaps (Fig. 4B). This demonstrated very that fer-4 and myc234 showed almost completely opposite responses: While the common genes were more strongly up- or down-regulated in fer-4 compared to Col-0, they showed attenuated responses in myc234. Hence, Feronia is an important negative regulator of a large part of the JA-response positively regulated by MYC2 MYC3 MYC4. The responsive genes are, however, not fully overlapping, so we were interested to see whether any specific differences occurred between myc234 and fer-4. Therefore, we performed a GO analysis of the 361 fer-4–specific and 307 myc234-specific genes (Fig. 4A and table S9). Twenty-one of 50 GO categories at P < 0.05 present among the fer-4–specific genes were also overrepresented among myc234-specific genes, indicating that generally similar cellular processes are affected by both regulators. Specifically to Feronia, we observed a clear overrepresentation of cell wall components and metal ion transport, which was not observed in the myc234-regulated genes. Conversely, GO categories related to programmed cell death, calcium-ion binding, and defense to fungi were only observed among myc234-regulated genes.
Fig. 4. Comparison of touch transcriptomes of myc2 myc3 myc4 with fer-4 and camta3-1.
(A) Venn diagrams showing overlaps between genes with differential touch response in fer-4 (411) and myc234 (357) mutants, and 82 genes defined as part of the MYC2-regulon. (B) Heatmap representing the transcript accumulation of the common 50 genes in fer-4 and myc234 mutants. Gene expression values were normalized per gene, with 1 indicating the highest expression level. Normalization between 0 and 1 was performed within each dataset (Col-0 versus fer-4 and Col-0 versus myc2 myc3 myc4). (C) Venn diagrams showing overlaps between genes with differential touch response in camta3-1 (79) and myc234 (357) mutants, and 82 genes defined as part of the MYC2-regulon. (D) Venn diagrams showing overlaps between genes with differential touch response in fer-4 (411) and camta3-1 (79) mutants.
Of the 79 genes with differential touch response at 22 min in camta3-1 (Fig. 3), 10 were overlapping with the myc234 357 DEGs (Fig. 4C and table S10), including bHLH19, UGT74E1, CA2+-DEPENDENT MODULATOR OF ICR1, and ethylene response factors ERF6/98/104. Twenty-nine genes were overlapping between the 79 camta3-1 and 411 fer-4 touch DEGs (Fig. 4D). The CAMTA3 transcript itself was 2.6× fold-induced by touching at 22 min in Col-0, with no significant differences in myc234 or fer-4 compared to Col-0. Three of these genes overlapping between myc234 and camta3-1 (Fig. 4A) have been defined as part of the core “MYC2 regulon,” based on direct promoter binding and transcriptional regulation (18). Cell wall–related genes, including expansin like EXLA1, EXLA3, and EXL1, and cell wall-associated kinases WAK1 and WAK2 were present in the 69 camta3-specific genes. Other transcription factors such as WRKY48 and WRKY25, pathogenesis-related proteins, and defense-related secreted peptides STMP4 and STMP5 were generally positively regulated by CAMTA3. Three genes involved in vegetative stage to flowering transition were less induced after touch in the camta3-1 mutant including calmodulin-like 23, laccase 8, and far-red-elongated hypocotyl-like fhl. In contrast, no clear signs of JA-related signaling could be observed. To verify the JA-(in)dependent expression of the CAMTA3-regulated genes, we assessed the expression of the 79 CAMTA3-regulated touch-responsive genes in a detailed RNA-seq analysis looking at Arabidopsis seedlings dipped into MeJA or mock solutions (48). Only bHLH19 showed an early and consistent up-regulation by dipping into MeJA solution from 15 min to 8 hours of treatment (fig. S8). Jacalin-related lectin 33 (JAL33) was induced by JA after approximately 2 to 3 hours only. We also checked their expression in a recent RNA-seq transcriptomics series that uses volatile methyl-jasmonate application (MeJA), thereby avoiding touch-induced effects (49). All three genes (bHLH19, Ca2+-dependent modulator of ICR1 CMI1, and ERF104) that were common to the MYC2 regulon and camta3-1–regulated touch-responsive genes (Fig. 4A) were induced by MeJA within 15 min (table S11). Only 3 of 69 camta3-1–specific genes (EXLA3, AT2G36650, and AT2G01300) were induced after 15 min of MeJA, further indicating that CAMTA3 is part of the JA-independent touch response network.

CAMTA1, CAMTA2, and CAMTA3 redundantly regulate touch-responsive gene expression of JA-independent touch marker genes

To investigate whether constitutive overexpression of CAMTA3 could affect JA-dependent and JA-independent touch response marker gene expression, we obtained two p35S:CAMTA3-YFP (OX) lines (Table 1) (50). We also included camta3-1 and a second T-DNA allele (camta3-2) as controls (Table 1) (50). Already under untreated conditions (Fig. 5), JA-independent touch marker genes TCH2 and CRK41 were significantly higher expressed in both CAMTA3 OX lines. JA marker gene JAZ8 showed normal expression in the OX lines, however. After touching, TCH2, CRK41, and bHLH19 showed significantly less induction in both camta3-1 and camta3-2 mutants, confirming the role of CAMTA3 in their induction. TCH2, CRK41, bHLH19, and PP2A5, however, showed a higher touch-induced expression in yellow fluorescent protein (YFP)–CAMTA3 overexpression plants. TCH4 showed a slightly higher basal expression in the CAMTA3 OX lines, while camta3-2 showed slightly less touch responsiveness for TCH4, although these changes did not meet the statistics cutoffs (Fig. 5). In contrast, genes representing JA-responsive genes such as JAZ8 and CML39 showed a wild-type–like touch response in both CAMTA3 loss- and gain-of-function lines.
Fig. 5. qPCR analysis of selected touch-responsive genes in CAMTA3 overexpression (OX) and T-DNA mutants at 0 and 22 min after gentle brushing.
The y axis represents fold inductions relative to untouched (0 min) Col-0 value (set to 1). Error bars designate SEM (n = 3 to 4). Statistical significance was determined by two-way ANOVA followed by post hoc Tukey tests (letters show significant difference between all samples at P < 0.05).
Although many mutants have been assessed for deregulation of touch-responsive gene expression, only very few have been reproducibly shown to have significant effects on the transcriptional response (our data and studies cited above). The strongest effects found so far in, e.g., myc234, jar1, and coi1 mutants were on genes that were induced by the JA-dependent pathway. In contrast, these mutants showed no effect on the “JA-independent” touch-responsive TCH2-4 genes (18). Moreover, mutants previously suggested to have reduced induction of TCH2-4 (fer-4 and ein6) (16, 30) showed wild-type–like responses to touching of green tissues in our study (Fig. 1 and fig. S4). Our findings suggested that in camta3-1 mutants TCH2 expression was less induced after touching than in wild-type plants (Fig. 1). As CAMTA3 is part of a small gene family in Arabidopsis, we hypothesized that CAMTA3 may cooperate with related factors to regulate gene expression of TCH genes. We therefore obtained a set of multiple mutants for CAMTA1, CAMTA2, and CAMTA3. The developmental effects in single camta3 mutants, including smaller rosette size and faster senescence, were more pronounced than in camta1 camta2 mutants, suggesting that CAMTA3 has the largest individual contribution as observed previously (fig. S9) (51). Double camta1 camta3 mutants looked relatively similar to camta3-1 mutants, while camta2 camta3 (camta2/3) mutants showed a stronger phenotype than single camta3-1 mutants. Last, the camta1 camta2 camta3 (camta1/2/3) mutant initially germinated and established itself relatively normally but then further growth became strongly reduced, resulting in severe dwarfism (fig. S9). As camta2/3 and camta1/2/3 mutants showed the strongest developmental effects, we assessed their capacity to induce touch-responsive gene expression of JA-dependent and JA-independent genes (Fig. 6). Strikingly, the camta1/2/3 mutants showed a significant inhibition of touch-induced expression of bHLH19, CML23, CRK41, WRKY48, PP2A5, EXLA3, TCH2, and TCH4 (Fig. 6), while camta2/3 and camta3-1 mutants showed an intermediate but also significantly diminished touch responsiveness for most of these genes. Thus, the loss of multiple CAMTA1/2/3 members resulted in a progressively stronger loss of touch responsiveness for JA-independent touch marker genes. In contrast, genes representing JA-dependent touch responses such as JAZ8 and CML39 showed a wild-type–like touch response in the camta3-1, camta2/3, and camta1/2/3 mutants. TCH3 and WRKY33 actually showed a higher basal expression in the camta2/3 and camta1/2/3 mutants and an enhanced touch responsiveness, suggesting that they may also be regulated by a third pathway.
Fig. 6. qPCR analysis of selected touch-responsive genes in seedlings of Col-0, camta3-1, camta2/3, and camta1/2/3 at 0 and 22 min after gentle brushing.
The y axis represents fold inductions relative to untouched (0 min) Col-0 value (set to 1). Error bars designate SEM (n = 3 to 4). Statistical significance was determined by two-way ANOVA followed by post hoc Tukey tests (letters show significant difference between all samples at P < 0.05).
Detailed analysis of the promoter regions of TCH2, TCH4, bHLH19, and EXLA3 showed the presence of CGCGT-related CAMTA1-3 binding motifs (52), suggesting direct interaction of CAMTA1-3 with their promoters (table S12). To assess whether CAMTA3-related transcription factors directly regulate the expression of TCH2, TCH4, bHLH19, and EXLA3, we assessed their binding using a DNA affinity purification sequencing (DAP-seq) dataset (53). Although CAMTA3 and CAMTA2 were not assessed in the DAP-seq study, CAMTA1 and related CAMTA5 were included in the assays, showing a clear and significant enrichment for CAMTA1 binding in the TCH2, TCH4, EXLA3, and bHLH19 promoters (Fig. 7A). Binding by CAMTA1 to the bHLH19 promoter appeared to be relatively weaker compared to TCH2 and TCH4. CAMTA5 binding could only be observed to the TCH2 promoter.
Fig. 7. CAMTA transcription factors bind directly to touch-induced promoters.
(A) DAP-seq analysis showing direct binding of CAMTA1 and/or CAMTA5 to the promoter regions of selected target genes. Peak heights are relative to number of reads mapped to a position. (B) EMSAs of CAMTA3 (1–153) to probes designed against specific regions of the indicated promoters. The sequences show 30 bp of the 40-bp probes; regions in red indicate CGCGT-related CAMTA3 binding sites, which were mutated in the mutated probes. Unlabeled excess competitor probe was added to outcompete CAMTA3 (1–153) binding to the radiolabeled probe.
To validate these interactions, we recombinantly expressed and purified a truncated 6xHis-tagged version of CAMTA3, containing the DNA binding domain present in the first 153 amino acids [CAMTA3 (1–153)], as the full-length 116-kDa protein was previously found not to be suitable for electromobility shift assays (EMSAs) (52). The binding of CAMTA3 (1–153) to the putative CGCGT binding sites present within the promoter regions that showed CAMTA1/5 binding in the DAP-seq data was assessed by EMSAs. For this purpose, 40–base pair (bp) 32P-radiolabeled probes were created containing the wild-type sequences or with specific mutations in the CGCGT-like motifs (Fig. 7B and table S12). The TCH2 promoter contained two adjacent motifs that were contained within the same probe, while the EXLA3 and bHLH19 promoters contained two and four CGCGT-like motifs, respectively. Strong binding of CAMTA3 (1–153) could be observed to probes for TCH2, TCH4, and EXLA3 (probe EXLA3-1 only). Two shifted bands of slightly different size could be observed in the TCH2 promoter, suggesting that both CGCGT motifs can be bound by CAMTA3 (1–153). The binding to the radiolabeled probes could be strongly reduced by addition of excess unlabeled probe. Specific mutation of the CGCGT sites in the probes completely abolished binding by CAMTA3 (1–153), further confirming the interaction is sequence-specific. Unexpectedly, no clear binding of CAMTA3 (1–153) to four different probes containing CGCGT-like sites in the bHLH19 promoter could be observed (Fig. 7B and fig. S10). Touch treatment did not affect the subcellular localization of CAMTA3-YFP, with a clear nuclear localization in both untouched seedlings and seedlings 22 min after touching (fig. S11). In conclusion, our results show that CAMTA1/3 can directly bind the promoters of TCH2, TCH4, and EXLA3 and perhaps weakly to the promoter of bHLH19.

CAMTA3 is required for thigmomorphogenesis

Regular touch treatment has been shown to result in reduced rosette size and delayed flowering in plants (7). As our transcriptomic analyses indicated altered touch responsivity at the gene expression level, we assessed the impact of fer-4, camta3, and piezo mutations on thigmomorphogenesis (Fig. 8 and figs. S12 and S13). Two-week-old soil-grown seedlings were exposed to mild touching with a brush twice daily until all plants had bolted. Regularly touched Col-0 plants showed a clear delay in bolting time, with the average untouched plant flowering around 27 days after sowing, while touched Col-0 plants only bolted around 31.5 days (Fig. 8, A to C). In addition, rosette size at day 35 after sowing was reduced by more than 40% in touched compared to untouched Col-0 (Fig. 8, D and E). A similar effect of regular touching was observed in piezo mutants (fig. S12), further confirming that Piezo mechanosensitive channels do not contribute strongly to touch responses in aboveground tissues. As a control for JA signaling-defective mutants, myc234 mutants were grown alongside camta3-1 mutants and showed a clear insensitivity to touch treatment, with no significant difference in bolting time in touched compared to untouched myc234 mutants, as previously reported (fig. S13) (18). The fer-4 mutant showed a strong reduction in overall growth compared to Col-0 even under untreated conditions as previously reported (54), with a three times smaller rosette area on day 35 under untouched conditions (fig. S12). Despite the general growth retardation, fer-4 plants were still very responsive to touch-induced delays in flowering and rosette size (fig. S12). In addition, camta3-1 plants showed a moderate reduction in rosette size under untreated conditions compared to Col-0 but flowered around the same time as Col-0 under untouched conditions, 27 days after sowing (Fig. 8, A to E). In contrast, camta3-1 mutants showed an almost complete insensitivity to regular touching, with no significant difference in bolting time and rosette size in touched compared to untouched camta3-1 mutants (Fig. 8, A to E). We also performed a similar experiment on the camta3-2 mutant, confirming the loss of thigmomorphogenesis was comparable to that observed in the camta3-1 mutant (fig. S14). In conclusion, CAMTA3 plays a critical role in thigmomorphogenesis in plants.
Fig. 8. Camta3 mutants show a loss of thigmomorphogenesis.
(A) Bolting time of camta3-1 mutant and the wild type in response to 18 days twice-daily touch treatment. (B) Line graphs show the percentage of bolting plants over the growth period (days after sowing). (C) Box and whisker plots show the comparison of average bolting day between the control group and the touched group. (D) Rosette size in camta3-1 mutants and the wild type after 18 days of twice-daily touch treatment. (E) Bar graph showing the significant reduction of Col-0 rosette area after touch treatment; no significant reduction in rosette size of camta3-1 mutants after touch treatment was observed. Means ± SE are shown. Statistical analysis was performed by a Student’s t test. The *** and n.s. represent P < 0.001 and P > 0.05, respectively.


Despite many years of research on how transcriptional responses to mechanical stimulation in plants are controlled, only few regulators have been identified and consistently validated. At least two signaling pathways contribute to the touch response (7, 18). One pathway appears to be controlled by a rapid release of JA and JA-Ile within 10 min, leading to degradation of JAZ repressors via ubiquitination by the SKP/Cullin/COI1 (SCFCOI) JA-receptor (7, 55, 56), allowing the activation of MYC2-related transcription factors. MYC2 and its partners then further activate JA/JA-Ile biosynthesis by directly regulating JA biosynthetic genes, resulting in a positive feedback loop (18). Many other transcription factors such as ORA47, bHLH19, and ERF109 are directly activated by MYC2, resulting in a broad transcriptional network, ultimately contributing to thigmomorphogenesis upon repeat touch treatments. In contrast, genes thought to be regulated by the largely uncharacterized JA-independent touch response pathway, such as TCH2, TCH4, CRK41, CML23, and WRKY48 still displayed a normal touch responsiveness in the myc2 myc3 myc4 (myc5) mutants (18), underlining the separation of these signaling pathways. In agreement, TCH2, TCH4, CRK41, CML23, and WRKY48 were found to be not differentially expressed by MeJA treatment (48).
Our analysis shows that the MYC2- to MYC5-dependent JA pathway is kept in a repressed state by the Feronia kinase. Furthermore, after touch treatment, fer-4 is needed to keep the JA-dependent signaling response in check (Figs. 1, 2, and 4). As the fer-4 mutant is severely affected in general growth even under normal conditions, it can be concluded that repressing the MYC2/JA pathway is needed for optimal growth under favorable conditions (19). As in MYC2-related mutants, the expression of JA-independent touch-responsive genes TCH2/4 was unaffected in fer-4 mutants. This repressive role in the regulation of JA-dependent touch responses and a lack of role in regulation of TCH2-4 genes in our experiments is different to previous findings that FER is required for mechanical signal transduction (16). A potential explanation may be that our study focused on whole seedlings (which contain far more shoot than root biomass) responding to “classical” mechanical stimulation by gently brushing, while the other study specifically looked at root tissues and used hypo-osmotic shock to cause mechanical stress. Our finding that fer-4 mutants—despite their already very small size—still display a very pronounced reduction in rosette size and delay in flowering upon regular touching (fig. S12), further suggesting that, in aboveground tissues, Feronia is unlikely to act as an activator of touch responses. A recent study showed a direct interaction between Feronia and MYC2, showing that MYC2 phosphorylation by Feronia leads to an inactivation and destabilization of MYC2 (57). Together with our findings, it appears that Feronia acts as a kinase repressing MYC2 already under normal conditions and preventing an exaggerated touch response upon mechanical stimulation. The Feronia-related kinase Theseus1 and the OXI1 kinase, however, do not appear to play a clear role in mechanical signal transduction.
Although at the start of our work the Arabidopsis Piezo homolog was completely uncharacterized, very recently, it was confirmed to be an MS channel in root tips (58). The piezo mutants showed no visible growth defects in aboveground tissues under normal condition, as also reported recently (38, 58, 59). Despite constitutive deregulation of 160 genes, whole piezo seedlings showed largely wild-type–like transcriptional touch response and thigmomorphogenesis. Other studies observed that Piezo is expressed in root cap cells and is needed for normal penetration of denser barriers in the substrate possibly via affecting Ca2+ signaling (38, 58). Mutants in plasma membrane Ca2+-permeable mechanosensitive channels MCA1 and MCA2 showed similar defects in the root penetration of dense agar as observed in piezo mutants (60), yet like here the transcriptional response to touching of aboveground tissues seemed unaffected (23). Therefore, it appears that mechanical sensing to guide root growth is likely to be regulated differently to thigmomorphogenesis in aerial tissues.
Although a few potential regulators of the JA-independent touch response (represented by e.g., TCH2-4 and EXLA3) have been suggested in the past, including EIN6 and Feronia (30), we could not confirm these observations in seedlings mechanically stimulated by brushing. Through detailed analysis of the promoters of highly touch-responsive genes, we identified CAMTA3 and its homologs CAMTA1/CAMTA2 as crucial regulators of JA-independent touch signaling. Already in the single camta3-1 mutant, a moderate loss of induction for TCH2 at 22 min after brushing could be observed (Figs. 1 and 5). In-depth RNA-seq analysis revealed that a largely different set of touch-responsive genes was affected in the camta3-1 mutant compared to fer-4 and myc2 myc3 myc4 mutants. The promoters of these genes were also exclusively overrepresented in CAMTA3-binding sites, but not, e.g., MYC2 G-boxes. Furthermore, the CAMTA3-specific genes showed either no or weak increase in expression when treated with MeJA (48, 49). Transcript analysis of the camta1/2/3 triple mutant revealed that they cooperate to induce the expression of TCH2 and TCH4 upon touch treatment, which appears to occur via direct binding of their promoters. TCH3 and WRKY33 were oppositely regulated, showing higher basal expression in the triple mutants as previously reported (51). In addition, after touching, WRKY33 and TCH3 where more highly induced in camta1/2/3 mutants. WRKY33 and TCH3 are positively regulated by SA (61), so their elevated expression is likely explained by the very high levels of SA in camta2 camta3/camta1/2/3 (51). Full bHLH19 touch-induced expression relies on both the MYC2 MYC3 MYC4 and CAMTA1 CAMTA2 CAMTA3 pathways. This may be due to additive effects with both sets of transcription factors contributing to bHLH19 expression, or that some cooperative regulation is in place between CAMTAs and MYC234, possibly by synergistic binding of the bHLH19 promoter.
Such a dualistic role of CAMTA1-3 has been previously reported, being repressors for SA-dependent signaling and immunity while being activators of cold-induced gene expression (51, 62). As no SA induction could be observed by mechanical stimulation (18), we hypothesize that TCH3 touch responsiveness is likely regulated by CAMTA1-3 in wild-type plants, but that in the triple mutant, this is overruled by the excessive amounts of SA. CAMTA3 has a complex protein structure with an N-terminal repression module (NRM) thought to repress SA signaling, which acts independently to the C-terminal IQ and calmodulin-binding domain (CaMD). Recent evidence suggests that rapid Ca2+ release by, e.g., cold treatment causes calmodulin to bind the CAMTA3 IQ and/or CaMD domains, thereby inactivating the SA-repression by the NRM domain (63). It has also been suggested the CAMTA3 can “decode” different Ca2+ signatures released by various stresses (64). It is therefore tempting to speculate that the specific rapid Ca2+ spike(s) released upon mechanical stimulation trigger calmodulin-binding of CAMTA1-3, thereby stimulating their positive role in gene expression of a specific subset of touch-responsive genes. As TCH2 encodes a calmodulin-like (CML24) protein, it could be involved in amplifying the touch response in a positive feedback loop.
Besides transcriptional effects on touch-responsive gene expression in camta3 mutants, we observed a notable insensitivity to touch-induced effects on plant growth and flowering. This indicates that CAMTA3 plays the major role of the CAMTA1-3 homologs with regard to touch signaling, as also observed for instance in their normal growth phenotype and repression of SA signaling (51). Previous work showed that mutants with impaired JA-dependent touch responses, such as aos and myc2 myc3 myc4, display impaired thigmomorphogenesis (7, 18). Our results show that camta1/2/3 mutants specifically fail to induce JA-independent touch signaling genes TCH2, TCH4, and CML23. Even single camta3 mutants have impaired thigmomorphogenesis. This suggests that both the JA-dependent and JA-independent pathways may need to be activated simultaneously to repress growth and flowering. How both pathways would interact and form a logic “AND” gate to repress flowering (possibly by regulating a complementary set of genes) will require further research. Mutants with impaired touch response do not necessarily appear to have severe growth defect phenotypes under normal conditions as observed for myc2 myc3 myc4 mutants (18), although camta3 and especially camta1/2/3 have progressively stronger growth defects.
While we now have a much better view of the transcription factors that induce touch-responsive gene expression, we still have no clear understanding of how the touch-induced mRNAs are so rapidly removed within 10 to 20 min after the peak of expression. The vcs, xrn4, and caf1a caf1b mutants showed normal mRNA induction and degradation patterns, indicating that they are either not important or that additional (redundant) factors cooperate. For instance, there are 11 Caf1a-related genes in the Arabidopsis genome, while VCS and VCS-related are neighboring genes on the Arabidopsis chromosome 3.
In conclusion, how touch responses are activated at a transcriptional level is getting more defined, with key regulators of the JA-dependent and JA-independent pathways now identified (Fig. 9). However, the mechanisms of the upstream events that lead to the JA release and CAMTA1-3 activation, likely involving mechanically activated channels and Ca2+ transients, are less well known. In addition, understanding how touch-responses are switched off rapidly and lead to growth reduction upon regular treatments requires further investigation.
Fig. 9. A model of touch-induced gene expression pathways.
Mechanical stimulation results in physical deformation of the cell wall and plasma membrane. Several plasma membrane–associated proteins are important in regulation of downstream signaling and gene expression. Mechanically activated Ca2+ channels allow influx of Ca2+ ions from the apoplast to the intracellular space. Ca2+ may be captured by calmodulins, which, in turn, may modulate the activity of downstream regulators such as the transcription factors CAMTA1-3. Next, CAMTA1-3 activate the expression of JA-independent touch-responsive genes, e.g., cell wall modifiers (EXLA1/3 and TCH4) and other calmodulins (TCH2/3 and CML23), potentially acting as a positive feedback loop. Simultaneously, mechanical stimulation also results in activation of a JA-dependent signaling pathway mediated by MYC2/3/4/5 transcription factors, activating downstream genes involved in defense and JA biosynthesis [such as 13-lipoxygenases (LOX), 12-oxophytodienoic acid (OPDA) reductases, and allene oxide cyclases], also acting as a positive feedback loop. Kinases such as calcium-dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPKs) are likely involved in mediating signaling from, e.g., the plasma membrane to downstream regulators such as TREPH1 and transcription factors. Feronia (FER) receptor–like kinase negatively regulates excessive JA-dependent touch signaling, likely by inhibitory phosphorylation of MYC2/3/4/5. FER may also regulate part of the touch response independently of JA/MYC2 pathways. Some overlap between the JA-dependent and JA-independent signaling pathways also occurs, as, for instance, bHLH19 requires both pathways to be fully touch-induced. Collectively, all these changes can translate into various thigmomorphogenic and defense-related responses.


Plant materials and touch treatment

To study touch-mediated molecular responses, Arabidopsis thaliana wild-type (Col-0 and Ler-0) and mutant (listed Table 1) plants were grown as follows. Arabidopsis seeds were surface sterilized with chlorine gas. The seeds were sown in plates containing 1× MS with vitamins, 1% sucrose, 1% phytoagar, MES (0.5 g/liter), and pH adjusted to 5.8. The plates were stratified at 4°C for 3 days before transferring to standard growth condition (21°C, 16-hour light/8-hour dark cycle, 100 μmol m−2 s−1). To study the touch-mediated molecular responses, 12-day-old wild-type and mutant seedlings were gently brushed with a soft paint brush for 10 times, taking about 30 s per plate (see movie S1). For transcript analysis, samples were collected 0, 22, and 40 min after touch treatment and then snap-frozen in liquid N2 and stored at −80°C until use. Approximately 10 to 12 seedlings were harvested to represent one biological replication. Four to five biological replicates were used for statistical analysis unless otherwise stated. The list of mutants used in this study is available in Table 1. Mutants were either obtained from the Nottingham Arabidopsis Stock Centre (NASC) or requested from other laboratories, and then, genotypes were confirmed using PCR. Myc2 myc3 myc4 myc5 was obtained from G. Howe (Michigan State University); caf1a caf1b and camta3-1 were obtained from K. Dehesh (University of California Riverside); fer-4 and the1 were obtained from B. Dotson (Lund University); vcs-7 was obtained from L. Sieburth (University of Utah); vcs-8 and xrn4 were gifted by C. Bailly (Sorbonne Université); glr3.3 glr3.6 was gifted by E. Farmer (University of Lausanne); wrky18 wrky40 wrky60 were gifted by D.-P. Zhang (Tsinghua University); camta1/2/3 multiple mutants were gifted by M. Thomashow (Michigan State University); and camta3-2, p35s::CAMTA3-YFP lines; 19-5 (OX1) and 32-2 (OX2) were gifted by J. Lee (Leibniz Institute of Plant Biochemistry).

RNA extraction, cDNA synthesis, and RT-qPCR

RNA extraction, cDNA synthesis, and RT-qPCR were performed as previously described by (65). Primers are shown in table S12. Briefly, frozen tissue samples were ground in a fine powder using a Qiagen Tissuelyser II bead mill, and total RNA was isolated using a Spectrum Plant Total RNA kit (Sigma-Aldrich, STRN250-1KT) with On-Column deoxyribonuclease (DNase) treatment (Sigma-Aldrich, DNASE70) according to the manufacturer’s instructions. A total of 500 ng of total RNA was used for cDNA synthesis using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). RT-qPCR was performed using diluted cDNA (1:7) and SYBR green fluorescent dye (SsoAdvanced Universal Sybr green mix, Bio-Rad). A list of primers used for the RT-qPCR is available in table S12. For normalization, ubiquitin was used as internal control gene. The relative transcript level of each transcript was calculated by comparing touched versus control plants according to (66). Data were analyzed using two-way ANOVA (genotype × treatment) with post hoc Tukey tests (letters show significant difference between all genotypes in the different time points at P < 0.05). Because of the dynamic range of touch induced gene expression, the values were not always normally distributed, as indicated by the Kolmogorov-Smirnov test. For those genes, the expression values were log2-transformed to obtain normal distribution before ANOVA analysis.

RNA-seq library preparation and differential gene expression analysis

For RNA-seq library preparation, total RNA was treated with Ambion Turbo DNase (Thermo Fisher Scientific, AM1907) and quantified using the Qubit RNA BR Assay Kit (Invitrogen, Q10210). Four biological replicates (n = 4) were used for 0-min samples of Col-0, camta3-1, fer-4, and piezo and 22-min samples of Col-0 and piezo. Three biological replicates (n = 3) were used for 22-min samples of camta3-1 and fer-4. Then, 500 ng of RNA was used for library preparation using Illumina TruSeq mRNA (poly-A selection) and TruSeq RNA UD Indexes for up to 96 samples (Illumina, 20022371). Samples were pooled and sequenced on a half Illumina NovaSeq6000 S4 lane, 2 × 150 bp reads, incl Xp kit. Data were processed using demultiplexing and quality controlled with FastQC. Alignment of reads was performed against the TAIR11 annotation using STAR (67). On average, 32 million reads per sample were generated. Counts were assigned to genes using featureCounts (68), and analysis of DEGs was performed with DeSEQ2 (69). Transcripts were considered differentially expressed if padj < 0.05 and fold change ≥ 1.5 or fold change ≤ −1.5. Raw RNA-seq data files are available on ArrayExpress (accession number E-MTAB- 10920). GO enrichment analysis was performed using TF2Network and GOrilla (70).

Thigmomorphogenesis analysis

Seeds were stratified at 4°C for 2 days before grown in soil mixture (soil, perlite, and vermiculate 4:1:1) under the standard growth conditions (22°C, 16-hour light/8-hour dark, 100 μmol m−2 s−1). Two-week-old seedlings were gently brushed twice daily approximately 10 times with a soft art paint brush. Touch treatment was applied for 18 to 26 days until all plants had bolted depending on the genotypes. Plants were considered to have bolted when the inflorescence stock reached to 1-cm height, and percentage of bolting was calculated as previously described (25). The rosette area of 31- to 35-day-old plants was measured using ImageJ.

Electromobility shift assays

The DNA binding domain of CAMTA3 (amino acids 1 to 153) was cloned into pDEST17 6xHisfusion vector and recombinantly expressed in Rosetta 2 (DE3) cells as previously described (71). In brief, a 50-ml culture was grown overnight at 28°C. The culture to was diluted in 1 liter of LB media to an optical density at 600 nm (OD600) of 0.1 and subsequently grown to OD600 0.6 at 28°C. Protein expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside and incubated at 28°C for 6 hours. Cells were pelleted by centrifugation and resuspended in 2 × 10 ml of lysis buffer containing one tablet of Roche cOmplete EDTA-free mini protease inhibitor cocktail and lysozyme, followed by sonication for 2 min in 5-s blocks and 10-s intervals. Purification was performed using GE Healthcare HisTrap FF columns according to the manufacturer’s instructions. Oligonucleotide probes (40 bp; table S12) were annealed and phosphorylated using NEB T4 polynucleotide kinase with gamma 32P-ATP (PerkinElmer) and purified using G-25 Sepharose Quick spin columns (Roche). Binding reactions were performed as previously described (71), separated on 6% acrylamide 0.5× tris-borate EDTA gels, dried, and imaged using a phosphorimager.

Confocal microscopy

Arabidopsis seedlings expressing CAMTA3-YFP were grown vertically on MS plates for 5 days, then subjected to control (untouched) or touch treatment, and stained with 4′,6-diamidino-2-phenylindole (DAPI). Root cells of the stained plants were used to detect the fluorescence under a Leica SP8 TCS confocal microscope. Samples were imaged with laser excitation at 514 nm and emission at 500 to 570 nm for YFP, while DAPI nuclear fluorescence was observed with excitation at 405 nm and emission at 415 to 476 nm.

Accession numbers

RNA-seq data files are available from ArrayExpress with accession number E-MTAB-10920. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.


This work is dedicated to the memory of Alex Van Moerkercke (1979-2021). We thank the National Genomics Infrastructure for assistance with RNA-seq analysis. We thank F. Paul for laboratory assistance.
Funding: O.V.A. was supported by the Swedish Research Council (Vetenskapsrådet, 2017-03854 and 2021-04358), Crafoord Foundation (20170862), Carl Trygger Foundation (CTS 17: 487), Carl Tesdorpf Stiftelse, PlantLink Seed Money, and NovoNordiskFonden (NNF18OC0034822). R.G. was supported by the Sven and Lilly Lawski Foundation (N2020-0033). O.V.A and K.K. were supported by the Wenner-Gren foundation (UPD2019-0211). A.V.M. (39908), E.D., H.C.T., and K.K. (41776) were supported by the Royal Physiographic Society of Lund. M.B. was supported by an IPRS scholarship at University of Western Australia, funded by the Australian government.
Author contributions: O.V.A., E.D., and R.G. conceived and planned the project. E.D., R.G., A.O.-C., K.K., M.P., M.B., H.C.T., L.D.M., A.V.M., and O.V.A. performed experiments. E.D., O.V.A., R.G., A.V.M., and A.G. analyzed data. O.V.A., E.D., and R.G. wrote the manuscript with input from the coauthors.
Competing interests: The authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S14

Other Supplementary Material for this manuscript includes the following:

Tables S1 to S12
Movie S1


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Science Advances
Volume 8 | Issue 20
May 2022

Submission history

Received: 3 September 2021
Accepted: 7 April 2022


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This work is dedicated to the memory of Alex Van Moerkercke (1979-2021). We thank the National Genomics Infrastructure for assistance with RNA-seq analysis. We thank F. Paul for laboratory assistance.
Funding: O.V.A. was supported by the Swedish Research Council (Vetenskapsrådet, 2017-03854 and 2021-04358), Crafoord Foundation (20170862), Carl Trygger Foundation (CTS 17: 487), Carl Tesdorpf Stiftelse, PlantLink Seed Money, and NovoNordiskFonden (NNF18OC0034822). R.G. was supported by the Sven and Lilly Lawski Foundation (N2020-0033). O.V.A and K.K. were supported by the Wenner-Gren foundation (UPD2019-0211). A.V.M. (39908), E.D., H.C.T., and K.K. (41776) were supported by the Royal Physiographic Society of Lund. M.B. was supported by an IPRS scholarship at University of Western Australia, funded by the Australian government.
Author contributions: O.V.A., E.D., and R.G. conceived and planned the project. E.D., R.G., A.O.-C., K.K., M.P., M.B., H.C.T., L.D.M., A.V.M., and O.V.A. performed experiments. E.D., O.V.A., R.G., A.V.M., and A.G. analyzed data. O.V.A., E.D., and R.G. wrote the manuscript with input from the coauthors.
Competing interests: The authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.



Department of Biology, Lund University, Lund, Sweden.
Plant Physiology Section, Agricultural Botany Department, Faculty of Agriculture, Cairo University, Egypt.
Roles: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Department of Biology, Lund University, Lund, Sweden.
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, and Writing - original draft.
Abraham Ontiveros-Cisneros
Department of Biology, Lund University, Lund, Sweden.
Roles: Investigation and Resources.
Department of Biology, Lund University, Lund, Sweden.
Role: Investigation.
Department of Biology, Lund University, Lund, Sweden.
Roles: Formal analysis, Investigation, and Validation.
Liesbeth De Milde
Department of Plant Biotechnology and Bioinformatics, Ghent University, Gent, Belgium.
VIB Center for Plant Systems Biology, Gent, Belgium.
Role: Validation.
ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, University of Western Australia, Perth, Australia.
Roles: Data curation, Investigation, and Resources.
Department of Plant Biotechnology and Bioinformatics, Ghent University, Gent, Belgium.
VIB Center for Plant Systems Biology, Gent, Belgium.
Roles: Conceptualization, Project administration, Resources, Supervision, Validation, and Writing - review & editing.
Alex Van Moerkercke
Department of Biology, Lund University, Lund, Sweden.
Department of Biology, Lund University, Lund, Sweden.
Roles: Conceptualization, Formal analysis, Investigation, Methodology, Validation, and Writing - review & editing.
Department of Biology, Lund University, Lund, Sweden.
Roles: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.

Funding Information

Vetenskapsrådet: 2017-03854
Vetenskapsrådet: 2021-04358
Sven and Lilly Lawski Foundation: N2017-0079
Novo Nordisk Fonden: NNF18OC0034822
Carl Tesdorpf Stiftelse
Sven and Lilly Laswki Foundation: N2020-0033


Corresponding author. Email: [email protected]

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