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

Introduction

When developing new products, particularly pesticides or medicines, it is essential to study the relationship between structure and activity to improve the potency of the principal chemicals. As a source of candidates for the development of agrochemicals, natural organic chemicals, including the secondary metabolites of plants, have often been exploited. The herbicide mesotrione was based on the structure of leptospermone from the lemon bottlebrush, Callistemon speciosus.¹⁾ Likewise, the structures of pyrethroid derivatives, used as insecticides, were derived from pyrethrins biosynthesized by Chrysanthemum cinerariaefolium.²⁾

1-O-cis-Cinnamoyl-β-D-glucopyranose is a potent allelochemical isolated from the Spiraea species that shows strong growth-inhibitory activity against some plants.^3,4) This property is attributed to the cis-cinnamate moiety because the strength of the root growth-inhibitory activity of 1-O-cis-cinnamoyl-β-D-glucopyranose is the same as that of cis-cinnamic acid (cis-CA; 1).⁵⁾ Compound 1 has long been thought to be an auxin agonist because its structure and physiological effects are similar to those of auxin.⁶⁾ In a study using auxin-insensitive mutants (aux1 and axr2), however, Wong et al. suggested that the mode of action of 1 was different from that of auxin.⁷⁾ Guo et al. further reported that treatment with 1 induced two Arabidopsis genes, MLPL1 (AT2G01520) and MLPL2 (AT2G01530), which are considered to be functional in the regulation of bolting by proteome and histochemical analysis.⁸⁾ Wasano et al. examined early cis-CA responsive genes by DNA microarray and reported that root-specific upregulation of the early auxin-responsive genes AUXIN/INDOLEACETIC ACID (Aux/IAA), GRETCHEN HAGEN-3 (GH3), and LATERAL ORGAN BOUNDARY DOMAIN (LBD) occurred within 2 hr after an exogenous treatment of 1.⁹⁾ As yet, little is known about the physiological functions and mode of action of 1 in plants.

Chemical derivatization of 1 has been conducted to intensify the plant growth-inhibitory activity. Abe et al. constructed a series of cis-CA derivatives to clarify the key features of 1 for lettuce root growth inhibition, and found that a plain ring, the cis-configuration of alkene, and carboxylic acid were essential for the activity.¹⁰⁾ Nishikawa et al. found that cis-CA analogues possessing the meta-iodo, meta-methoxy, and meta-trifluoromethyl groups on the aromatic ring were more potent than 1.¹¹⁾ Furthermore, they constructed conformationally-constrained cis-CA analogues, in which the aromatic ring and cis-olefin were connected by a carbon bridge, showing that the inhibitory activities of the five-membered and six-membered bridged compounds were 10 times stronger than 1.¹²⁾ While these cis-CA analogues might serve as the lead chemicals in developing new herbicides, the reasons for their ability to enhance growth-inhibitory activity need to be clarified through the use of molecular biology.

DNA microarray analysis is an effective tool for investigating the global gene response of model plants.^13,14) It has been used to monitor changes in transcript levels in response to various allelochemical stresses on a whole-genome scale.^9,15,16) Presently, new agrochemicals are required to control weed strains that are resistant to commercial herbicides, but evaluating promising chemical derivatives is labor intensive and time consuming. DNA microarray analysis is potentially an efficient and rapid tool for evaluating the bioactivities of chemical derivatives.

In the present study, we examined a rapid evaluation method of substituent effects on plant growth inhibition at the molecular level. We selected three cis-CA analogues, (Z)-2-[3,4-dihydronaphthalen-1(2H)-ylidene]acetic acid (2), 2-(3,4-dihydronaphthalen-1-yl)acetic acid (3), and (Z)-3-(3-iodophenyl)acrylic acid (4), which have been reported to possess stronger growth-inhibitory activity than 1 against lettuce roots.^11,12) Two of them are a conformationally constrained cis-CA analogue connecting the aromatic ring and cis-olefin by a six-membered carbon bridge and its double-bond isomer; the other is meta-iodo-substituted cis-CA analogue (Fig. 1). First, we conducted short- and long-term bioassays of these chemicals against Arabidopsis. Then, we monitored gene responses to the tested compounds using a DNA microarray. Finally, we estimated the physiological changes of Arabidopsis seedlings from the Gene Ontology analysis data.

Fig. 1. Structures of cis-cinnamic acid (cis-CA; 1) and the cis-CA analogues (2–4) used in this study.

Materials and Methods

1. Chemicals

The synthesis of 1 was performed by the Z-selective olefination 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 (Col-0, Inplanta Innovations Inc., Yokohama, Japan) were sterilized in 70% ethanol for 1 min, sterilized in 2% sodium hypochlorite with 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 8 min, and then rinsed three times with sterilized distilled water. The sterilized seeds were placed on 0.8% agar (Nakarai Tesque, Inc., Kyoto, Japan) with 0.5× Murashige and Skoog Plant Salt Mixture (Nihon Pharmaceutical Co. Ltd., Tokyo, Japan) and 1% sucrose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a sterilized Petri dish (90-mm diameter). The seeds were held at 4°C overnight in darkness and then transferred to a growth chamber. Emerging plants were maintained in the growth chamber under a schedule of 16 hr light (22°C).

3. Root growth inhibition assay

The root growth inhibition assay was performed on agar plates (2% agar with 0.5× Murashige and Skoog Plant Salt Mixture and 1% sucrose) containing various concentrations of cis-CA analogues (1–4), and the sterilized seeds described above were transferred to vertically oriented agar plates. Six days after sowing in the growth chamber under a schedule of 16 hr light (22°C) and 8 hr dark (20°C), the root lengths were measured using an SZH dissecting microscope (Olympus, Tokyo, Japan) and SensivMeasure image measuring software (Mitani, Fukui, Japan). The effective concentration required for half of the maximum inhibition (EC₅₀) was calculated using the probit method with SPSS for Windows ver. 11.0.1J statistical software (SPSS Japan Inc., Tokyo, Japan).

To evaluate the persistency of the bioactivity of these cis-CA analogues, Arabidopsis seeds were sown on 80 mL of medium (0.5× Murashige and Skoog Plant Salt Mixture, 1% sucrose, and 0.4% gellan gum) containing 10 µM of cis-CA analogues in Agripots (Kirin Co. Ltd., Tokyo, Japan). Each pot contained 15 seeds, and they were grown in the same conditions as described above. Two weeks after sowing, the total weight of the seedlings was measured. A statistical analysis (one-way ANOVA and Tukey–Kramer test) was performed using SPSS software.

4. DNA microarray analysis

Plants were treated with 20 µM of 2, 3, or 4. Control plants were not treated. The plants were sampled at 6 hr post treatment and immediately frozen in liquid nitrogen. Total RNA was extracted from seedlings with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quantity and quality of the extracted RNAs were checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The two-color spike mix was added to the total RNA, and the RNA was labeled with a Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s two-color protocol. Fluorescent cRNA was generated from the total RNA. Briefly, 500 ng of RNA was reverse transcribed using MMLV reverse transcriptase and an oligo(dT) primer containing the T7 promoter. It was subsequently transcribed in vitro using T7 RNA polymerase, resulting in Cy3-labeled (control) and Cy5-labeled (cis-CA-treated) cRNAs. The cRNAs were purified using RNeasy Mini Spin columns (Qiagen) and then quantified with a NanoDrop ND-1000 UV–VIS spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Mixtures of 825 ng of Cy3-labeled and Cy5-labeled cRNAs were co-hybridized at 65°C for 17 hr on an Agilent Technologies 4×44 K Arabidopsis (v4) 60-mer oligo-microarray. The slides were then washed and the fluorescence intensity detected using an Agilent G2505B Scanner. Two independent biological replicates were assayed. The fluorescence intensity of individual spots on scanned images was quantified and corrected for background noise using Feature Extraction software (Agilent Technologies). To ensure a high quality of analysis, only features that passed three criteria were analyzed. Features flagged in the Feature Extraction software as non-uniform (IsFeatNonUnifOL and IsFeatPopnOL), saturated (IsSaturated), or low signal (IsWellAboveBG) were omitted, and the remaining features were further filtered by the Feature Significance test at p<0.01. Total RNA profiling data were normalized via linear and lowess methods followed by spike-in normalization using a Two-Color RNA Spike-In Kit (Agilent Technologies). The ratio of the intensity in the cis-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 are GSE37862, GSE37899, and GSE51400.

Results and Discussion

1. Bioactivities of cis-CA analogues

We observed the concentration-dependent root growth-inhibitory activities of the cis-CA analogues against Arabidopsis. As shown in Table 1, all of the cis-CA analogues tested (2–4) showed stronger activities than 1, which is consistent with previous studies using lettuce seedlings.^10–12) When the bioactivity was evaluated 6 days after chemical exposure, the results reflected an acute toxicity. Thus, a long-term bioassay was necessary to evaluate the cis-CA analogues as candidates for lead compounds in developing herbicides. We observed the growth-inhibitory activity of these chemicals after a two-week exposure to a cis-CA analogue. The growth-inhibitory activities of 1 and 2 decreased drastically two weeks after chemical exposure (Fig. 2). In contrast, 3 and 4 inhibited seedling growth after two weeks. Based on the results of the long-term bioassay, 3 and 4 are apparently more promising candidates for herbicide development than 2 (Fig. 2), although the three compounds (2–4) showed an equivalent level of activity in the short-term bioassay (Table 1).

Tested compound	EC50 (µM)	The fiducial limit at 95% level
1	2.98	2.43–3.82
2	1.26	1.02–1.53
3	0.72	0.64–0.80
4	0.49	0.43–0.56

Fig. 2. Effects of cis-cinnamic acid (cis-CA) analogues (1–4) on the 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.

Comparison of the root growth-inhibitory activity against Arabidopsis between 1, IAA, and its artificial analogue, 1-naphthaleneacetic acid (NAA), has been performed by Wong et al. (2005).⁷⁾ The EC₅₀ value of 1 (3.5 µM) in this report is equivalent to our data shown in Table 1. IAA and NAA are more active than 1, as they reportedly showed lower EC₅₀ values (0.018 µM and 0.18 µM, respectively).⁷⁾ Thus, the above-mentioned candidates (3 and 4) are more active than 1 and less active than IAA and NAA.

2. DNA microarray analysis

2.1 Gene response and Gene Ontology analysis

Table 2 shows the number of responsive genes whose transcript levels changed more than threefold 6 hr post exposure to the cis-CA analogues. Treatment with 4, as compared with 1, resulted in a drastic increase in the number of responsive genes. More than 1,000 genes were upregulated after a 6-hr exposure to 4. Singular Enrichment Analysis (SEA) of the upregulated genes identified 51 GO terms as significant, and the genes associated with auxin stimulus response (GO: 0009733) were overrepresented at a high level of significance, together with responses to heat (GO: 00009408), toxin catabolic processes (GO: 00009407), and responses to jasmonic acid stimulus (GO: 0009753). GO analysis based on the molecular function revealed that the UDP-glucosyltransferase (GO: 0035251) category was overrepresented (Supplemental Table S1).

Tested compound	Number of responsive genes		Reference
	Upregulated	Downregulated
1	383	364	Wasano et al. (2013)
2	220	306	This study
3	413	437	This study
4	1170	978	This study
IAA	403	472	Wasano et al. (2013)

After exogenous treatment with 3, Arabidopsis seedlings exhibited 413 upregulated and 437 downregulated genes at levels equivalent to those after treatments with 1 and IAA (Table 2). Of the 413 upregulated genes, 407 were annotated in the query list. SEA identified 17 GO terms as significant (Supplemental Table S2); the top seven overrepresented GO terms, ranked by their p-values, were all related to auxin response.

Treatment with 2, as compared with 1, induced a smaller number of responsive genes. Of the 220 upregulated genes, 213 were annotated in the query list. SEA identified only two GO terms as significant: lipid localization (GO: 0010876) and endomembrane system (GO: 0012505). Although the GO term “response to auxin stimulus” was not significantly overrepresented after treatment with 2, a more than 30-fold upregulation of GH3 family genes (GH3.1, GH3.2, and GH3.3) and a more than fivefold upregulation of LBD family genes (LBD16, LBD17, LBD18, LBD29, and LBD33) were observed as responses to treatment with 1. The upregulation of GH3 genes might contribute to seedling growth regulation because some gain-of-function mutation studies of GH3 genes caused negative effects on plant growth.^18,19) No additional distinguishable gene responses between treatments with 1 and 2 were observed.

2.2 Estimation of the physiological changes in cis-CA-analogue-treated Arabidopsis seedlings from the GO analysis data

By using a hierarchical tree diagram of overrepresented GO terms based on the biological process category,¹⁷⁾ the activated physiological functions after exposure to cis-CA analogues are predictable. GO terms involved in activated physiological functions formed clusters of high significance level GO terms, as shown by the high degree of color saturation of a box (Supplemental Fig. S1). Two hierarchical tree diagrams of overrepresented GO terms were constructed on the basis of the biological process category.

Graphical results of the genes upregulated in response to 4 are shown in Supplemental Fig. S1A. Four GO term clusters with high significance levels were divided into two groups: one was categorized as “response to auxin,” and the other “response to stresses,” which included the GO terms “response to jasmonic acid,” “responses to heat,” and “toxin catabolic process.” The upregulated genes belonging to the GO category “responses to heat” (Supplemental Table S3) were mainly comprised of heat shock proteins, which act as molecular chaperones to prevent protein misfolding caused by reactive oxygen species (ROS).²⁰⁾ Eleven of the genes listed in Supplemental Table S3 also belong to the GO term “response to hydrogen peroxide (GO: 0042542),” implying that the meta-substitution of an iodine group on the aromatic ring of cis-CA added a function that generated ROS. Interestingly, one of the heat-responsive genes, AtBAG6 (AT2G46240), is included in Supplemental Table S3. BAG (BCL2-associated athanogene) is a gene family that was originally identified in mammals that encodes calmodulin-binding proteins known to associate with the anti-apoptotic protein, BCL2. Kang et al. reported that the expression of AtBAG6 was strongly associated with the induction of programmed cell death in yeast and plants.²¹⁾ Therefore, the induction of AtBAG6 expression might partially contribute to enhanced growth-inhibitory activities. Past studies have demonstrated that xenobiotics induce putative detoxification pathways involving glutathione S-transferases (GSTs), cytochrome P450s (CYP450s), and uridine diphosphate glucosyltransferases (UGTs) in plants.²²⁾ In our DNA microarray results, 14 GST genes, 18 cytochrome P450 genes, and 18 UGT genes were upregulated in response to treatments with 4 (Supplemental Table S4). ATP-binding cassette (ABC) proteins were originally identified in the detoxification process as transporters of glutathione S-conjugated chemicals to vacuoles.²³⁾ In the present study, three ABC transporters were upregulated after treatment with 4, including a 60-fold upregulation of AtPDR12, indicating that the genes related to detoxification and elimination of xenobiotics were strongly upregulated in response to 4.

Graphical results of the genes upregulated in response to 3 are represented in Supplemental Fig. S1B. Sixty of the genes belong to the GO term “response to auxin stimulus,” and most of them were classified in early auxin-responsive gene families (Aux/IAA, GH3, and SAUR). It is generally accepted that the three major classes (Aux/IAA, GH3, and SAUR) respond to auxin stimulus within five to 60 min.^24,25) Furthermore, Wasano et al. suggested that the physiological function of 1 is distinguishable from that of auxin.⁹⁾ The response of auxin-responsive genes to 3 was different from that to 1. Additionally, the number of upregulated SAUR genes was at a level comparable to that of an IAA response (Table 3). Moreover, the transcript levels of Aux/IAA and GH3 induced by 3 were almost equal to those induced by IAA. These results indicate that exogenous 3 and IAA trigger transcriptomic induction in a similar manner in Arabidopsis.

Gene family	Number of upregulated genes by chemical treatment
	1	2	3	4	IAA
Aux/IAA	5	2	10	11	11
GH3	5	3	5	5	5
SAUR-related	4	0	21	31	22

3. Evaluation of cis-CA analogues as candidates for developing agrochemicals

We selected three candidates (2–4) from a series of cis-CA derivatives based on information from previous structure-bioactivity studies in lettuce seedlings,^10–12) and showed that the bioactivity of 2 decreased in a long-term bioassay using Arabidopsis seedlings (Fig. 2). The other cis-CA analogues, 3 and 4, had equivalent growth-inhibitory activities in this bioassay, which were stronger than those of 1 and 2. However, a global transcriptomic analysis indicated that there are differences in the responsive genes between of 3 and 4. The gene response to treatment with 3 was almost identical to that with IAA, while that with 4 was far greater than those with 1 and IAA (Table 2). In addition to the response to auxin stimulus, 4 upregulated genes related to biotic and abiotic stresses. Thus, the DNA microarray analysis method is effective in evaluating the enhanced plant growth-inhibitory activity caused by modifications of the principal chemicals.

It should be noted that 3 is a double-bond isomer of 2 (Fig. 3). The results of the bioassay and the transcriptomic analysis showed a clear difference between the two compounds. Although it is possible that this is attributed to the efficiency of absorption and/or transport, it is also possible, based on the structural similarity between 3 and NAA, that cis-CA analogues bind to an auxin receptor. IAA and the artificial auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) and NAA, bind to the base of the pocket of an F-box protein, transport inhibitor response 1 (TIR1), via an important functional moiety, the side-chain carboxyl group.^26,27) The carboxyl group of IAA anchors the plant hormone to the bottom of the TIR1 pocket by forming a salt bridge and two hydrogen bonds with two residues from the pocket floor (Arg 403 and Ser 438). Because 3 possesses a single C–C bond connecting the aromatic ring, its carboxyl group is able to rotate with a low-energy barrier such as IAA and NAA (Fig. 3). In contrast, the rotation of the carboxyl group of 2 is limited by the position of the double C–C bond. This implies that cis-CA analogues with a constrained carboxyl group could show lower binding affinities to TIR1, and the transcriptomic analysis in the present study supports this idea.

Fig. 3. Difference in the rotatability of the carboxyl group between the endo-alkenyl analogue 3 and its double-bond isomer 2.

Physiological experiments using antagonists of auxin or genetically modified Arabidopsis mutants are necessary to elucidate the molecular mechanism responsible for the bioactivity of the cis-CA analogues. We showed, however, that a DNA microarray could be a powerful tool for the rapid evaluation of structurally modified derivatives of allelochemicals. In this context, the analyzed data will be useful in subsequent studies, such as the design and synthesis of molecular probes for new potent inhibitors that target new types of agrochemicals.

Acknowledgment

We thank Dr. Sayaka Morita, Dr. Tomoko Takemura, and John S. Maninang for constructive discussions. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN), Tokyo, Japan.

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