The Effect of Gibberellin and an Ethylene Inhibitor on Twining of Vine Cuttings in Japanese Morning Glory (Ipomoea nil (L.) Roth)

Abstract

“Twining” along other objects is an important morphogenetic survival response of vine plants. The twining response, induced by the stimulus of touching a supporting object, is a form of “thigmomorphogenesis”. Ethylene is thought to play an important role in thigmomorphogenesis in higher plants, so it is likely to be involved in vine twining. However, the relationship between ethylene and vine twining is not well understood. We used vine cuttings excised from morning glory (Ipomoea nil (L.) Roth ‘Violet’) plants in order to investigate the effect of an ethylene inhibitor on elongation and twining. The vine cuttings required gibberellin for elongation and twining. In the vine cuttings with elongation and twining induced by gibberellin treatment, treatment with 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, significantly decreased the angle of rotation and the spiral pitch of the twining. These results suggest that ethylene may be involved in either sensing a pole via touch and the morphological changes during vine twining or both. We also selected one of the genes for 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which is a key enzyme in the ethylene biosynthetic pathway, with higher expression in tissues on the side touching the pole in twining vines as one of the candidate genes thought to be involved in vine twining in morning glory.

Introduction

Higher plants obtain energy through photosynthesis; therefore, it is important for them to expand their leaves at a more elevated position than surrounding plants to receive enough sunlight. To win the competition for survival, vine plants adopt a strategy of climbing along supporting objects using their long stems, rather than expend energy to develop firm stems with supporting tissues. Therefore, the morphogenetic response of “twining” to objects is crucial for vine plant survival.

“Twining of vine plants” is a classic theme that even caught the attention of Charles Darwin, famous for his theory of evolution (Darwin, 1865). Plants generally exhibit circumnutation, a circular or elliptic movement of the growing stem (Darwin and Darwin, 1880). Vine plants search for environmental cues around them by relatively extensive circumnutation, and when they find a suitable object for climbing, they grow upward by twining their long stem, called a vine, along it. It is thought that the stimulus of touching a supporting object is involved in the induction of the twining response in vines, but the detailed mechanisms of the process are poorly understood.

The phenomenon of plants changing their morphology due to mechanical stimuli, such as touch is defined as “thigmomorphogenesis” (Jaffe, 1981; Jaffe and Forbes, 1993). As a typical example of thigmomorphogenesis, mechanical stress stimulates inhibition of elongation and promotion of lateral expansion of stems in plants, resulting in shorter and stronger stems in terms of plant morphology (Jaffe and Forbes, 1993). It has been reported that multiple plant hormones are involved in the process of thigmomorphogenesis (Jaffe and Forbes, 1993; Telewski, 2021). Among these plant hormones, ethylene, for which biosynthesis is promoted by mechanical stimuli (Biro and Jaffe, 1984; Jaffe and Biro, 1979; Telewski, 2021), is generally thought to play an important role in the early stages of thigmomorphogenesis in higher plants (Jaffe and Forbes, 1993). However, the relationship between ethylene and the twining response caused by touch stimuli in vines has not yet been demonstrated.

In higher plants, ethylene is produced from methionine via S-adenosyl-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) (Argueso et al., 2007; Kende, 1993). It is thought that the conversion of SAM to ACC is a rate-limiting step during the ethylene biosynthetic pathway; in other words, ACC synthase (ACS), which catalyzes this reaction, is the key enzyme (Adams and Yang, 1979; Lürssen et al., 1979; Yoshii et al., 1980). Based on the assumption that ethylene is involved in the vine twining response caused by touch stimuli, we hypothesized that there may be ACS genes for which expression changes accompany the twining response. In recent years, the whole genome sequence of morning glory (Ipomoea nil (L.) Roth), the only model organism among vine plants, has been determined (Hoshino et al., 2016). According to the database, there are 13 kinds of ACS homologs in the whole genome sequence of morning glory. However, the relationship between these ACS genes and vine twining has not been clarified.

In studying the role of plant hormones in the mechanism of vine twining, exogenous treatment with chemicals such as hormone inhibitors is an effective method. However, vine plants generally have long stems and some of them are relatively large, so there is a problem that the use of whole plants for investigating the effects of chemicals on vine twining requires large spaces. To address this problem, an experimental system using vine cuttings excised from plants would be useful. However, in seedlings of the standard morning glory ‘Violet’, it was reported that a direct precursor of active gibberellin is transported from cotyledons to stems, where it is converted to active gibberellin and utilized for elongation (Yang et al., 1995, 1996). Also, the dwarf morning glory ‘Kidachi’ does not twine because its vines do not grow long. It was reported that the dwarf phenotype of ‘Kidachi’ is associated with a deficiency in endogenous gibberellins, and that exogenous treatment with gibberellin restores this phenotype (Barendse and Lang, 1972; King et al., 1994; Simmons and Coulter, 1979). Therefore, exogenous application of gibberellins could be necessary to induce a twining response in an experimental system using vine cuttings excised from plants.

In this study, we attempted to induce a twining response in vine cuttings by treatment with a test solution containing gibberellin. Furthermore, we investigated the effect of an ethylene inhibitor on twining in an experimental system using vine cuttings in which elongation and twining were induced by gibberellin treatment. We also searched for ACS genes with expressions related to twining of vines in morning glory.

Materials and Methods

Plant materials

Morning glory seeds (Ipomoea nil (L.) Roth: standard strain ‘Violet’ and dwarf strain ‘Kidachi’) were obtained from the National BioResource Project (NBRP, Kyushu University, Fukuoka, Japan). The morning glory seeds were scratched to promote water absorbance and sown in plastic pots filled with culture soil (Yasai-no-Baiyodo; TAKII & Co., Ltd., Kyoto, Japan) containing a liquid fertilizer (Hyponex liquid fertilizer; HYPONeX JAPAN, Corp., Ltd., Osaka, Japan). Plants were grown under continuous light (80 μmol·m⁻²·s⁻¹, white fluorescent light, FL40SSW/37; NEC Corporation, Tokyo, Japan) at 27°C and were used for treatments and RNA extraction.

Treatment of the dwarf morning glory ‘Kidachi’ plants with gibberellin

Each morning glory ‘Kidachi’ plant was given 200 mL of water with or without 140 μM gibberellic acid (GA₃) at 1, 2, and 4 weeks after sowing. When the vines started to elongate in GA₃-treated plants, a pole was set near to them. Three plants were used for each treatment of either water alone or water with 140 μM GA₃.

Treatment of vine cuttings with gibberellin and an ethylene inhibitor in morning glory ‘Violet’

Vine cuttings were taken from 18 cm below the shoot tip from morning glory ‘Violet’ plants of which the length between the cotyledonary node and the shoot tip was 20 to 35 cm for a test gibberellin dose or 25 to 35 cm for a test ethylene inhibitor. Leaves longer than 1 cm were removed from vine cuttings at the base of petioles.

Vine cuttings were treated by immersing the basal end in 4 mL of the test solution, which contained 2% (w/v) sucrose in a 10 mM sodium phosphate buffer (pH 6.2) with or without GA₃ at concentrations of 0.01 to 1,000 μM, in a 15 mL-centrifuge tube (product number 430053; Corning Inc., NY, USA). A bamboo skewer as a supporting pole was inserted into the centrifuge tube with the vine cutting, then a small amount of cotton was placed into the upper end of the centrifuge tube to secure them. The cuttings were incubated under continuous light at 27°C. After 24 h of incubation, the elongation and angle of rotation of vine twining were measured. The angle of rotation was determined from the position of the vine at the upper end of the centrifuge tube in relation to that of the movement locus of the shoot tip around the pole when viewed from above (“α°” in Fig. 1). Six vine cuttings were used for treatment using each concentration of GA₃.

Fig. 1

Schematic diagram of the measurement of the shoot apex rotational degrees after treatment of vine cuttings. (A) A top view of a vine cutting. (B) A side view of a vine cutting. The angle of rotation (α°) was measured from the position of the vine at the upper end of the centrifuge tube in relation to that of the movement locus of the shoot tip around the pole when viewed from above. The spiral pitch of the twining was calculated by dividing the rotational degrees by length of the vine above the upper end of the centrifuge tube (α/β degrees·cm⁻¹).

1-methylcyclopropene (1-MCP) was used as an ethylene action inhibitor. Vine cuttings immersed in 4 mL of the basal medium, which contained 100 μM GA₃ and 2% (w/v) sucrose in a 10 mM sodium phosphate buffer (pH 6.2), at the basal end were fumigated with 1.0 ppm 1-MCP in a sealed glass tank (volume 58 L) under continuous light at 27°C. After 24 h of treatment, the elongation and angle of rotation of vine twining were measured (“α°” in Fig. 1). We also calculated the spiral pitch of twining, which represents the rotational degrees (“α°” in Fig. 1) per centimeter of length of the vine above the upper end of the centrifuge tube (“β cm” in Fig. 1). Twelve vine cuttings were used for each tank treatment of either the basal medium alone or the basal medium plus 1-MCP fumigation.

Molecular phylogenetic analysis of ACS homologs

The database of U.S. National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) was searched for the genes of the whole genome of the morning glory ‘Tokyo Kokei Standard’ which have similarities in the deduced amino acid sequence to the ACS genes of other plants as well as ACS homologs derived from Arabidopsis thaliana (L.) Heynh and tomato (Solanum lycopersicum L.). Genes that were registered as ACS in the morning glory whole genome database based on sequence homology, excluding those with apparently short sequences considered as junk genes, were selected as morning glory ACS homologs. The sequences were aligned using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) in the GenomeNet server (Kyoto University Bioinformatics Center). A phylogenetic tree was constructed using the maximum likelihood method (Jones et al., 1992) on MEGAX (Kumar et al., 2018). Bootstrap analysis (1,000 replicates) was used to determine statistical reliability (Felsenstein, 1985).

RNA extraction

A pole, 8 mm in diameter and 50 cm or 35 cm long, was set near to 14-day-old morning glory ‘Violet’ plants. Subsequently, these plants were grown under continuous light. To compare gene expressions in vines twining along a pole to those without twining, vine segments with twining were obtained from the uppermost 5 cm portion of the intact vines that twined up the 50 cm pole; vine segments without twining were obtained from the same portion of vines that were grown until they were at least 5 cm taller than the 35 cm pole. Five segments of vines were gathered per sample, and four samples per condition were used for RNA extraction. To compare gene expressions on the side touching the pole to those on the non-touching side in twining vines, vine segments were obtained by cutting the vines at the upper end of the vine area touching the pole as well as 5 cm below, and then the segments were split longitudinally in half to separate the touching and non-touching sides. Five tissues from each side were collected per sample, and three samples per side were used for RNA extraction.

Extraction of total RNA was performed using phenol-chloroform extraction. In detail, tissues were immediately frozen in liquid nitrogen and homogenized using a mortar and pestle or a beads cell disrupter (Micro Smash MS-100; TOMY SEIKO Co., Ltd., Tokyo, Japan) with stainless steel beads (3.2 mm diameter) at 3,500 rpm for 30 s three times. A mixture of extraction buffer [100 mM LiCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v) sodium dodecyl sulfate (SDS), 100 mM Tris-HCl, pH 8.0]: phenol (1:1, v/v) at 80°C was added to the homogenates, which were further homogenized using a beads cell disrupter at 4,000 rpm for 60 s. A half volume of chloroform containing 4% (v/v) isoamyl alcohol was added, and samples were further mixed using the beads cell disrupter at 3,500 rpm for 30 s. This mixture was centrifuged at 15,000 rpm for 15 min at 4°C. The collected upper aqueous layer was mixed with an equal volume of 4 M LiCl and kept at −70°C for 1 h. After melting, the solution was centrifuged at 15,000 rpm for 15 min at 4°C. The resultant pellet containing total RNA was further purified using the ethanol precipitation method, and used as a template of cDNA synthesis.

cDNA synthesis

Before the cDNA synthesis reaction, total RNA was treated with 20 U·mL⁻¹ of DNase I (New England Biolabs Inc., Ipswich, MA, USA) at 37°C for 10 min to remove contaminant genomic DNA. First strand cDNA was synthesized from the total RNA using 0.25 pmol·μL⁻¹ oligo (dT)₂₀ primer and the reverse transcriptase ReverTra Ace (TOYOBO Co., Ltd., Osaka, Japan) according to the manufacturer’s manual. The synthesized cDNA was used for semi-quantitative PCR.

Semi-quantitative RT-PCR analysis

The cDNA fragments with partial sequences of 13 ACS homologs were amplified using their respective gene-specific primers (Table S1) designed according to sequences which corresponded to regions other than their homologous regions obtained from the database of the whole genome sequence of morning glory. As an internal control, a DNA fragment with a partial sequence of cDNA of the ubiquitin gene in morning glory was amplified using its gene-specific primers (Table S1). PCR was performed using Taq DNA polymerase (New England Biolabs Inc.) under the following conditions: 20, 25, 30, 33, or 36 cycles of 95°C for 30 s, 56°C for 30 s, and 68°C for 30 s. The amplified DNA fragments, which were sufficient for detection but which did not reach a plateau, were used to compare the transcript levels, which meant that the cDNA fragments for the 13 ACS homologs and the ubiquitin gene were amplified at 33 cycles and at 25 cycles, respectively, and were then separated by electrophoresis on a 2% (w/v) agarose gel. After staining with GelRed (Biotium Inc., Fremont, CA, USA), the amplified DNA fragments were detected with a Luminoimage Analyzer (LAS-1000UVmini; FUJIFILM Corporation, Tokyo, Japan).

Photographs and movies

Photographs of plants were taken with a digital camera (PENTAX K-7; Ricoh Imaging Company Ltd., Tokyo, Japan). The movement of vine cuttings was recorded with a time-lapse camera (TLC200; Brinno Inc., Taipei, Taiwan) at 20 min intervals from the side. Photographs and movies were processed using Photoshop Elements 15 (Adobe Inc., San Jose, CA, USA) and Premiere Elements 15 (Adobe), respectively.

Results

The effect of gibberellin on plant growth in the dwarf morning glory ‘Kidachi’

The vines of dwarf morning glory ‘Kidachi’ plants treated with water did not elongate and had stems which were not long enough to twine even five weeks after sowing (Fig. 2). The vine elongation of ‘Kidachi’ plants was promoted by treatment with GA₃ solution, and their elongated vines twined along a pole (Fig. 2).

Fig. 2

Dwarf morning glory ‘Kidachi’ plants treated with water (Control) or GA₃ solution (+ GA₃). Each plant was given 200 mL of water with or without 140 μM GA₃ at 1, 2, and 4 weeks after sowing. (A) The 1-week-old plants. (B) The 5-week-old plants. Scale bars: 10 cm.

The effect of gibberellin on twining of morning glory ‘Violet’ cuttings

Vine cuttings excised from morning glory ‘Violet’ grew 18 ± 3 mm longer and minimal twining up poles occurred when they were treated with 2% (w/v) sucrose in 10 mM sodium phosphate buffer (pH 6.2) for 24 h (Figs. 3 and 4). Elongation and twining of vine cuttings were enhanced and varied according to the concentration of GA₃ used (Figs. 3 and 4); twining was promoted significantly when 1 μM or more of GA₃ was added (P < 0.05; Tukey’s HSD test).

Fig. 3

Morning glory ‘Violet’ vine cuttings treated with the test solution with 100 μM GA₃ (+ GA₃) or without GA₃ (−). (A, B) Shortly after the start of treatment. (C, D) After 24 h of treatment. (A, C) Treatment with the test solution without GA₃. (B, D) Treatment with the test solution containing 100 μM GA₃. Scale bars: 1 cm.

Fig. 4

The effects of GA₃ on elongation and twining of vine cuttings in the morning glory ‘Violet’. The elongation (A) and angle of rotation (B) of the twining vines after 24 h of treatment are shown. Data are averages of six cuttings with SEs (bars). Different letters indicate significant differences at P < 0.05 by Tukey’s HSD test.

The effect of 1-MCP on twining in vine cuttings of the morning glory ‘Violet’

To elucidate the relationship between vine twining of morning glory and the action of ethylene, vine cuttings of the morning glory ‘Violet’ were treated with 1-MCP, an ethylene action inhibitor, during the induction of twining caused by GA₃. Elongation of the vine cuttings which were fumigated with 1.0 ppm 1-MCP for 24 h during treatment with the basal medium containing 100 μM GA₃ in a sealed tank was 46 ± 3 mm (Table 1). This did not differ significantly from the 47 ± 3 mm elongation of vine cuttings treated with the basal medium without 1-MCP fumigation in a sealed tank (Table 1; Fig. 5). However, the angle of twining rotation of vine cuttings treated with the basal medium plus 1-MCP fumigation was 415 ± 36°, which was significantly reduced compared to cuttings treated with the basal medium without 1-MCP fumigation at 565 ± 31° (P < 0.01; Student’s t-test) (Table 1; Fig. 5). Correspondingly, the rotation angles per centimeter of vine length for cuttings in the basal medium treatment and the basal medium plus 1-MCP fumigation treatment were 52.1 ± 2.9°·cm⁻¹ and 38.3 ± 3.1°·cm⁻¹, respectively (Table 1). Vine twining loosened significantly with 1-MCP treatment (P < 0.01; Student’s t-test) (Table 1). Additionally, vine cuttings treated with 1-MCP did not often begin to twine even when they touched a pole due to circumnutation (Fig. 6).

Table 1

The effects of treatment with 1-MCP for 24 h on elongation and twining of vine cuttings in the morning glory ‘Violet’.

Fig. 5

Morning glory ‘Violet’ vine cuttings treated with a basal medium (Control) or the basal medium plus 1.0 ppm 1-MCP fumigation (+ 1-MCP). (A, B) Shortly after the start of treatment. (C, D) After 24 h of treatment. (A, C) The basal medium, which contained 100 μM GA₃, treatment. (B, D) The basal medium plus 1-MCP treatment. Scale bars: 1 cm.

Fig. 6

Time lapse analysis of movement in morning glory ‘Violet’ vine cuttings, which were treated with a basal medium (A) or the basal medium plus 1.0 ppm 1-MCP fumigation (B). The circumnutational and twining movement of the vine cutting was recorded at 20 min intervals from the side with a time-lapse camera. The elapsed time from the start of treatment is shown in the upper left corner of each image. The closed arrowhead indicates the position where the vine began to touch the pole. Scale bars: 1 cm.

Transcript levels of ACS genes in morning glory ‘Violet’ vines

According to the whole genome sequence of morning glory (Hoshino et al., 2016), there are 15 kinds of morning glory genes that have similar deduced amino acid sequence to ACS genes of other plants. We performed a phylogenetic analysis based on the deduced amino acid sequence of these 15 morning glory genes and ACS homologs derived from A. thaliana and S. lycopersicum (Fig. S1). Among the morning glory genes, INIL02g17176 and INIL15g14228 are included in a clade which consists of aminotransferase genes of unknown function (Fig. S1, clades II). Therefore, we excluded INIL02g17176 and INIL15g14228, then analyzed the transcript levels of the remaining 13 ACS homologs of morning glory and examined their relationships with vine twining.

We performed a semi-quantitative RT-PCR analysis for RNA obtained from the intact vines with or without twining, and compared the transcript levels of 13 ACS homologs between them (Fig. S2). As a result, we could not identify an ACS gene with a higher transcript level in twining vines than in non-twining vines (Fig. S2). Further, we performed a semi-quantitative RT-PCR analysis for RNA obtained from the tissues on the side touching the pole and those on the other, non-touching, side in twining vines, and compared the transcript levels of 13 ACS homologs between these tissues (Figs. 7 and S3). In all analyzed samples, the transcript level of INIL05g09675 in the tissues on the side touching the pole was higher than on the non-touching side (Fig. 7). Some of the analyzed ACS genes exhibited different transcript levels in each sample independent of twining and touching (Figs. S2 and S3).

Fig. 7

Expression analysis of an ACS homolog (accession number: INIL05g09675) in the tissues on the side touching a pole and those on the other, non-touching, side in twining vines established by semi-quantitative RT-PCR. The gel electrophoresis profiles of the DNA fragments, which were amplified from cDNA for INIL05g09675 and the ubiquitin gene (accession number: INIL03g17366) of morning glory by PCR, are shown. The vine segments were obtained from morning glory ‘Violet’ plants by cutting the vines at the upper end of the area touching the pole and 5 cm below, and they were then split in half, separating the touching (The side in touch with a pole) and non-touching sides (The side not in touch with a pole). Five tissues from each side were collected for each sample. Samples 1–3: the expressions in the tissues on the non-touching side in twining vines; Samples 4–6: the expressions in the tissues on the side touching a pole in twining vines.

Discussion

Requirement of gibberellin for elongation and twining in vine cuttings of morning glory

It is sometimes difficult for researchers to use whole plants, which are too large, to investigate the mechanism of vine twining in a laboratory with limited space. Therefore, an experiment using vine cuttings excised from vine plants may resolve this problem, and could be a useful and simple system for quantitative assessment (Fig. 1). In this study, applying gibberellin was necessary for vine cuttings from the morning glory ‘Violet’ plants to elongate and twine along a pole (Figs. 3 and 4). The dwarf morning glory ‘Kidachi’, which is partially deficient in synthesis of gibberellins, does not twine because its vines do not grow long (Fig. 2). Exogenous treatment with gibberellin restores the dwarf phenotype of ‘Kidachi’ (Barendse and Lang, 1972; King et al., 1994; Simmons and Coulter, 1979) and its ability to twine along a pole (Fig. 2). This indicates that gibberellins are required for vine elongation and this gibberellin-induced elongation is necessary for vine twining in morning glory. Moreover, in seedlings of the morning glory ‘Violet’, it was reported that GA₂₀, a direct precursor of active GA, is transported from cotyledons to stems, where GA₂₀ is converted to GA₁, an active GA in morning glory, and utilized (Yang et al., 1995, 1996). When vine cuttings excised from plants of the morning glory ‘Violet’ were treated without GA₃, elongation and twining along a pole were minimal (Figs. 3 and 4). This result may indicate that the supply of active gibberellins or their precursors from other organs, i.e. cotyledons, hypocotyls, and/or roots, were cut off, and that the level of gibberellins in the vine cutting was insufficient to enable the elongation necessary for twining. Hence, gibberellin supply is necessary to induce a twining response in vines excised from morning glory plants.

Involvement of ethylene in sensing touch of a pole and/or in the morphological changes in vine twining of morning glory

“Twining” to other objects is an important morphogenetic response for vine plant survival. Although twining has been researched for over a century (Darwin, 1865), detailed mechanisms of the twining process have not been clarified. It is thought that the stimulus of touching a supporting object is involved in the induction of the twining response in vines. Therefore, the twining response can be considered a form of “thigmomorphogenesis”. Ethylene is generally thought to be involved in thigmomorphogenesis in higher plants (Jaffe and Forbes, 1993). 1-MCP irreversibly inhibits the function of ethylene receptors by strongly binding to them and maintaining the inhibitory effect until a turnover of the ethylene receptors (Blankenship and Dole, 2003; Sisler and Serek, 1997). The angle of rotation along a pole and the spiral pitch of the twining were significantly suppressed by 1-MCP fumigation in vine cuttings of the morning glory ‘Violet’ (Table 1; Fig. 5). Moreover, it appeared that the 1-MCP treated vine cuttings failed to cling and twine despite touching a pole several times by circumnutation (Fig. 6). These results suggest that ethylene could be involved in sensing the touch of a pole and/or in morphological changes during vine twining in morning glory.

The approximate directionality of vine twining in morning glory is determined by circumnutation. In the morning glory mutant ‘weeping’, which is defective in endodermal cells required for gravity sensing in the stem, circumnutation and twining are abnormal and severely limited (Hatakeda et al., 2003; Kitazawa et al., 2005). Additionally, circumnutation of the morning glory ‘Violet’ vines was potently inhibited by an auxin transport inhibitor (Hatakeda et al., 2003). It has therefore been suggested that gravity sensing and auxin redistribution are important for circumnutation (Hatakeda et al., 2003; Kitazawa et al., 2005). In contrast, the morning glory ‘Violet’ vine cuttings treated with an ethylene action inhibitor exhibited normal circumnutation (Fig. 6), which suggests that ethylene is unlikely to be involved in circumnutation in morning glory. Although the role of ethylene during the twining response appears to promote the morphological change along a pole through touch and to make the vine cling tightly to a pole (Table 1; Figs. 5 and 6), further investigation into the process is necessary.

An ACS gene related to vine twining

We looked for ACS genes with expressions related to twining of vines in morning glory. Although our data imply involvement of ethylene in vine twining (Table 1; Figs. 5 and 6), among the 13 ACS homologs in morning glory, there were no genes with higher transcript levels in twining than non-twining vines (Fig. S2). On the other hand, the INIL05g09675 transcript level differed between tissues on the side touching a pole and those on the non-touching side in all twining vine samples, and its transcript levels were relatively high in the tissues on the touching side (Fig. 7). We selected the ACS gene INIL05g09675 as a candidate gene involved in vine twining in morning glory. Ethylene production caused by the differential expression of INIL05g09675 in tissues on the side touching a pole in twining vines may change the vine’s shape to adjust to a pole, and consequently promote twining. This hypothesis is supported by the observation of loosened vine twining caused by an ethylene action inhibitor (Table 1; Fig. 5).

We also found that the transcript level of INIL02g40697 in vines without twining was higher than in those that twined up poles in all analyzed samples (Fig. S2). Although further investigation is necessary, the increased expression of INIL02g40697 may promote ethylene production in response to the stress caused by the lack of supporting objects. Among analyzed ACS genes, some of them exhibited different transcript levels in each sample in the same experimental group (Figs. S2 and S3). Although the regulatory mechanisms for the expressions of these genes have not been clarified, factors other than vine twining may control them.

In vivo roles of only a few ACS homologs of morning glory are known; for instance, the expressions of INIL05g09675 and INIL05g24005 in the hypocotyls were stimulated by auxin application to the cotyledons (Frankowski et al., 2009; Kęsy et al., 2010). However, the change in expression of ACS homologs related to vine twining has not been reported previously. In morning glory, methods for generating not only transformants, but also genome-edited plants have recently been identified (Kikuchi et al., 2005; Ono et al., 2000; Shibuya et al., 2018; Shimizu et al., 2003; Watanabe et al., 2017, 2018). An assay for localizing the expression using promoter-reporter-gene introduced transformants and for identifying the function of the ACS genes by genome editing technology would be effective to clarify the role of ethylene in vine twining in morning glory.

Acknowledgements

We are grateful to the National BioResource Project (NBRP) “Morning glory” for providing seeds of I. nil. We would also like to thank Uni-edit (https://uni-edit.net/) for editing and proofreading this manuscript.

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