Transcriptomic Evaluation of the Enhanced Plant Growth-Inhibitory Activity Caused by Derivatization of Cis-Cinnamic Acid

J. Pestic. Sci. 39(2), 1–6 (2014) DOI: 10.1584/jpestics.D13-090

Original Article

Transcriptomic evaluation of the enhanced plant growth-inhibitory activity caused by derivatization of cis-cinnamic acid

Naoya Wasano,1 Mami Sugano,1 Keisuke Nishikawa,2 Katsuhiro Okuda,2 Mitsuru Shindo,2 So-Young Park,1 Syuntaro Hiradate,1 Tsunashi Kamo1,* and Yoshiharu Fujii1

1 Biodiversity Division, National Institute for Agro-Environmental Sciences, 3–1–3 Kannondai, Tsukuba, Ibaraki 305–8604 Japan 2 Institute for Materials Chemistry and Engineering, Kyushu University, 6–1 Kasuga-Koen, Kasuga, Fukuoka 816–8580, Japan (Received December 25, 2013; Accepted February 3, 2014)

To establish a rapid high-throughput evaluation system for the enhanced plant growth-inhibitory activity caused by modifications of cis-cinnamic acid’s (cis-CA’s) chemical structure, a DNA microarray assay was used to analyze the changes in early gene responses of Arabidopsis thaliana seedlings. After a 6-h exposure to (Z)-3-(3-iodophenyl)acrylic acid, we observed an upregulation in three classes of early auxin-responsive genes, which was similar to the transcriptional response to indole-3-acetic acid (IAA), together with an upregulation of the genes related to environmental stress and toxin detoxification responses. Gene responses to 2-(3,4-dihydronaphthalen-1-yl)acetic acid were similar to those to IAA. In contrast, fewer genes were upregulated in response to its double-bond isomer, (Z)-2-[3,4-dihydronaphthalen-1(2H)-ylidene]acetic acid, than to cis-CA. DNA microarray data suggest that the structurally different cis-CA analogues trigger diverse gene responses. © Pesticide Science Society of Japan Keywords: cis-cinnamic acid analogues, Arabidopsis thaliana, DNA microarray, Gene Ontology analysis, structure-bioactivity relationship, auxin. Electronic supplementary materials: The online version of this article contains supplementary materials (Supplemental Figure S1 and Supplemental Tables S1–S4), which is available at http://www.jstage.jst.go.jp/browse/jpestics/.

agonist because its structure and physiological effects are similar Introduction to those of auxin.6) In a study using auxin-insensitive mutants When developing new products, particularly pesticides or medi- (aux1 and axr2), however, Wong et al. suggested that the mode cines, it is essential to study the relationship between structure of action of 1 was different from that of auxin.7) Guo et al. fur- and activity to improve the potency of the principal chemicals. ther reported that treatment with 1 induced two Arabidopsis As a source of candidates for the development of agrochemicals, genes, MLPL1 (AT2G01520) and MLPL2 (AT2G01530), which natural organic chemicals, including the secondary metabolites are considered to be functional in the regulation of bolting by of plants, have often been exploited. The herbicide mesotrione proteome and histochemical analysis.8) Wasano et al. examined was based on the structure of leptospermone from the lemon early cis-CA responsive genes by DNA microarray and report- bottlebrush, Callistemon speciosus.1) Likewise, the structures of ed that root-specific upregulation of the early auxin-responsive pyrethroid derivatives, used as insecticides, were derived from genes AUXIN/INDOLEACETIC ACID (Aux/IAA), GRETCHEN pyrethrins biosynthesized by Chrysanthemum cinerariaefolium.2) HAGEN-3 (GH3), and LATERAL ORGAN BOUNDARY 1-O-cis-Cinnamoyl-β-d-glucopyranose is a potent allelo- DOMAIN (LBD) occurred within 2 hr after anexogenous treat- chemical isolated from the Spiraea species that shows strong ment of 1.9) As yet, little is known about the physiological func- growth-inhibitory activity against some plants.3,4) This property tions and mode of action of 1 in plants. is attributed to the cis-cinnamate moiety because the strength Chemical derivatization of 1 has been conducted to intensi- of the root growth-inhibitory activity of 1-O-cis-cinnamoyl-β- fy the plant growth-inhibitory activity. Abe et al. constructed d-glucopyranose is the same as that of cis-cinnamic acid (cis- a series of cis-CA derivatives to clarify the key features of 1 for CA; 1).5) Compound 1 has long been thought to be an auxin lettuce root growth inhibition, and found that a plain ring, the cis-configuration of alkene, and carboxylic acid were essential 10) * To whom correspondence should be addressed. for the activity. Nishikawa et al. found that cis-CA analogues E-mail: [email protected] possessing the meta-iodo, meta-methoxy, and meta-trifluoro- 11) Published online ●●● ●●●, ●●● methyl groups on the aromatic ring were more potent than 1. © Pesticide Science Society of Japan Furthermore, they constructed conformationally-constrained 2 N. Wasano et al. Journal of Pesticide Science

tion of benzaldehyde, followed by hydrolysis of the ester of cis- olefin, in accordance with the literature.10) The meta-iodo analogue 4 was also prepared by using the procedure described above from commercially available 2-iodobenzaldehyde.11) The conformationally constrained analogue 2 was synthesized via the Horner–Wadsworth–Emmons reaction of α-tetralone and following hydrolysis.12) The endo-alkenyl analogue 3 was synthesized through the Reformatsky reaction of α-tetralone with the bromoacetic acid ethyl ester, dehydration, and hydrolysis.12) IAA was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

2. Plant materials and growth conditions Seeds of Arabidopsis thaliana L. (Col-0, Inplanta Innovations Fig. 1. Structures of cis-cinnamic acid (cis-CA; 1) and the cis-CA ana- Inc., Yokohama, Japan) were sterilized in 70% ethanol for 1 min, logues (2–4) used in this study. sterilized in 2% sodium hypochlorite with 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 8 min, and then rinsed cis-CA analogues, in which the aromatic ring and cis-olefin three times with sterilized distilled water. The sterilized seeds were connected by a carbon bridge, showing that the inhibito- were placed on 0.8% agar (Nakarai Tesque, Inc., Kyoto, Japan) ry activities of the five-membered and six-membered bridged with 0.5× Murashige and Skoog Plant Salt Mixture (Nihon compounds were 10 times stronger than 1.12) While these cis- Pharmaceutical Co. Ltd., Tokyo, Japan) and 1% sucrose (Wako CA analogues might serve as the lead chemicals in developing Pure Chemical Industries, Ltd., Osaka, Japan) in a sterilized new herbicides, the reasons for their ability to enhance growth- Petri dish (90-mm diameter). The seeds were held at 4°C over- inhibitory activity need to be clarified through the use of mo- night in darkness and then transferred to a growth chamber. lecular biology. Emerging plants were maintained in the growth chamber under DNA microarray analysis is an effective tool for investigating a schedule of 16 hr light (22°C). the global gene response of model plants.13,14) It has been used to monitor changes in transcript levels in response to various 3. Root growth inhibition assay allelochemical stresses on a whole-genome scale.9,15,16) Present- The root growth inhibition assay was performed on agar plates ly, new agrochemicals are required to control weed strains that (2% agar with 0.5× Murashige and Skoog Plant Salt Mixture are resistant to commercial herbicides, but evaluating promis- and 1% sucrose) containing various concentrations of cis-CA ing chemical derivatives is labor intensive and time consuming. analogues (1–4), and the sterilized seeds described above were DNA microarray analysis is potentially an efficient and rapid transferred to vertically oriented agar plates. Six days after sow- tool for evaluating the bioactivities of chemical derivatives. ing in the growth chamber under a schedule of 16 hr light (22°C) In the present study, we examined a rapid evaluation and 8 hr dark (20°C), the root lengths were measured using an method of substituent effects on plant growth inhibition SZH dissecting microscope (Olympus, Tokyo, Japan) and Sen- at the molecular level. We selected three cis-CA analogues, sivMeasure image measuring software (Mitani, Fukui, Japan). (Z)-2-[3,4-dihydronaphthalen-1(2H)-ylidene]acetic acid The effective concentration required for half of the maximum

(2), 2-(3,4-dihydronaphthalen-1-yl)acetic acid (3), and (Z)-3- inhibition (EC50) was calculated using the probit method with (3-iodophenyl)acrylic acid (4), which have been reported to SPSS for Windows ver. 11.0.1J statistical software (SPSS Japan possess stronger growth-inhibitory activity than 1 against let- Inc., Tokyo, Japan). tuce roots.11,12) Two of them are a conformationally constrained To evaluate the persistency of the bioactivity of these cis-CA cis-CA analogue connecting the aromatic ring and cis-olefin by analogues, Arabidopsis seeds were sown on 80 mL of medium a six-membered carbon bridge and its double-bond isomer; the (0.5× Murashige and Skoog Plant Salt Mixture, 1% sucrose, other is meta-iodo-substituted cis-CA analogue (Fig. 1). First, and 0.4% gellan gum) containing 10 µM of cis-CA analogues in we conducted short- and long-term bioassays of these chemicals Agripots (Kirin Co. Ltd., Tokyo, Japan). Each pot contained 15 against Arabidopsis. Then, we monitored gene responses to the seeds, and they were grown in the same conditions as described tested compounds using a DNA microarray. Finally, we estimat- above. Two weeks after sowing, the total weight of the seed- ed the physiological changes of Arabidopsis seedlings from the lings was measured. A statistical analysis (one-way ANOVA and Gene Ontology analysis data. Tukey–Kramer test) was performed using SPSS software. Materials and Methods 4. DNA microarray analysis 1. Chemicals Plants were treated with 20 µM of 2, 3, or 4. Control plants were The synthesis of 1 was performed by the Z-selective olefina- not treated. The plants were sampled at 6 hr post treatment and Vol. 39, No. 2, 000–6 (2014) Transcriptomic evaluation of plant’s response to cis-cinnamic acid analogs 3 immediately frozen in liquid nitrogen. Total RNA was extracted Results and Discussion from seedlings with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The 1. Bioactivities of cis-CA analogues quantity and quality of the extracted RNAs were checked with We observed the concentration-dependent root growth-inhib- an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, itory activities of the cis-CA analogues against Arabidopsis. CA, USA). The two-color spike mix was added to the total RNA, As shown in Table 1, all of the cis-CA analogues tested (2–4) and the RNA was labeled with a Quick Amp Labeling Kit (Agi- showed stronger activities than 1, which is consistent with pre- lent Technologies) according to the manufacturer’s two-color vious studies using lettuce seedlings.10–12) When the bioactivity protocol. Fluorescent cRNA was generated from the total RNA. was evaluated 6 days after chemical exposure, the results reflect- Briefly, 500 ng of RNA was reverse transcribed using MMLV ed an acute toxicity. Thus, a long-term bioassay was necessary to reverse transcriptase and an oligo(dT) primer containing the evaluate the cis-CA analogues as candidates for lead compounds T7 promoter. It was subsequently transcribed in vitro using T7 in developing herbicides. We observed the growth-inhibitory RNA polymerase, resulting in Cy3-labeled (control) and Cy5- activity of these chemicals after a two-week exposure to a cis- labeled (cis-CA-treated) cRNAs. The cRNAs were purified using CA analogue. The growth-inhibitory activities of 1 and 2 de- RNeasy Mini Spin columns (Qiagen) and then quantified with creased drastically two weeks after chemical exposure (Fig. 2). a NanoDrop ND-1000 UV–VIS spectrophotometer (NanoDrop In contrast, 3 and 4 inhibited seedling growth after two weeks. Technologies, Wilmington, DE, USA). Mixtures of 825 ng of Based on the results of the long-term bioassay, 3 and 4 are ap- Cy3-labeled and Cy5-labeled cRNAs were co-hybridized at parently more promising candidates for herbicide development 65°C for 17 hr on an Agilent Technologies 4×44 K Arabidop- than 2 (Fig. 2), although the three compounds (2–4) showed an sis (v4) 60-mer oligo-microarray. The slides were then washed equivalent level of activity in the short-term bioassay (Table 1). and the fluorescence intensity detected using an Agilent G2505B Comparison of the root growth-inhibitory activity against Scanner. Two independent biological replicates were assayed. Arabidopsis between 1, IAA, and its artificial analogue, 1-naph- The fluorescence intensity of individual spots on scanned -im thaleneacetic acid (NAA), has been performed by Wong et al. ages was quantified and corrected for background noise using Feature Extraction software (Agilent Technologies). To ensure Table 1. Arabidopsis root growth-inhibitory activity of cis-cinnamic a high quality of analysis, only features that passed three cri- acid (cis-CA; 1) and its analogues (2–4) teria were analyzed. Features flagged in the Feature Extraction The fiducial limit at Tested compound EC (µM) software as non-uniform (IsFeatNonUnifOL and IsFeatPop- 50 95% level nOL), saturated (IsSaturated), or low signal (IsWellAboveBG) 1 2.98 2.43–3.82 were omitted, and the remaining features were further filtered by the Feature Significance test at p<0.01. Total RNA profiling 2 1.26 1.02–1.53 data were normalized via linear and lowess methods followed 3 0.72 0.64–0.80 by spike-in normalization using a Two-Color RNA Spike-In Kit 4 0.49 0.43–0.56 (Agilent Technologies). The ratio of the intensity in thecis -CA- treated sample (Cy5) to that in the control sample (Cy3) was calculated for each gene. We defined a gene as responsive when the ratio of both biological replicates was greater than three to one. Ratios shown are the averages of the two independent experiments. In this manuscript, the gene ID number and the gene names are from The Arabidopsis Information Resource website (TAIR: http://www.arabidopsis.org/). We used the web-based toolkit agriGO (http://bioinfo.cau.edu.cn/agriGO/index.php) for the Gene Ontology (GO) enrichment analysis.17) Each expression pattern was analyzed with the SEA program and tested by a binomial test model with a false discovery rate of <0.01. In the graphical results of the GO analysis, the degree of color saturation of a box is positively correlated to the enrichment level of the GO term. Microarray data are available through the Gene Expression Omnibus (GEO) database at NCBI (http://www.ncbi. nlm.nih.gov/geo/). The accession numbers in the GEO databases Fig. 2. Effects of cis-cinnamic acid (cis-CA) analogues (1–4) on the are GSE37862, GSE37899, and GSE51400. biomass of Arabidopsis seedlings. Fifteen seeds were planted on 80 mL of Murashige & Skoog medium containing 1% sucrose and 10 µM of cis- CA analogue. Two weeks after sowing the total weights of the seedlings were measured. Means with different letters are significantly different by Tukey–Kramer HSD test (p<0.05) after a one-way ANOVA. 4 N. Wasano et al. Journal of Pesticide Science

7) (2005). The EC50 value of 1 (3.5 µM) in this report is equivalent tion mutation studies of GH3 genes caused negative effects on to our data shown in Table 1. IAA and NAA are more active plant growth.18,19) No additional distinguishable gene responses than 1, as they reportedly showed lower EC50 values (0.018 µM between treatments with 1 and 2 were observed. and 0.18 µM, respectively).7) Thus, the above-mentioned candi- 2.2 Estimation of the physiological changes in cis-CA- dates (3 and 4) are more active than 1 and less active than IAA analogue-treated Arabidopsis seedlings from the GO analy- and NAA. sis data By using a hierarchical tree diagram of overrepresented GO 2. DNA microarray analysis terms based on the biological process category,17) the activated 2.1 Gene response and Gene Ontology analysis physiological functions after exposure to cis-CA analogues are Table 2 shows the number of responsive genes whose transcript predictable. GO terms involved in activated physiological func- levels changed more than threefold 6 hr post exposure to the cis- tions formed clusters of high significance level GO terms, as CA analogues. Treatment with 4, as compared with 1, resulted shown by the high degree of color saturation of a box (Supple- in a drastic increase in the number of responsive genes. More mental Fig. S1). Two hierarchical tree diagrams of overrepre- than 1,000 genes were upregulated after a 6-hr exposure to 4. sented GO terms were constructed on the basis of the biological Singular Enrichment Analysis (SEA) of the upregulated genes process category. identified 51 GO terms as significant, and the genes associated Graphical results of the genes upregulated in response to 4 are with auxin stimulus response (GO: 0009733) were overrepre- shown in Supplemental Fig. S1A. Four GO term clusters with sented at a high level of significance, together with responses to high significance levels were divided into two groups: one was heat (GO: 00009408), toxin catabolic processes (GO: 00009407), categorized as “response to auxin,” and the other “response to and responses to jasmonic acid stimulus (GO: 0009753). GO stresses,” which included the GO terms “response to jasmon- analysis based on the molecular function revealed that the UDP- ic acid,” “responses to heat,” and “toxin catabolic process.” The glucosyltransferase (GO: 0035251) category was overrepresented upregulated genes belonging to the GO category “responses to (Supplemental Table S1). heat” (Supplemental Table S3) were mainly comprised of heat After exogenous treatment with 3, Arabidopsis seedlings ex- shock proteins, which act as molecular chaperones to prevent hibited 413 upregulated and 437 downregulated genes at levels protein misfolding caused by reactive oxygen species (ROS).20) equivalent to those after treatments with 1 and IAA (Table 2). Of Eleven of the genes listed in Supplemental Table S3 also belong the 413 upregulated genes, 407 were annotated in the query list. to the GO term “response to hydrogen peroxide (GO: 0042542),” SEA identified 17 GO terms as significant (Supplemental Table implying that the meta-substitution of an iodine group on S2); the top seven overrepresented GO terms, ranked by their p- the aromatic ring of cis-CA added a function that generated values, were all related to auxin response. ROS. Interestingly, one of the heat-responsive genes, AtBAG6 Treatment with 2, as compared with 1, induced a smaller (AT2G46240), is included in Supplemental Table S3. BAG number of responsive genes. Of the 220 upregulated genes, 213 (BCL2-associated athanogene) is a gene family that was origi- were annotated in the query list. SEA identified only two GO nally identified in mammals that encodes calmodulin-binding terms as significant: lipid localization (GO: 0010876) and en- proteins known to associate with the anti-apoptotic protein, domembrane system (GO: 0012505). Although the GO term BCL2. Kang et al. reported that the expression of AtBAG6 was “response to auxin stimulus” was not significantly overrepre- strongly associated with the induction of programmed cell death sented after treatment with 2, a more than 30-fold upregulation in yeast and plants.21) Therefore, the induction of AtBAG6 ex- of GH3 family genes (GH3.1, GH3.2, and GH3.3) and a more pression might partially contribute to enhanced growth-inhibi- than fivefold upregulation of LBD family genes (LBD16, LBD17, tory activities. Past studies have demonstrated that xenobiotics LBD18, LBD29, and LBD33) were observed as responses to induce putative detoxification pathways involving glutathione treatment with 1. The upregulation of GH3 genes might contrib- S-transferases (GSTs), cytochrome P450s (CYP450s), and uri- ute to seedling growth regulation because some gain-of-func- dine diphosphate glucosyltransferases (UGTs) in plants.22) In our DNA microarray results, 14 GST genes, 18 cytochrome P450 Table 2. Number of responsive genes whose transcript levels changed genes, and 18 UGT genes were upregulated in response to treat- more than threefold 6 hr post exposure to cis-cinnamic acid (cis-CA; 1), ments with 4 (Supplemental Table S4). ATP-binding cassette its analogues (2–4), and indole-3-acetic acid (IAA) (ABC) proteins were originally identified in the detoxification Tested Number of responsive genes process as transporters of glutathione S-conjugated chemicals Reference 23) compound Upregulated Downregulated to vacuoles. In the present study, three ABC transporters were upregulated after treatment with 4, including a 60-fold upregula- 1 383 364 Wasano et al. (2013) tion of AtPDR12, indicating that the genes related to detoxifica- 2 220 306 This study tion and elimination of xenobiotics were strongly upregulated in 3 413 437 This study response to 4. 4 1170 978 This study Graphical results of the genes upregulated in response to 3 IAA 403 472 Wasano et al. (2013) are represented in Supplemental Fig. S1B. Sixty of the genes Vol. 39, No. 2, 000–6 (2014) Transcriptomic evaluation of plant’s response to cis-cinnamic acid analogs 5

Table 3. Number of upregulated early auxin-responsive genes belonging to three gene families 6 hr post exposure to cis-cinnamic acid (cis-CA; 1), its analogues (2–4), and indole-3-acetic acid (IAA)

Number of upregulated genes by Gene family chemical treatment 1 2 3 4 IAA Aux/IAA 5 2 10 11 11 Fig. 3. Difference in the rotatability of the carboxyl group between the GH3 5 3 5 5 5 endo-alkenyl analogue 3 and its double-bond isomer 2. SAUR-related 4 0 21 31 22 chain carboxyl group.26,27) The carboxyl group of IAA anchors the plant hormone to the bottom of the TIR1 pocket by forming belong to the GO term “response to auxin stimulus,” and most a salt bridge and two hydrogen bonds with two residues from of them were classified in early auxin-responsive gene fami- the pocket floor (Arg 403 and Ser 438). Because 3 possesses lies (Aux/IAA, GH3, and SAUR). It is generally accepted that a single C–C bond connecting the aromatic ring, its carboxyl the three major classes (Aux/IAA, GH3, and SAUR) respond to group is able to rotate with a low-energy barrier such as IAA and auxin stimulus within five to 60 min.24,25) Furthermore, Wasano NAA (Fig. 3). In contrast, the rotation of the carboxyl group of 2 et al. suggested that the physiological function of 1 is distin- is limited by the position of the double C–C bond. This implies guishable from that of auxin.9) The response of auxin-respon- that cis-CA analogues with a constrained carboxyl group could sive genes to 3 was different from that to 1. Additionally, the show lower binding affinities to TIR1, and the transcriptomic number of upregulated SAUR genes was at a level comparable analysis in the present study supports this idea. to that of an IAA response (Table 3). Moreover, the transcript Physiological experiments using antagonists of auxin or ge- levels of Aux/IAA and GH3 induced by 3 were almost equal to netically modified Arabidopsis mutants are necessary to eluci- those induced by IAA. These results indicate that exogenous3 date the molecular mechanism responsible for the bioactivity of and IAA trigger transcriptomic induction in a similar manner in the cis-CA analogues. We showed, however, that a DNA micro- Arabidopsis. array could be a powerful tool for the rapid evaluation of structurally modified derivatives of allelochemicals. In this context, 3. Evaluation of cis-CA analogues as candidates for developing the analyzed data will be useful in subsequent studies, such as agrochemicals the design and synthesis of molecular probes for new potent We selected three candidates (2–4) from a series of cis-CA inhibitors that target new types of agrochemicals. derivatives based on information from previous structure- Acknowledgements bioactivity studies in lettuce seedlings,10–12) and showed that the bioactivity of 2 decreased in a long-term bioassay using We thank Dr. Sayaka Morita, Dr. Tomoko Takemura, and John S. Mani- Arabidopsis seedlings (Fig. 2). The other cis-CA analogues, 3 and nang for constructive discussions. This work was supported by the Pro- 4, had equivalent growth-inhibitory activities in this bioassay, gramme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN), Tokyo, Japan. which were stronger than those of 1 and 2. However, a global transcriptomic analysis indicated that there are differences in the References responsive genes between of 3 and 4. The gene response to treat- 1) G. Mitchell, D. W. Bartlett, T. E. M. Fraser, T. R. Hawkes, D. C. Holt, ment with 3 was almost identical to that with IAA, while that J. K. Townson and R. A. Wichert: Pest Manag. Sci. 57, 120–128 with 4 was far greater than those with 1 and IAA (Table 2). In (2001). addition to the response to auxin stimulus, 4 upregulated genes 2) F. Tattersfield, R. P. Hobson and C. T. Gimingham: J. Agric. Sci. 19, related to biotic and abiotic stresses. Thus, the DNA microar- 266–296 (1929). ray analysis method is effective in evaluating the enhanced plant 3) S. Hiradate, S. 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Supplemental Table S1. Singular Enrichment Analysis of upregulated genes in Arabidopsis in response to the meta-iodo analogue 4.

GO term Ontology Description Number in Number in p-value FDR

input list BG/Ref

GO:0042221 P response to chemical stimulus 200 2085 1.20E-45 2.10E-42

GO:0050896 P response to stimulus 274 4057 9.40E-37 8.50E-34

GO:0010033 P response to organic substance 141 1342 1.10E-35 6.50E-33

GO:0009719 P response to endogenous stimulus 121 1068 2.10E-33 9.40E-31

GO:0009725 P response to hormone stimulus 102 982 1.60E-25 5.60E-23

GO:0009733 P response to auxin stimulus 60 360 2.10E-24 6.30E-22

GO:0009408 P response to heat 30 161 5.40E-14 1.40E-11

GO:0006950 P response to stress 134 2320 1.30E-12 2.90E-10

GO:0019748 P secondary metabolic process 48 489 7.80E-12 1.60E-09

GO:0009611 P response to wounding 29 197 2.50E-11 4.50E-09

GO:0009753 P response to jasmonic acid stimulus 29 215 1.60E-10 2.70E-08

GO:0009266 P response to temperature stimulus 43 485 1.70E-09 2.60E-07

GO:0009404 P toxin metabolic process 14 53 6.40E-09 8.30E-07

GO:0009407 P toxin catabolic process 14 53 6.40E-09 8.30E-07

GO:0010200 P response to chitin 22 151 7.30E-09 8.80E-07

GO:0006979 P response to oxidative stress 33 332 1.10E-08 1.20E-06

GO:0009628 P response to abiotic stimulus 86 1471 1.10E-08 1.20E-06

GO:0009605 P response to external stimulus 38 429 1.50E-08 1.50E-06

GO:0009743 P response to carbohydrate stimulus 27 240 2.30E-08 2.20E-06

GO:0009642 P response to light intensity 16 90 7.20E-08 6.50E-06

GO:0009644 P response to high light intensity 13 57 9.80E-08 8.50E-06

GO:0042542 P response to hydrogen peroxide 12 53 3.30E-07 2.70E-05

GO:0010035 P response to inorganic substance 27 279 3.70E-07 2.90E-05

GO:0009695 P jasmonic acid biosynthetic process 9 29 1.10E-06 7.80E-05

GO:0000302 P response to reactive oxygen species 14 85 1.10E-06 7.80E-05

GO:0031407 P oxylipin metabolic process 10 39 1.20E-06 8.30E-05

GO:0031408 P oxylipin biosynthetic process 9 32 2.10E-06 0.00014

GO:0009694 P jasmonic acid metabolic process 9 34 3.30E-06 0.00021

GO:0008610 P lipid biosynthetic process 32 439 9.60E-06 0.0006

GO:0009607 P response to biotic stimulus 41 638 1.00E-05 0.00063

GO:0051707 P response to other organism 39 599 1.30E-05 0.00075

GO:0010876 P lipid localization 7 24 2.40E-05 0.0014

GO:0006790 P sulfur metabolic process 20 220 2.70E-05 0.0015

GO:0006629 P lipid metabolic process 47 841 6.70E-05 0.0036

GO:0070887 P cellular response to chemical stimulus 30 452 9.00E-05 0.0046

GO:0051704 P multi-organism process 43 776 0.00016 0.0081

GO:0009755 P hormone-mediated signaling pathway 23 321 0.00021 0.0099

GO:0032870 P cellular response to hormone stimulus 23 321 0.00021 0.0099

GO:0046527 F glucosyltransferase activity 21 136 6.40E-09 2.20E-06

GO:0035251 F UDP-glucosyltransferase activity 18 94 4.00E-09 2.20E-06

GO:0004364 F glutathione transferase activity 14 57 1.40E-08 3.30E-06

GO:0008194 F UDP-glycosyltransferase activity 25 219 5.80E-08 1.00E-05

GO:0016758 F transferase activity, transferring hexosyl 31 340 1.80E-07 2.50E-05

groups

GO:0003700 F transcription factor activity 108 2173 4.10E-07 4.80E-05

GO:0016491 F oxidoreductase activity 79 1463 8.80E-07 8.80E-05

GO:0080044 F quercetin 7-O-glucosyltransferase activity 8 22 1.60E-06 0.00014

GO:0080043 F quercetin 3-O-glucosyltransferase activity 8 26 4.50E-06 0.00035

GO:0030528 F transcription regulator activity 112 2417 5.70E-06 0.0004

GO:0016757 F transferase activity, transferring glycosyl 36 544 2.00E-05 0.0013

groups

GO:0019825 F oxygen binding 22 255 2.30E-05 0.0013

GO:0016765 F transferase activity, transferring alkyl or aryl 16 158 5.20E-05 0.0028

(other than methyl) groups

Supplemental Table S2. Singular Enrichment Analysis of upregulated genes in Arabidopsis in response to the endo-alkenyl analogue 3.

GO term Ontology Description Number in Number in p-value FDR

input list BG/Ref

GO:0009733 P response to auxin stimulus 47 360 5.00E-34 4.00E-31

GO:0009719 P response to endogenous stimulus 60 1068 1.00E-24 4.00E-22

GO:0009725 P response to hormone stimulus 57 982 3.70E-24 9.70E-22

GO:0050896 P response to stimulus 117 4057 3.00E-23 5.90E-21

GO:0010033 P response to organic substance 63 1342 4.80E-22 7.70E-20

GO:0042221 P response to chemical stimulus 78 2085 1.90E-21 2.60E-19

GO:0010252 P auxin homeostasis 5 21 7.40E-06 0.00083

GO:0009416 P response to light stimulus 19 596 4.10E-05 0.0033

GO:0009404 P toxin metabolic process 6 53 4.00E-05 0.0033

GO:0009407 P toxin catabolic process 6 53 4.00E-05 0.0033

GO:0009314 P response to radiation 19 613 6.00E-05 0.0043

GO:0009886 P post-embryonic morphogenesis 5 35 6.50E-05 0.0043

GO:0009408 P response to heat 9 161 9.50E-05 0.0058

GO:0048878 P chemical homeostasis 8 136 0.00017 0.0094

GO:0003700 F transcription factor activity 48 2173 3.00E-06 0.00093

GO:0030528 F transcription regulator activity 50 2417 1.00E-05 0.0017

GO:0004364 F glutathione transferase activity 6 57 5.80E-05 0.006

Supplemental Table S3. The list of the genes from Arabidopsis belonging to the overrepresented GO term “response to heat” in 6 h after treatment with the meta-iodo analogue 4.

GO term ID No. Gene Name Foldchange on

treatment with 4

Response to heat AT3G46230 ATHSP17.4 76.8

(GO:00009408) AT4G27670 HSP21 41.1

AT5G12020 HSP17.6II 39.8

AT1G52560 26.5 kDa class I small heat shock protein-like 34.6

AT5G12030 AT-HSP17.6A 27.3

AT4G25200 ATHSP23.6-MITO 24.6

AT1G09080 BIP3 22.9

AT1G53540 HSP17.6C-CI 22.4

AT1G54050 HSP17.4-CIII 18.9

AT1G16030 HSP70B 18.8

AT1G74310 ATHSP101 15.1

AT1G07400 HSP17.8-CI 13.0

AT5G59820 RHL41 12.5

AT2G29500 HSP17.6B-CI 11.3

AT4G10250 ATHSP22.0 11.1

AT3G24500 ATMBF1C/MBF1C 11.0

AT5G51440 HSP23.5-M 9.63

AT2G46240 BAG6 9.58

AT5G57050 ABI2 9.19

AT3G12580 HSP70 8.99

AT1G59860 HSP17.6A-CI 7.71

Supplemental Table S4. A list of the genes in Arabidopsis related to detoxification of chemicals in 6 h after treatment with the meta-iodo analogue 4.

Functional category ID No. Gene name Foldchange on

(GO term) treatment with 4

Toxin catabolic process AT1G69930 ATGSTU11 42.8

(GO:00009407) AT1G17170 ATGSTU24 20.0

AT2G29470 ATGSTU3 17.6

AT2G29480 ATGSTU2 14.4

AT1G78380 ATGSTU19 14.1

AT2G29440 ATGSTU6 11.4

AT2G29420 ATGSTU7 10.9

AT1G17180 ATGSTU25 8.49

AT2G29460 ATGSTU4 7.32

AT1G78370 ATGSTU20 6.38

AT2G29490 ATGSTU1 6.35

AT1G27130 ATGSTU13 5.67

AT2G29450 ATGSTU5 5.32

AT2G47730 ATGSTF8 4.88

UDP-glucosyl- AT1G05680 UDP-glucosyl transferase family protein 36.3 transferase activity AT3G46660 UDP-glucosyl transferase family protein 18.4

(GO:0035251) AT1G05560 UGT1 15.2

AT5G59580 UDP-glucosyl transferase family protein 14.7

AT2G36790 UGT73C6 12.3

AT2G36750 UGT73C1 11.6

AT4G01070 GT72B1 10.1

AT2G15480 UGT73B5 8.75

AT2G36800 DOGT1 8.49

AT1G05530 UGT2/UGT75B2 8.08

AT1G22400 UGT85A1 7.61

AT4G34131 UGT73B3 7.50

AT4G34135 UGT73B2 7.23

AT2G15490 UGT73B4 7.06

AT2G43820 GT/UGT74F2 6.47

AT1G73880 UDP-glucosyl transferase family protein 5.76

AT3G43190 SUS4 4.15 AT3G46670 UDP-glucosyl transferase family protein 3.40

Cytochrome P450 AT3G25180 CYP82G1 31.5

AT3G14660 CYP72A13 21.8

AT5G52320 CYP96A4 18.5

AT1G16410 CYP79F1 15.0

AT1G74110 CYP78A10 14.7

AT1G19630 CYP722A1 10.8

AT2G46950 CYP709B2 9.70

AT5G52400 CYP715A1 8.30

AT5G09970 CYP78A7 8.10

AT4G39500 CYP96A11 8.00

AT4G37370 CYP81D8 7.97

AT2G34500 CYP710A1 6.80

AT5G42590 CYP71A16 5.38

AT5G61320 CYP89A3 5.03

AT1G64950 CYP89A5 4.77

AT1G64940 CYP89A6 4.08

AT2G45570 CYP76C2 3.93

AT5G04660 CYP77A4 3.39

ABC transporter AT1G15520 ATPDR12 60.0

AT3G59140 ATMRP14 7.82

AT3G13080 ATMRP3 5.45