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Article

Distribution of Plant Hormones and Their Precursors in Cambial Region Tissues of Quercus myrsinifolia and Castanopsis cuspidata var.sieboldii after Bending Stems or Applying Ethylene precursor

by
Yoshio Kijidani
1,*,
Taku Tsuyama
1 and
Yuji Tokumoto
2
1
Division of Forest and Environmental Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
2
Institute for Tenure Track Promotion, University of Miyazaki, Miyazaki 889-2192, Japan
*
Author to whom correspondence should be addressed.
Forests 2023, 14(4), 813; https://doi.org/10.3390/f14040813
Submission received: 27 February 2023 / Revised: 12 April 2023 / Accepted: 13 April 2023 / Published: 15 April 2023
(This article belongs to the Special Issue Intrinsic Regulation of Diameter Growth in Woody Plants)
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Abstract

:
The role of plant hormones in tension wood (TW) formation has been studied but is still unclear. IAA, ABA, ACC, tZ, tZR, iP, and iPR in cambial region tissues were identified and quantified by liquid chromatography/mass spectrometry (LC/MS). We examined the distribution of plant hormones and their precursors in the stems of Quercus myrsinifolia Blume and Castanopsis cuspidata var.sieboldii Nakai after bending the stems or applying an ethylene precursor (ACC). After 3 weeks of bending, though not after 1 week of bending, the auxin (IAA) and abscisic acid (ABA) amounts were larger on the TW side than on the opposite wood (OW) side and in upright trees. After 2 weeks of bending, the peak concentrations of IAA in cambium on the TW side were obviously higher than those on the OW side. After 1 week of bending, the ACC amounts on both sides were larger than in upright trees, but after 3 weeks of bending, they were smaller than in upright trees. Applied ACC did not enhance TW formation but induced axical parenchyma and phloem formation in C. cuspidata var.sieboldii. These results indicated that the distribution patterns of IAA and ABA might have important roles in TW formation in these two species. The role of ACC might be limited in the early stages of TW formation.

1. Introduction

To utilize wood efficiently, more information is needed on how cambium regulates diameter growth in woody plants. Softwood and hardwood are among the most important renewable resources in the world. They are mainly used as component materials in wooden structures, furniture, and wood flooring. Therefore, it is important to well understand and manage the variation of mechanical properties and dimensional stability of lumber from plantations. It has been reported that the wood density and microfibril angle (MFA) in the S2 layer of the cell wall can explain variations in the mechanical properties of commercially important softwoods and hardwoods [1,2,3,4]. Wood density and MFA are also important parameters in determining the dimensional stability of softwood [5]. Tracheids and wood fibers form through diameter growth in the cambium (cell division and differentiation). Therefore, regulation of diameter growth by cambium could affect the cell wall structures and thus the mechanical properties and dimensional stability of lumber. Although various research topics on diameter growth in woody plants have been studied, reaction wood formation remains unclear and might be a very interesting topic in this research field because it can cause drastic changes in MFA and chemical components in the cell wall. The presence of reaction wood has negative effects on the performance of component materials in wooden structures, furniture, and wood flooring. Reaction wood is formed in an inclined and bent stem as a gravitropic response. Compression wood or tension wood (TW) forms on the lower or upper side of the stems in softwoods or hardwoods, respectively. However, quantitative studies on endogenous plant hormones during reaction wood formation are scarce. Particularly, more quantitative studies on TW formation in hardwoods are needed.
Many studies focused on the balance of auxin (IAA) amounts in TW and opposite wood (OW) sides during TW formation. Studies involving the application of IAA revealed that TW forms in the region with the lowest IAA concentration [6]. Application of IAA to the upper side of hardwood stems during bending inhibited TW formation [7]. Application of IAA transport inhibitor around upright stems induced TW formation at the axially lower part from the application part [8,9]. A quantitative study demonstrated larger amounts of IAA in the cambial region tissues on the TW side than on the opposite side, but there was no difference from those in upright trees [10]. The peak concentrations of IAA in cambium were also higher on the TW side than on the OW side after 15 days of bending, but the difference between TW and OW in the peak concentration of IAA in cambium was less dramatic than that in the total amounts of IAA [10]. A study on a gene family related to the auxin signal transduction pathway in hybrid aspen showed that TW formation is accompanied by expression changes of these genes and not by changes in IAA amounts in the TW side after 11 days of bending [11].
Gibberellins (GAs) and brassinosteroids (BRs) might play roles in TW formation. Application of the GA3 or GAs biosynthesis inhibitors enhanced or suppressed TW formation, respectively [12]. The amount of GA3 was enriched in the xylem of the TW side, accompanied by a reduced level of a poplar DELLA protein [13]. However, the GAs biosynthesis inhibitor did not inhibit the formation of tension wood fibers [14]. Application of BRs or a BRs biosynthesis inhibitor delayed or dramatically enhanced G-fiber maturation, respectively [15]. However, a key BRs biosynthesis gene was predominantly expressed in TW, accompanied by a higher BR (castasterone) amount than in OW [16].
Recent studies have focused on ethylene biosynthesis during TW formation. During stem tilting, the rates of ethylene evolution increased to high levels, xylem production was accelerated, and TW formation was induced on the TW side [17]. However, application experiments showed a stimulating effect of ethylene on wood formation, but TW was not formed [18]. Gravitational stimulation resulted in TW formation, strong induction of ethylene precursor (1-amino-1-cyclopropanecarboxylic acid; ACC) oxidase gene expression, and ACC oxidase activity; however, ACC levels were higher in the OW side than in the TW side [19]. Application of ACC and ethylene to wild-type and ethylene-insensitive hybrid aspen trees induced G-layers and altered the fiber cell wall cellulose microfibril angle [20]. Ethylene-insensitive hybrid aspen trees indicate that, although G-fibers form, the cellulose microfibril angle in the G-fiber secondary cell wall decreases, the chemical compositions of the secondary cell wall and G-layers change, and the characteristic asymmetric growth and reduction of vessel density are suppressed in comparison with those in wild-type TW [21].
From these applications and quantitative studies on plant hormones, conflicting results were obtained (Table 1). The changes in IAA sensitivity might affect TW formation. However, quantitative studies on plant hormones are scarce, and important information on TW formation was obtained from hybrid aspens [10,11,13,15,16,19,20,21]. Therefore, to understand TW formation, more quantitative information on IAA, ACC, and other plant hormones and the precursors of those other plant hormones will be needed in various types of hardwoods.
In this study, we reported the distribution of IAA, ACC, abscisic acid (ABA), cytokinins, and cytokinin precursors (trans-zeatin (tZ), tZ riboside (tZR), isopentenyladenine (iP), and iP riboside (iPR)) in cambial region tissues in the stems of Quercus myrsinifolia and Castanopsis cuspidata var.sieboldii after bending treatment (1, 2, and 3 weeks of bending) or application of ACC. Many studies have been conducted on diffuse porous trees (hybrid aspens). In this study, we focused on ring porous (C. cuspidata var.sieboldii) and radial porous (Q. myrsinifolia) trees.

2. Materials and Methods

2.1. Sample Trees and Stem Bending Procedure

The 5- to 6-year-old Q. myrsinifolia and C. cuspidata var.sieboldii shown in Table 2 were used as sample trees for the stem bending treatments (Experiment 1, Figure 1). For each 1- and 3-week bending treatment for the analysis of plant hormones, 3 bending and 2 upright Q. myrsinifolia were used (10 trees in total). For a 2-week bending treatment for the analysis of plant hormones, 4 Q. myrsinifolia (2 bending and 2 upright trees) and 4 C. sieboldii (2 bending and 2 upright trees) were used (8 trees in total). In addition, for observation of wood anatomy, 2 Q. myrsinifolia (1 bending and 1 upright tree) and 2 C. cuspidata var.sieboldii (1 bending and 1 upright tree) were used (4 trees in total). These sample trees were grown in the nursery in the experimental forest of the University of Miyazaki. No silvicultural practices were carried out in the nursery. The nursery used in this study is located in the western part of Miyazaki City. The average annual temperature and precipitation during the experiments in the experimental forest were 16.8 °C and 3820 mm, respectively. The altitude of the nursery was 196 m. The diameter of the trees at breast height (DBH) and the tree height were measured with a tape measure (Table 2).
As shown in Figure 1, sample trees were inclined with stainless wire and a weight for fixing. The maximum angles of inclination ranged from 30.7 to 43.6 degrees (Table 2). At the beginning of treatment, a pin was inserted into the cambium of each sample tree for the observation of wood anatomy. To measure the levels of plant hormones in cambial region tissues after bending for 1, 2, or 3 weeks, samples (2 cm (T) × 3 cm (L) × 1 cm (R)) of cambial region tissues sandwiched between the outer bark and the outermost xylem were obtained from the TW side and the OW side at the height position that provided the maximum inclination angle of the bending trees. Upright trees were sampled at the same height as the bending trees. Immediately after collection, the samples were stored in a deep freezer (−80 °C) before extraction was performed. To examine the wood anatomy of the bending trees and upright trees, samples of cambial region tissues sandwiched between the outer bark and the outermost xylem were obtained from the same positions used for the plant hormone analysis in bending and upright trees, and the samples for the pinning method [22] were cut from sample trees after the cessation of xylem formation.

2.2. Sample Seedlings and Treatment Procedure for ACC Application

The 2-year-old C. cuspidata var.sieboldii trees shown in Table 3 were used as sample seedlings for ACC application (Experiment 2, Figure 1). As shown in Table 3, 10 potted seedlings (5 lanolin-applied (control) and 5 ACC/lanolin-applied seedlings) were used for the analysis of plant hormones, and 4 potted seedlings (2 lanolin-applied (control) and 2 ACC/lanolin-applied) were used for the observation of wood anatomy (14 seedlings in total). These sample seedlings were grown on the campus of the University of Miyazaki. The average annual temperature and precipitation during Experiment 2 in Miyazaki city were 18.4 °C and 3046 mm, respectively. The altitude of the University of Miyazaki campus is 28.8 m. The diameters of seedlings at the base point and their heights were measured with a tape measure (Table 3).
As shown in Figure 1, we prepared seedlings for control and treatment. The area of lanolin application was 1 cm2 (0.5 cm (T) × 2.0 cm (L)) in each seedling. In control seedlings, 0.5 g of lanolin was applied to each of the 2 positions in control (control in experiment 2, Figure 1). As shown in treatment of Figure 1, 1.5% ACC in 0.5 g lanolin and 0.5 g lanolin without ACC were applied to each seedling. To start the treatments, a pin was inserted into the cambium of each seedling for observation of wood anatomy. To measure the levels of plant hormones in cambial region tissues after 1 week of application, samples (0.5 cm (T) × 2.0 cm (L) × 0.5 cm (R)) of cambial region tissues sandwiched between the outer bark and the outermost xylem were obtained from the lanolin- or ACC-applied positions of sample seedlings. The samples were stored in a deep freezer (−80 °C) before extraction was performed. To examine the wood anatomy of control and ACC-applied seedlings, samples of cambial region tissues sandwiched between the outer bark and the outermost xylem were obtained from the same positions used for the plant hormone analysis in seedlings, and the samples for the pinning method [22] were cut from seedlings after the cessation of xylem formation.

2.3. Observation of Wood Anatomy

To examine the anatomical characteristics of wood formed after stem bending or ACC application, we embedded the samples in epoxy resin and prepared 6-μm-thick cross sections. Wood fibers in TW were characterized by a G-layer and small amounts of lignin. Therefore, the cross sections were stained with a zinc chloride-iodine solution with phloroglucinol-HCl (G-layer) or Mäule reagents (lignin distribution) [23]. Radial sections for the examination of axial parenchyma development were stained with Toluidin blue O (Sigma-Aldrich, St. Louis, MI, USA).

2.4. Quantification of Plant Hormones and Its Precursor

IAA, ABA, ACC, tZ, tZR, iP, and iPR in cambial region tissues were identified and quantified by liquid chromatography/mass spectrometry (LC/MS). Samples were extracted by solution (methanol: 75%, distilled water: 20%, formic acid: 5% (v/v)) with deuterium-labeled plant hormones and its precursors (D2-IAA (97% content; Sigma, St. Louis, MI, USA), D6-ABA (98% content; Olchemim, Olomouc, Czechia), D4-ACC (98% content; Olchemim, Olomouc, Czechia), D5-tZ (98% content; Olchemim, Olomouc, Czechia), D5-tZR (97% content; Olchemim, Olomouc, Czechia), D6-iP (98% content; Olchemim, Olomouc, Czechia), and D6-iPR (98% content; Olchemim, Olomouc, Czechia)) as internal standards (50 ng). The extracts were purified using reverse-phase cartridges (Sep-pack cartridge, C18 500 mg, Waters, Milford, MA, USA) and separated into an acidic fraction (IAA and ABA) and a basic fraction (ACC, tZ, tZR, iP, and iPR) using mixed-mode solid-phase extraction (Oasis MCX, 6 cc (150 mg), Waters, Milford, MA, USA) and then quantified by LC/MS (Ultimate 3000, Q-exactive, Thermo Fisher Scientific, Waltham, MA, USA). For both IAA and ABA analyses, the column was an ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, MA, USA); as the mobile phase, acetonitrile containing 0.1% (v/v) formic acid and distilled water containing 0.1% (v/v) formic acid was used, and the flow rate was 0.3 mL/min. For ACC analysis, the column was a CORTECS UPLC HILIC (2.1 mm × 100 mm, 1.6 μm, Waters, Milford, MA, USA); as the mobile phase, acetonitrile containing 0.1% (v/v) acetic acid and distilled water containing 0.1% (v/v) acetic acid was used, and the flow rate was 0.3 mL/min. For tZ, tZR, iP, and iPR analyses, the column was a CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm, Waters, Milford, MA, USA); as the mobile phase, methanol containing 0.1% (v/v) formic acid and distilled water containing 0.1% (v/v) formic acid was used, and the flow rate was 0.3 mL/min. Detection and quantification were carried out using Q-exactive operated in the positive (IAA, tZ, tZR, iP, and iPR) and negative (ABA, ACC) ion, targeted-SIM (selected ion monitoring) mode using calibration curves with deuterium-labeled plant hormones and its precursors as internal standards. As described in a previous report [24], the levels of plant hormones and their precursors in the cambial region tissues are shown as amounts (ng) per cambium area (L × T cm2) (ng/cm2).

2.5. Analysis of Radial Distribution of IAA with Cryo-Sectioning

According to a previous study using GC/MS [25], we analyzed the radial distribution of IAA using LC/MS combined with cryo-sectioning. Two Q. myrsinifolia trees (one upright and one bending tree) and four C. cuspidata var.sieboldii trees (two upright and two bending trees) were selected from the sample trees for experiment 1 in Figure 1 for this analysis. The samples (1 cm (T) × 1.5 cm (L) × 0.5 cm (R)) of cambial region tissues sandwiched between the outer bark and the outermost xylem were obtained from the TW side and the OW side at the height position that provided the maximum inclination angle of each bending tree. The samples were obtained at the same height for the upright trees as for the bending trees. The 30-μm-thick tangential cryosections were obtained continuously using a cryotome (Leica CM1850, Leica Microsystems, Wetzlar, Germany). From each sample, we obtained 100 cryosections from bark to xylem. Cross sections were obtained before continuous tangential cryosectioning. We determined the types of tissues in each cryosection based on the distance from the outermost bark measured in cross sections using image analysis and the cell types observed in tangential cryosections after extraction using a light microscope. We divided each section into phloem, including the developing zone, cambium, and xylem, including the developing zone. The extracts of three sections classified as cambium were combined, and the extracts of five sections classified as phloem and xylem were combined, purified, and quantified by the same procedures as described above for the analysis of the radial distribution of IAA.

2.6. Statistical Analysis

As shown in experiment 1 in Figure 1, we tried to examine the distribution of plant hormones and their precursors in cambial region tissues in the stems of two species after bending treatments (1, 2, and 3 weeks of bending). It was assumed that levels of plant hormones in the sample trees varied depending on the sampling date and the microenvironment of the growth site. Therefore, we prepared the upright trees (control trees) for 1-, 2-, and 3-week bending treatments. We evaluated the effects of bending treatment on the amounts of plant hormones based on the ratios of plant hormones in the bending trees to those in the upright trees (means of replicates of upright trees) obtained at the same sampling date. We assumed that the levels of plant hormones varied with the duration of the bending treatment as well as the sampling position (TW or OW side). To detect variation of levels of plant hormones exactly, we examined the significant difference in the amount ratios of plant hormones between TW side and OW side after 1, 2, and 3 weeks of bending (multiple comparison test, Tukey HSD, p < 0.05).
As shown in experiment 2 in Figure 1, we tried to examine the distribution of plant hormones and their precursors in cambial region tissues in the stems of C. cuspidata var.sieboldii after application of ACC. Previous studies concluded the positive role of ACC; however, ACC levels were higher in the OW side than in the TW side [19]. We assumed the possibility of transporting ACC. To detect variation of levels of plant hormones and transport of ACC exactly, we examined the significant difference among the level of plant hormones in 2 lanolin-applied positions of control seedlings and lanolin- or ACC-applied positions of ACC-applied seedlings (multiple comparison test, Tukey HSD, p < 0.05).

3. Results

3.1. Wood Anatomy Formed after Bending Treatment

The wood anatomy formed after bending treatment in sample trees (Q. myrsinifolia and C. cuspidata var.sieboldii) was examined (Table 2). Fibers on the TW side of bending trees were stained purple (A and B, Figure 1), in contrast to upright trees, which were stained yellow using a zinc chloride-iodine solution with phloroglucinol-HCl. Fibers on the TW side of bending trees were not stained red (C and D, Figure 1), in contrast to upright trees, which were stained red with Mäule reagents. From the results in Figure 1, it was recognized that the bending treatment had formed TW with G-fiber on the TW side of the stem in these species, as shown in Figure 1.

3.2. Distribution of Plant Hormones and Their Precursor after Bending Treatment

Each data of 1- and 3-week bending treatments were obtained from 3 replicate trees (Q. myrsinifolia) in Figure 2. Each data of 2-week bending treatments were obtained from 4 replicate trees (2 Q. myrsinifolia and 2 C. cuspidata var.sieboldii). We combined these two species for the analysis of plant hormones because TW formation was induced in both species by stem bending treatment. Based on the amount ratios in Figure 2, the following results were obtained: The means of IAA and ABA amounts in the sample trees after the 1-week bending on both the TW and OW sides were smaller than those of the upright trees. The means of IAA and ABA amounts in the TW side of the sample trees after the 2-week bending were larger than those in the OW side but smaller than those in the upright trees. These results on IAA were consistent with those of previous studies of hybrid aspen [10,19]. However, the means of IAA and ABA amounts in the TW side of the sample trees after the 3-week bending were higher than those in both the OW side and the upright trees. In the 3-week bending, significant differences in IAA amount ratios were recognized between the TW and OW sides. A significant difference in the ABA amount ratio was recognized between the TW side after the 3-week bending and both sides after 1- and 2-week bending. Therefore, it was recognized that the amount ratios of IAA and ABA on the TW side increased significantly after the 3-week bending. The mean ACC amounts in both the TW and OW sides after 1-week bending were larger than those of the upright trees. The mean ACC amounts in the OW side of the sample trees after the 2-week bending were larger than those of both the TW side and the upright trees. A significant difference in the ACC amount ratio was recognized between the TW side and the OW side after the 2-week bending. These ACC results were consistent with those of a previous study of hybrid aspen [19]. However, the means of the ACC amounts in both the TW and OW sides after the 3-week bending were not higher than those of the upright trees. Therefore, it was recognized that the means of the ACC amount ratios on the TW side decreased gradually from the 1-week bending to the 3-week bending. Although there was no significant difference in the amount ratios of cytokinins and their precursors among the examined samples, those in the TW side were larger than those in the OW side after the 2-week and 3-week bending.

3.3. Peak Concentrations of IAA in Cambium after 2-Week Bending

Total amounts of IAA were larger on the TW side than on the OW side and smaller than those in the upright trees (2 weeks in Figure 2). However, the peak concentrations of IAA in cambium might show a different tendency from the total amounts of IAA. Therefore, we examined the peak concentrations of IAA in cambium in the upright trees and in the 2-week bending trees by LC/MS analysis combined with cryosectioning. As shown in Figure 3, the peak concentrations of IAA in cambium were higher in the TW side than in the OW side and lower than those of the upright trees. Especially, small peak concentrations of IAA in cambium on the OW side of C. cuspidata var.sieboldii were recognized and were different from the results of a previous study of hybrid aspens [10]. It was difficult to cryosection accurate tangential sections for Q. myrsinifolia because the presence of aggregate ray parenchyma curved the cambium arrangement in tangential directions. Therefore, the peak concentrations of IAA in the cambium were examined only in 1 upright tree and 1 bending tree in Q. myrsinifolia.

3.4. Wood Anatomy and the Distribution of Plant Hormones and Their Precursors after ACC Application in C. cuspidata var.sieboldii Seedlings

Cross-sections of wood formed after ACC application were shown in Figure 4A–D. As shown in Figure 4, the application of 1.5% ACC in lanolin and lanolin with no ACC did not induce TW formation with G-fibers. However, application of 1.5% ACC in lanolin enhanced phloem formation and axial parenchyma in both the application side and the opposite side (Figure 4A,B and Figure 5). Before ACC application, TW formation was observed in some seedlings. It is very difficult to prepare seedlings without TW. However, clear TW formation induced by ACC application was not observed in experiment 2 (Figure 1). The amounts of plant hormones and their precursors after ACC application are shown in Figure 6. ACC amounts were significantly higher on the ACC-applied side of the stem in treated seedlings than on the lanolin-applied side in control seedlings. In treated seedlings, ACC application did not affect IAA or ABA amounts but induced significantly larger amounts of tZR and iPR on the side opposite the ACC-applied side. ACC amounts were also higher (without significance) on the lanolin-applied side of the stem in treated seedlings than on the lanolin-applied side in control seedlings. ACC might be transported to the side opposite the ACC-applied side and then affect the amounts of tZR and iPR.

4. Discussion

4.1. The Role of IAA in TW Formation

Our study showed IAA amounts in the TW side increased gradually from 1-week to 3-weeks of bending, and IAA amounts were higher in the TW side after 3 weeks of bending trees than in upright trees (Figure 2). On the other hand, IAA amounts on the OW side gradually decreased from 1 to 3 weeks of bending (Figure 2). Therefore, the largest difference in IAA amounts between the TW side and the OW side occurred in the 3-week bending trees. Our previous study on softwoods showed that IAA’s role in xylem formation might be the promotion of cell division [24,26]. The fact that the largest difference in IAA amounts between the TW and OW sides occurred after 3 weeks of bending clearly explained the asymmetric growth pattern in TW formation. A previous quantitative study of hybrid aspen showed smaller amounts of IAA in the TW side of 2-week bending trees than in that of upright trees and concluded that TW formation did not need large amounts of IAA [10]. Since the previous results were similar to the present results for the 2-week bending treatment, a 3-week bending treatment in hybrid aspen might show similar results to the present study. The amount of IAA in bending trees could be changed along with TW formation.
Our study demonstrated that the peak concentrations of IAA in cambium on the OW side were much smaller than those on the TW side and upright trees, especially in C. cuspidata var.sieboldii (Figure 3). This result, although not consistent with the results of the previous study [10], also clearly explained the suppressed xylem formation on the OW side. TW formation in a majority of hardwoods involves asymmetric growth, G-fiber formation, and decreased numbers and diameters of vessels. A recent study of nst/snd triple-knockout or quadruple-knockout aspens revealed that the signals related to asymmetric growth and vessel formation might differ from those related to G-fiber and secondary cell wall formation [27]. From our results on IAA, we hypothesized that IAA distribution patterns might induce asymmetric growth during TW formation. How did the peak concentrations of IAA on the OW side decrease? Its synthesis, transport, and metabolism would affect the amounts of IAA. In a future study, we should examine the effects of stem bending on the synthesis, transport, and metabolism of IAA on the OW side. In this study, Q. myrsinifolia was used for the 1-, 2-, and 3-week bending treatments. However, C. cuspidata var.sieboldii was used only for the 2-week treatment. To elucidate TW formation, more sample trees of C. cuspidata var.sieboldii and other hardwood species will be needed for a study of the distribution of plant hormones after 3 weeks of bending.

4.2. The Role of ABA in TW Formation

ABA, as well as IAA, increased gradually from 1 to 3 weeks of bending, and the amount of ABA on the TW side was 2.4 times that in upright trees (Figure 2). Asymmetric distributions of ABA amounts were recognized after the 2- and 3-week bending treatments. A review of plant hormones during TW formation showed no relationship between ABA and TW formation [28]. ABA was found to have an important role in drought sensitivity in beech (Fagus sylvatica) [29]. The attenuation of ABA responses compromised photoperiodic control of bud dormancy and did not affect growth cessation [30]. A study on circadian regulation in Arabidopsis thaliana showed that more than 40% of ABA-induced genes are circadian regulated [31]. In eucommia trees (Eucommia ulmoides), ABA displayed an annual distribution pattern opposite those of IAA and cambial activity [32], although ABA showed the same pattern as IAA in our study. In TW formation, transcript profiling by RNA-seq showed that ABA-related genes encoding protein kinase/phosphatase, ABA-responsive element binding factor, and ABA receptor were all downregulated in upright trees compared with the TW or OW side [33]. Therefore, the signaling induced by larger amounts of ABA might change the cambium activity from normal xylem formation under circadian regulation to TW formation.

4.3. The Roles of ACC and Cytokinins in TW Formation

The ACC amounts in the OW side after 2 weeks of bending were larger than those in both the upright trees and the TW side (Figure 2). Our result for ACC after 2 weeks of bending was consistent with a previous study on hybrid aspen [19]. However, the ACC amounts in both the TW and OW sides after 3 weeks of bending were not greater than those of the upright trees (Figure 2), and this result conflicted with a previous study on hybrid aspen [19]. As shown in Figure 1, the bending treatment used in our study induced TW formation. In Q. myrsinifolia, the effects of ACC on TW formation might be smaller than those of IAA and ABA. In addition, as shown in Figure 4, Figure 5 and Figure 6, the application of ACC did not obviously induce TW formation but did enhance phloem development and axial parenchyma and change the amounts of endogenous precursors of cytokinins in C. cuspidata var.sieboldii. Figure 2 and Figure 6 showed a similar trend of decreased cytokinins on the side of increased ACC in the OW and ACC treatments. Our results (Figure 4), which showed no induction of TW formation, were consistent with a previous study on the effect of ethylene [18]. An increase in ACC might affect the development of both phloem and axial parenchyma. As shown in Figure 2, there was a slight, but not significant, increase in cytokinins on the TW side after 2 and 3 weeks of bending. The genes involved in hormone biosynthesis, such as cytokinin oxidase related to cytokinin, were downregulated in TW and in upright trees compared with the OW side [33]. Cytokinins as well as ACC might be involved in the gravitational response and in the control of differentiation in phloem and axial parenchyma.

5. Conclusions

In this study, we examined the distribution of plant hormones and its precursors in the stems of Q. myrsinifolia and C. cuspidata var.sieboldii after bending the stems or applying ACC. We obtained the following results on IAA and ABA: After 3 weeks of bending, though not after 1 week of bending, IAA and ABA amounts were larger on the TW side than on the OW side and in upright trees. After 2 weeks of bending, the peak concentrations of IAA in cambium on the TW side were obviously higher than those on the OW side. Based on these results, we hypothesized that the difference in IAA amounts between the TW and OW sides induced the asymmetric growth pattern in TW formation. In addition, the signaling induced by larger amounts of ABA on the TW side might change the cambium activity from normal xylem formation under circadian regulation to TW formation. We also obtained the following results on ACC and cytokinins: After 1 week of bending, the ACC amounts on both sides were larger than in upright trees, but after 3 weeks of bending, they were smaller than in upright trees. The application of ACC did not obviously induce TW formation but did enhance phloem development and axial parenchyma and change the amounts of endogenous cytokinins and their precursors. Cytokinins as well as ACC might be involved in the gravitational response and in the control of differentiation in phloem and axial parenchyma.

Author Contributions

Conceptualization, Y.K.; methodology, Y.K.; formal analysis, Y.K.; writing—original draft preparation, T.T. and Y.T.; writing—review and editing, T.T. and Y.T.; visualization, Y.K.; funding acquisition, Y.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was mainly funded by a Grant-in Aid for Scientific Research (C), 2019–2021 (19K06172, Yoshio Kijidani) from the Ministry of Education, Science, Sports, and Culture of Japan; and a part of this research was funded by a Grant-in Aid for Challenging Research (Pioneering), 2021–2024 (21K18226, Satoshi Ito) from the Ministry of Education, Science, Sports, and Culture of Japan.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank Azusa Tomiie for her contribution to data collection.

Conflicts of Interest

The authors declare no conflict of interest.

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Forests 14 00813 g001 550
Figure 1. Bending treatment and application of ACC. (a) Experiment 1 for bending treatment; (b) Experiment 2 for application treatment of ACC. (A,B) were stained with a zinc chloride–iodine solution with phloroglucinol-HCl (G-layer). (C,D) were stained with Mäule reagents (lignin distribution).
Figure 1. Bending treatment and application of ACC. (a) Experiment 1 for bending treatment; (b) Experiment 2 for application treatment of ACC. (A,B) were stained with a zinc chloride–iodine solution with phloroglucinol-HCl (G-layer). (C,D) were stained with Mäule reagents (lignin distribution).
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Figure 2. Distributions of plant hormones in sample trees for 1-week, 2-week, and 3-week bending treatments are shown in Table 2. TW and OW are the upper and lower sides of the stem in each bending tree, respectively. Each value is the ratio of plant hormones in treated trees to those in upright trees (control trees). Each value of TW and OW is the mean of bending trees (n = 3 or 4). Different characters show a significant difference (Tukey HSD, p < 0.05). There was no significant difference in cytokinins among the samples.
Figure 2. Distributions of plant hormones in sample trees for 1-week, 2-week, and 3-week bending treatments are shown in Table 2. TW and OW are the upper and lower sides of the stem in each bending tree, respectively. Each value is the ratio of plant hormones in treated trees to those in upright trees (control trees). Each value of TW and OW is the mean of bending trees (n = 3 or 4). Different characters show a significant difference (Tukey HSD, p < 0.05). There was no significant difference in cytokinins among the samples.
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Figure 3. Radial distributions of IAA amounts from phloem to xylem in six sample trees after 2-week bending. TW and OW are upper and lower side of the stem in each bending tree, respectively. Upright is control tree without bending treatment. Yellow colored area–bark; Green colored area–phloem; Pink colored area–cambium; Blue colored area–xylem. Data in Cambium was IAA amounts in three sections classified as cambium. Each data of Bark, Phloem and Xylem was IAA amounts in five sections classified as Bark, Phloem and Xylem, respectively.
Figure 3. Radial distributions of IAA amounts from phloem to xylem in six sample trees after 2-week bending. TW and OW are upper and lower side of the stem in each bending tree, respectively. Upright is control tree without bending treatment. Yellow colored area–bark; Green colored area–phloem; Pink colored area–cambium; Blue colored area–xylem. Data in Cambium was IAA amounts in three sections classified as cambium. Each data of Bark, Phloem and Xylem was IAA amounts in five sections classified as Bark, Phloem and Xylem, respectively.
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Figure 4. Wood anatomy formed after ACC application in sample trees (C. cuspidata var.sieboldiii). The Xylem formed after treatment was determined by the pinning method (arrows). (A,D) were cross-sections stained with Mäule reagents (lignin distribution). (C) were cross-sections stained with a zinc chloride-iodine solution with phloroglucinol-HCl (G-layer). (B) was a radial section stained with Toluidin blue O.
Figure 4. Wood anatomy formed after ACC application in sample trees (C. cuspidata var.sieboldiii). The Xylem formed after treatment was determined by the pinning method (arrows). (A,D) were cross-sections stained with Mäule reagents (lignin distribution). (C) were cross-sections stained with a zinc chloride-iodine solution with phloroglucinol-HCl (G-layer). (B) was a radial section stained with Toluidin blue O.
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Figure 5. Phloem development in ACC-applied seedlings and control seedlings (C. cuspidata var.sieboldii). The stem diameters of the applied points of the ACC applied seedling were larger than those of the control seedlings (arrows), because of active phloem development. Cross-sections were stained with a zinc chloride–iodine solution with phloroglucinol HCl (G-layer).
Figure 5. Phloem development in ACC-applied seedlings and control seedlings (C. cuspidata var.sieboldii). The stem diameters of the applied points of the ACC applied seedling were larger than those of the control seedlings (arrows), because of active phloem development. Cross-sections were stained with a zinc chloride–iodine solution with phloroglucinol HCl (G-layer).
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Figure 6. Amounts of plant hormones in sample seedlings (C. cuspidata var.sieboldii) after 1 week of ACC application. ACC in treated seedlings includes endogenous and exogenous ACC. Each value is the mean of 5 sample seedlings in each treatment (n = 5, Table 3). Different characters show a significant difference (Tukey HSD, p < 0.05).
Figure 6. Amounts of plant hormones in sample seedlings (C. cuspidata var.sieboldii) after 1 week of ACC application. ACC in treated seedlings includes endogenous and exogenous ACC. Each value is the mean of 5 sample seedlings in each treatment (n = 5, Table 3). Different characters show a significant difference (Tukey HSD, p < 0.05).
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Table
Table 1. Comparison of the results from the literature on the role of plant hormones in TW formation.
Table 1. Comparison of the results from the literature on the role of plant hormones in TW formation.
Plant HormonesNo.Effects on TW Formation
IAA[6,7,8,9]Negative effects
[10,11]No or small effects
GA3[12,13]Positive effects
[14]No or small effects
BRs[15]Negative effects
Ethylene[16,20]Positive effects
[17,18]No or small effects
ACC[19,20]Positive effects
[21]No or small effects
No.: Reference number in reference section.
Table
Table 2. Sample trees for bending treatment.
Table 2. Sample trees for bending treatment.
SpeciesAgenH
(m)		D
(cm)		MBA
(degree)		Treatment-PeriodExperiment
Q. myrsinifolia (Radial-porous wood)634.264.1034.2Bending_1 WAnalysis of plant hormones
24.003.65 Upright_1 W
522.655.2537.8Bending_2 W
22.754.25 Upright_2 W
635.134.8330.7Bending_3 W
23.823.70 Upright_3 W
513.233.042.6BendingAnatomy
12.73.0 Upright
C. cuspidata var.sieboldii (Ring-porous wood)523.104.0043.6Bending_2 WAnalysis of plant hormones
23.756.25 Upright_2 W
513.146.040.4BendingAnatomy
13.606.0 Upright
H, D, and MBA are the means of two or three trees in each sample tree. n—number of trees in each sample tree; H—tree height; D—diameter at breast height (five or six-year-old trees) or diameter at the base of seedlings (two-year-old seedlings); MBA—maximum angle of inclination; 1 W, 2 W, and 3 W—one week, two weeks, and three weeks.
Table
Table 3. Sample seedlings for the application of ACC.
Table 3. Sample seedlings for the application of ACC.
SpeciesAgenH
(m)		D
(cm)		Treatment-PeriodExperiment
C. cuspidata var.sieboldii (Ring-porous wood)250.650.76ACC/lanoline_1 WAnalysis of plant hormones
250.650.78Lanolin_1 W
220.660.90ACC/lanolineAnatomy
220.600.73Lanolin
H and D are the means of two or five trees in each sample seedlings. n—number of seedlings; H—seedling height; D—diameter at the base of seedlings.
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Kijidani, Y.; Tsuyama, T.; Tokumoto, Y. Distribution of Plant Hormones and Their Precursors in Cambial Region Tissues of Quercus myrsinifolia and Castanopsis cuspidata var.sieboldii after Bending Stems or Applying Ethylene precursor. Forests 2023, 14, 813. https://doi.org/10.3390/f14040813

AMA Style

Kijidani Y, Tsuyama T, Tokumoto Y. Distribution of Plant Hormones and Their Precursors in Cambial Region Tissues of Quercus myrsinifolia and Castanopsis cuspidata var.sieboldii after Bending Stems or Applying Ethylene precursor. Forests. 2023; 14(4):813. https://doi.org/10.3390/f14040813

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Kijidani, Yoshio, Taku Tsuyama, and Yuji Tokumoto. 2023. "Distribution of Plant Hormones and Their Precursors in Cambial Region Tissues of Quercus myrsinifolia and Castanopsis cuspidata var.sieboldii after Bending Stems or Applying Ethylene precursor" Forests 14, no. 4: 813. https://doi.org/10.3390/f14040813

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