原著論文
Changes in Endogenous Hormones in Leaves during Phase Transition in Malus hupehensis Seedlings
2025 年 94 巻 1 号 p. 40-47
詳細
2025 年 94 巻 1 号 p. 40-47
To accelerate the early development of superior apple varieties, it is crucial to establish techniques to expedite the transition from the juvenile phase to the adult phase. Clarifying the physiological mechanisms underlying this phase transition will contribute to the development of an apple-seedling growing system that ensures early phase transitions. Herein, the apomictic crabapple Malus hupehensis (Pamp.) Rehd. was hydroponically cultivated under controlled conditions to explore its phytohormone dynamics during the phase transition. Flowering was confirmed in 15 of 57 seedlings at approximately 10 months after germination. The flowering rate was 26.3%. The average height and average stem circumference of the flowered seedlings were 27 cm and 0.56 cm greater, respectively, than those of the unflowered seedlings. The ABA concentration in mature leaves at the main stem tip of the flowered seedlings exceeded that in the unflowered seedlings up to the 70th node, and then dropped below that of the unflowered seedlings by the 90th node’s developmental stage. The concentrations of GA4 and cytokinins in the mature leaves at the main stem tip showed no significant difference between the flowered and unflowered seedlings. These results suggest that employing hydroponics in a controlled setting is beneficial for facilitating early phase transition in M. hupehensis. Furthermore, maintaining low ABA concentration levels in the mature leaves at the main stem tip could stimulate phase transitions in M. hupehensis.
Phase transition from the juvenile to the adult phase is an important phenomenon in the development of fruit trees. Apple breeding always takes a long time because apple seedlings undergo a phase transition that enables them to respond to flowering stimuli only after a 5- to 12-year-long juvenile period from germination to first flowering (Zimmerman, 1973), followed by the adult phase in which stable flowering occurs every year. This long juvenile phase has been an obstacle to apple breeding research.
Research has revealed that the duration of the juvenile phase in woody plants is determined by both the growing environment and genetic factors (Hackett, 2011). Several studies have explored methods to reduce the juvenile period of apple seedlings. Some researchers boosted the vegetative growth of apple seedlings under optimal environmental conditions. For apples and pears, good vitality of the seedlings can shorten the juvenile phase (Visser et al., 1976). Zimmerman (1971) grew seedlings of an apomictic crabapple, Malus hupehensis (Pamp.) Rehd., continuously in a greenhouse; the first flowering was observed 12 months after germination. Aldwinckle (1975) reported that Malus spp. seedlings flowered 16–20 months after germination with manual defoliation in a greenhouse maintained at 17–21°C with a 16-h photoperiod. The optimization of conditions such as light, temperature, and nutrients can thus accelerate apple seedling growth, reducing the time from germination to flowering.
Transformation technology and viral vectors have also been used in attempts to achieve early flowering. Kotoda et al. (2006) generated transgenic apple trees expressing the apple TERMINAL FLOWER 1 gene (MdTFL1) antisense RNA, leading to flowering in a greenhouse eight months after grafting. Sasaki et al. (2011) inoculated apples with MdTFL1-introduced apple latent spherical virus (ALSV) vectors, resulting in the formation of flower buds approx. two months after grafting. Moreover, given the establishment of genome editing technology for apples (Nishitani et al., 2016), early flowering facilitated by this technology is anticipated to be achievable in the foreseeable future. However, the physiological mechanisms underlying the phase transition of apples are still unclear.
The morphological indicators of phase transition in fruit trees include tree height, number of nodes, stem circumference, branching pattern, leaf size, and root growth (Hackett, 2011; Zimmerman, 1973). Zimmerman (1971) considered that M. hupehensis seedlings undergo a phase change at a height of 2 m. In two segregating apple progenies, the phase change occurred around the 70th to 75th nodes and the 93rd to 104th nodes over two consecutive years (Atay, 2020). M. hupehensis may transition from the juvenile to the adult phase at a tree height of 1.8–2.0 m and the 75th to 80th nodes of the main stem node positions (Zimmerman, 1973). Zeng et al. (2009) reported that in growing shoots, different node positions (considered part of the phase transition) exhibited diverse amounts and characteristics of proteins.
Plant hormones are thought to participate in the phase transition (Barbosa and Dornelas, 2021; Guo et al., 2017; Song et al., 2020; Xing et al., 2014). In two-year-old hybrid seedlings of ‘Jonathan’ and ‘Golden Delicious’ apples, the dynamic of zeatin (Z) in the bark and dihydrozeatinriboside (DHZR) cytokinin in the leaves were identified as potential quantitative markers of the phase transition (Zhang et al., 2008). However, these results are not reproducible because the plant materials were hybrid seedlings of cultivated species. Given the high heterogeneity of cultivated apple varieties, it is not feasible to provide hybrid seedlings for phase transition tests, making it impossible to determine the exact phase transition time. To advance the study of phase transition, it is essential to determine the duration of the phase transition by establishing a reproducible seedling growing system; therefore, in the present study, we used the apomictic crabapple M. hupehensis (Olien, 1987), a wild species within the genus Malus that shares the same genotype as the seedlings. Xing et al. (2014) quantified the levels of phytohormones in the leaves of M. hupehensis seedlings in both the juvenile and adult phases. They found that the concentrations of auxin (IAA), cytokinin (CKs), and abscisic acid (ABA) were higher in the adult phase than in the juvenile phase, whereas gibberellin (GA) levels were higher in the juvenile phase than in the adult phase. In that study, however, the juvenile and mature shoots of six-year-old trees were compared, leaving it unclear how plant hormones shifted from the phase transition to the first flowering in younger apple seedlings.
The aims of the present study were to shorten the juvenile phase and clarify the changes in plant hormones during the phase transition of M. hupehensis. Hydroponics with round-the-clock light exposure and stable temperature conditions were employed as the cultivation method, given the ability of this setup to facilitate faster growth compared to traditional soil cultivation methods. We also investigated the changes in plant hormone levels in the leaves during the growth period associated with the phase transition that triggers the initial flowering of M. hupehensis seedlings, aiming to promote a more consistent artificial induction of the phase transition in seedlings using plant growth regulators in the future. The findings of this study will contribute to expand our knowledge of fruit tree breeding efficiency.
Nucellar seeds were collected from M. hupehensis growing at Takizawa Farm, Field Science Center of Agriculture, Iwate University, Japan. Flowers were covered by paper bags before blooming to prevent contamination, and the bags were removed after fruiting. The seeds were subjected to cold stratification under 4°C to break dormancy. After cold stratification, the germinated seeds were transferred to petri dishes containing moistened filter paper. These seedlings were cultivated hydroponically in a controlled environment room at 25°C under 24-h light conditions using a high-pressure sodium lamp (USHIO HILux Gro Super HPS600W; Ushio America, Inc. Cypress, CA, USA) throughout the experiment, which was conducted in 2019. PPFD levels were 370 μmol·m−2·s−1 and 130 μmol·m−2·s−1 at distances of 100 cm and 200 cm directly beneath the lamp.
Culture conditionsSixty seedlings that had germinated in the petri dishes were transplanted to a hydroponic container at 13–21 days after germination (DAG). A cell tray was placed in a container filled with water and pebbles for hydroponic cultivation. Approximately 30 days later, all seedlings were transferred to plastic pots (15 cm in diameter, 17 cm tall) with a slit and filled with hydro balls for support, with pairs of pots placed in each 9.5-L hydroponic cultivation container. For fertilizer management, we used a 500-fold diluted solution of Hyponex (N:P:K = 6.5:6:19) (HYPONeX Japan, Osaka, Japan). To promote growth in tree height, all shoots originating from the axillary vegetative bud were pruned, retaining only the main stem.
At 323 DAG, the hydroponic seedlings were transferred for soil cultivation into 21-cm pots. Volcanic ash soil collected from Takizawa Farm and sterilized in an autoclave was used.
Sampling for plant hormone analysisEight trees with relatively uniform growth were used for plant hormone analysis. Their leaves were numbered from the bottom node of the main stem as node 1, node 2, and so on. The leaves at nodes 49–51 were collected once the main stem had grown until the leaves at node 50 were completely expanded, which occurred at 104–107 DAG. Once the main stem had grown until the leaves at node 70 were completely expanded, the leaves at nodes 69–71 were collected at 153–157 DAG. Similarly, leaves at nodes 89–91 were collected once the seedlings had grown to the point where the leaves at node 90 were completely expanded at 175–185 DAG. Leaves at nodes 109–111 were collected when the leaves at node 110 were completely expanded at 214–235 DAG. The sampling timing is considered to include the transition points from the juvenile to adult phases in previous reports (Atay, 2020; Zimmerman, 1971). The height of the seedlings and the fresh weight of the leaves were measured for each sample. The tree stem circumference at 30 cm from the ground was measured, and the nodes of each tree were counted after the tree had stopped growing. The leaves were stored at −20°C until ABA, gibberellin A4 (GA4), trans-zeatin (Z), trans-zeatin riboside (ZR), isopentenyl adenine (iP), and isopentenyl adenosine (iPA) were analyzed. Auxin was also analyzed, but the measurement did not succeed technically.
Plant hormone analysisExtracts and purification procedures for ABA, GA4 and cytokinins were performed following the methods of Dobrev and Kaminek (2002), Opio et al. (2020), and Watanabe et al. (2021) with some modifications. The collected leaves per sample were macerated in cold 80% (v/v) methanol containing 100 mg·L−1 butyl hydroxytoluene (Wako Pure Chemical Industries, Osaka, Japan) with internal standards: 80 ng of d6-ABA, 10 ng of d2-GA4, 10 ng of d5-Z, 10 ng of d5-ZR, 10 ng of d6-iP, and 10 ng of d6-iPA (OlChemIm, Olomouc, Czech Republic). The sample was extracted and the homogenate was left at 4°C in darkness overnight. The homogenate was suction-filtered, and the filtrate was evaporated in vacuo at 40°C with a rotary evaporator, then dissolved in phosphate buffer (pH 8.5). Insoluble polyvinylpolypyrrolidone was added to the solution and it was shaken for 20 min. The solution was then centrifuged at 14,000 rpm at 4°C for 5 min and suction-filtered. The filtrate was evaporated in vacuo at 40°C and dissolved in distilled water. The solution was partitioned with petroleum ether three times, and the aqueous portion was evaporated in vacuo at 40°C and then dissolved in 1% (v/v) acetic acid.
The solution was loaded into a preconditioned Bond Elut LRC-C18 column (500 mg; Agilent Technologies, Santa Clara, CA, USA). The column was then washed with 1% acetic acid, and the hormone fraction was eluted with 1% acetic acid containing 80% (v/v) methanol. The eluate was evaporated in vacuo at 40°C and dissolved in 1 M formic acid. The solution was loaded into a preconditioned Oasis MCX column (150 mg; Waters, Milford, MA, USA). The ABA and GA fractions were then eluted with methanol after the column was washed with 1 M formic acid. The cytokinin (CK) fractions were eluted with a solution of 60% (v/v) methanol containing 0.35 M ammonia after being washed with 0.35 M ammonia, and then transferred to a microtube. After evaporation and transfer, the ABA and GA fractions were dissolved in methanol and loaded into a Bond Elut LRC-DEA column (500 mg; Agilent Technologies) that had been pretreated and washed with methanol. The solution was then eluted with 1% (v/v) acetic acid containing methanol, evaporated, and transferred to a microtube. The samples were concentrated to dryness (40°C) with a centrifugal evaporator and cryopreserved at −20°C.
For the separation and collection of ABA and GA fractions, we used a high-performance liquid chromatography (HPLC) system consisting of a system controller SCL-10A, liquid pump LC-10ATVP, degasser DGU-12A, UV detector SPD-10A, column oven CTO-10A, Chromatopack C-R6A, and fraction collector SF-2120.
The hormones were quantified using ultra-high-performance liquid chromatography coupled with a tandem mass spectrometer (UPLC-MS/MS) (Quattro Premier XE; Waters) equipped with a VanGuard BEHC 18 column (2.1 mm × 50 mm; Waters) in conjunction with a mass spectrometer. The concentration of each plant hormone was determined by calculating the area ratio of the peak area of natural and labeled ions using an internal standard.
Among the 60 seedlings grown in the hydroponics conditions, 57 survived and ceased growth at 7–8 months after germination. After the axillary shoots were removed and the growth ceased, some apical and axillary buds remaining on the main stem became flower buds. Flowers originating from axillary buds of the main stem were observed at 318 DAG and later. At this point, some of the seedlings had already grown fruit with an approx. diameter of 5-mm. As the flowering time prior to the investigation was unknown, the flowering date was estimated using the number of days required for fruits of the same diameter after flowering based on the present study; the earliest flowering time was estimated as 298 DAG, i.e., 9 months and 23 days after germination. Of the 57 seedlings, 15 flowered (Fig. 1), for a flowering rate of 26.3% (Table 1).
Tree height and flowering at 10 months (318 DAG) of M. hupehensis growth after sowing and starting hydroponics. Black bars: flowered seedlings. Solid line: tree height 200 cm. Dashed line: tree height 240 cm.
The numbers and growth data for the flowered and unflowered M. hupehensis seedlings at 318 DAG.
For all of the tested seedlings, the average tree height of the flowered seedlings was 246.6 cm, whereas that of the unflowered seedlings was 219.6 cm (Table 1). The average number of nodes of the flowered seedlings was 111.1, and was greater than that of the unflowered seedlings, which numbered 103.2. The average stem circumference of flowered seedlings at 30 cm from the ground was 3.64 cm, which was also greater than that of the unflowered seedlings at 3.08 cm. In a previous report, Zimmerman (1971) pointed out that M. hupehensis seedlings undergo a phase change at a height of 2 m. In the present study, all flowered seedlings had a tree height greater than 200 cm. Of the 57 seedlings, 47 grew to a height greater than 200 cm. For those 47 seedlings, the average tree height of the unflowered seedlings was 233.6 cm, still lower than that of the flowered seedlings (Table 1). The average stem circumference at 30 cm from the ground of the unflowered seedlings was 3.16 cm, which was lower than that of the flowered seedlings. The average number of nodes was 108.2, which was not significantly different from that of the flowered seedlings.
Among the flowered seedlings, the number of flowered nodes was 1–11 per tree, spanning from nodes 71 to 115. The node positions indicated that most of the flowers appeared between nodes 91 and 95 (Fig. 2A). The next most frequent positions were nodes 96–100, followed by nodes 81–85 and nodes 101–105. The distance from the ground to the flowered nodes ranged from 158 to 254 cm, with the most flowering observed between 210 to 220 cm, followed by 180 to 190 cm (Fig. 2B). The peak flowering period was between 320 to 329 DAG (Fig. 2C), followed by 310–319 DAG.
Number of flowered nodes at each node position (A), node height (B), and flowering date (C). Flowered node at 290–319 days after germination (DAG) is a presumed value based on flower and fruit formation.
When growth ceased, all eight seedlings sampled for plant hormone analysis were taller than 2 m, and four of the eight seedlings flowered approx. 10 months after germination. The tree height of the flowered and unflowered seedlings did not differ significantly until the collection of leaves at node 90, with the flowered seedlings being taller than the unflowered ones at the time point when the leaves of node 110 were collected (Fig. 3).
Tree height of the flowered and unflowered M. hupehensis seedlings at nodes 50, 70, 90, and 110. Solid dots: flowered seedlings. Open squares: unflowered seedlings. Values in parentheses indicate the number of days from germination to growth for the above nodes. * and NS indicate a significant difference at P < 0.05 and a nonsignificant difference by t-test, respectively.
The ABA and GA4 concentrations at node 50 could not be obtained due to technical issues, so the ABA and GA4 concentrations reported in this study were from nodes 70 to 110. The ABA concentration in the mature leaves at the tip of the main stem in the flowered seedlings was 173.3 ng·g−1 fresh weight (FW) when the trees reached node 70, then declined to 41.6 ng·g−1FW at node 90, and further decreased to 36.8 ng·g−1FW at node 110 (Fig. 4). The ABA concentrations in the unflowered seedlings were 87.1 ng·g−1FW, 182.9 ng·g−1FW, and 55.7 ng·g−1FW when the tree reached nodes 70, 90, and 110, respectively. ABA levels were significantly higher at node 70 in the flowered seedlings than in the unflowered ones and were lower at node 90 in the flowered seedlings compared to the unflowered ones.
ABA, GA4, Z, ZR iP, and iPA concentrations of the flowered and unflowered M. hupehensis trees at main stem nodes 50, 70, 90, and 110 (70, 90, and 110 in ABA and GA4). Solid dots: flowered seedlings. Open squares: unflowered seedlings. Vertical bars indicate SD (n = 4). * and NS indicate a significant difference at P < 0.05 and a nonsignificant difference by t-test, respectively.
The GA4 concentrations in the mature leaves at the tip of the main stem in the flowered seedlings were 1.12 ng·g−1FW, 0.80 ng·g−1FW, and 1.09 ng·g−1FW when the tree grew to nodes 70, 90, and 110, and were 0.86 ng·g−1FW, 1.12 ng·g−1FW, and 2.43 ng·g−1FW in the unflowered seedlings, respectively (Fig. 4). There was no significant difference in the GA4 concentration between the flowered and unflowered seedlings.
Figure 4 shows the concentrations of Z, ZR, iP, and iPA in the mature leaves at the tip of the main stem in the flowered and unflowered seedlings. The Z concentrations in the mature leaves at the tip of the main stem in the flowered seedlings were 0.47–0.88 ng·g−1FW when the tree reached nodes 50, 70, 90, and 110, and were 0.56–0.88 ng·g−1FW in the unflowered seedlings. The ZR concentrations were 2.46–4.06 ng·g−1FW in the flowered seedlings and 2.63–5.42 ng·g−1FW in the unflowered ones. The iP concentrations were 0.77–1.91 ng·g−1FW in the flowered seedlings and 0.68–1.37 ng·g−1FW in the unflowered ones. The iPA concentrations were 6.58–9.28 ng·g−1FW in the flowered seedlings and 9.30–12.53 ng·g−1FW in the unflowered ones. There were no significant differences between the flowered and unflowered seedlings in any of the cytokinins, which exhibited minor fluctuations as the trees progressed to nodes 50, 70, 90, and 110.
Zimmerman (1971) demonstrated that the earliest flowering of M. hupehensis apple seedlings cultivated in a greenhouse with long days and without the use of transformation techniques occurred 12 months after germination. The seedlings were over 300 cm tall at nine months after germination. In a study by Aldwinckle (1975), apple seedlings flowered 16 months after germination under optimum growing conditions in a greenhouse, and the seedlings were 250–300 cm tall at nine months after germination. In the present experiment, 47 of 57 seedlings reached heights greater than 200 cm within 10 months after germination (Fig. 1). Hyponex 500-fold diluted solution served as the culture solution. The tree height did not increase as rapidly as in the reports cited above, but based on the fruit size, we estimated that the initial flowering occurred at 298 DAG (Fig. 2C). In this experiment, the shortest period from germination to flowering was estimated to be nine months and 23 days. Flowering in this study occurred earlier than in previous studies. The seedlings showed peak flowering activity at 321–330 DAG.
Studies of the early flowering of apple seedlings have been conducted in both natural and greenhouse conditions (Visser, 1964, 1976; Way, 1971; Zimmerman, 1971). However, this study marks the first investigation conducted in a fully artificial environment with constant temperature and lighting conditions. This study was also the first phase-transition approach under hydroponic conditions. Our findings indicated that hydroponics in a controlled artificial environment was effective in promoting an early phase transition in M. hupehensis.
In reports on early flowering published to date, the seedlings that successfully underwent phase transition had undergone treatments such as extended photoperiods, defoliation, or exposure to low temperatures (Aldwinckle, 1975; Zimmerman, 1971). Under natural conditions, gibberellin inhibitors or artificial stress treatments were used to induce flowering (Zimmerman et al., 1985). In the present study, M. hupehensis seedlings were cultivated hydroponically in a stable environment without any induced stress. However, following the cessation of growth, leaf shedding occurred, potentially contributing to the flowering process.
The stem cross-sectional area was previously shown to be crucial for phase transition in apple seedlings (Atay, 2020). Here, the stem circumference was measured instead of the cross-sectional area, providing a comparable indicator. For trees taller than 200 cm, significant differences were observed in both the average tree height and average stem circumference between the flowered and unflowered seedlings; however, the average number of nodes showed no significant difference (Table 1). We thus inferred that tree height and stem circumference are key factors in promoting phase transition, irrespective of whether the trees are taller than 200 cm or include all seedlings.
According to Figure 2A, the lowest flowered node was between nodes 71–75, while the highest concentration of flowered nodes was observed between nodes 91–100. The maximum flowering height was 210–220 cm above the ground (Fig. 2B). Buds were collected from 10 out of 57 trees for another experiment, nine of which flowered. Therefore, the flower count at each node may have been higher if the buds had not been removed.
Phase transition is known to proceed from the apical part of the seedling stem to the basal part (Poethig, 1990). Zimmerman (1971) demonstrated that the earliest flowering of M. hupehensis apple seedlings was 12 months after germination, and the seedlings were more than 300 cm tall at nine months after germination, suggesting that phase change occurred within 7–10 months after seed germination. Zimmerman (1973) reported that the phase transition in M. hupehensis occurred at nodes 75–80, representing a height of 180–200 cm. Therefore, it is thought that M. hupehensis undergoes a phase transition during the development of the main stem several months before the flowering period.
In the present study, mature leaves at the tip of the main stem were collected when the M. hupehensis seedlings reached a main stem nodal stage that was expected to precede phase transition. We measured the concentrations of ABA, GA4, and CKs in the leaves at the tip of the main stem during this period. Seedling growth ceased when the leaves at nodes 109–111 were collected. Four of the eight seedlings that were tested flowered after growth ceased, and we concluded that the flowered seedlings had reached the transition phase. As a result, we posit that the time point at which the leaves were collected coincided with the phase transition.
Xing et al. (2014) reported that ABA levels in the leaves of 6-year-old M. hupehensis seedlings were lower in the juvenile phase than in the adult phase from March to May (when the shoots started to grow), but higher in July and August, when growth ceased. In the present study, the ABA concentration in the leaves of the flowering seedlings decreased as the main stem progressed from nodes 70 to 90, remaining constant until reaching node 110 (Fig. 4). In the unflowered seedlings, on the other hand, the ABA concentration increased from nodes 70 to 90 and then decreased at node 110. The ABA concentration of the flowered seedlings was lower than that in the unflowered seedlings at node 90, whereas at node 110 the ABA concentrations did not differ significantly. We speculate that the decline in ABA concentration in the leaves, followed by a period of stability, may be associated with phase transition. In the unflowered seedlings, the ABA concentration decreased as the main stem progressed from nodes 90 to 110, a pattern that occurred later than in the flowered seedlings. It is thus also possible that the unflowered seedlings experienced phase transition at a later time and flowered due to the ABA concentration remaining low. The timing of phase transition may vary even if the tree heights are the same. It may be possible to accelerate and stabilize phase transition by spraying an ABA synthesis inhibitor on apple seedlings. Song et al. (2020) reported that the ABA contents in fully expanded leaves of 6-year-old hybrid seedlings of Pyrus pyrifolia Nakai cv. Whangkeumbae and P. bretschneideri Rehd. cv. Zaosu were higher in the adult phase than in the juvenile phase. However, Hillman et al. (1974) found that the ABA concentration in Hedera helix L. leaves was higher in the juvenile phase than in the adult phase. The relationship between phase transition and ABA in woody plants remains unclear, although it is known that the ABA concentration in leaves increases under drought stress (Kondo et al., 2012). Since hydroponic cultivation always provides sufficient water, the cultivation method used here may have affected the ABA content in the leaves of M. hupehensis. Therefore, in future studies it will be necessary to compare the changes in ABA content in leaves during the phase transition between hydroponic and soil cultivation of M. hupehensis.
Xing et al. (2014) reported that the CK levels in the leaves of M. hupehensis seedlings (the specific CK type is unknown) decreased from March to August in both the juvenile and adult phases. From March to June, the CK levels in the leaves of the adult phase were higher than those in the juvenile phase. However, in the present experiment, there was no significant increase or decrease in the values of Z, ZR, iP, or iPA in the leaves at the tip of main stem during phase transition (Fig. 4). In addition, Xing et al. (2014) examined juvenile and adult segments from a 6-year-old seedlings that were already in the adult phase, although they did not specify which segments they examined. In the present study, apomictic seedlings that first bloomed about 10–12 months after germination were examined. The flowering node of each seedling was determined, indicating that phase transition likely occurred during the period when the leaves were being collected. We thus consider that the phytohormonal dynamics during the first flowering phase transition of apple seedlings can be more precisely explained compared to previous reports. The role of CK concentrations in the leaves at the tip of the main stem during the phase transition of M. hupehensis appears to be limited.
We observed that the GA4 concentration in the mature leaves at the tip of the main stem did not change substantially in the flowered seedlings as the node position increased (Fig. 4). The GA4 concentration in the unflowered seedlings did not differ significantly from that in the flowered seedlings at nodes 70, 90, and 110. It was reported that in Arabidopsis, GAs promoted phase transition, but inhibited flower differentiation (Yamaguchi et al., 2014). The gibberellin synthesis inhibitor SADH was reported to accelerate the flowering of apple seedlings after phase transition (Zimmerman, 1973). However, in the present study, the GA4 concentration in the leaves of the flowered seedlings did not differ significantly from that in the leaves of the unflowered seedlings, and it did not undergo a significant change due to phase transition. We thus assume that the GA4 concentration in the leaves at the tip of the main stem had no effect on phase transition. Here, the leaves at the tip of the main stem were examined during growth. In future studies, other parts, such as buds or stems, should also be examined. The dynamics of hormones from the conclusion of the growth phase to flowering should also be explored. It remains unclear whether the advancement of phase transition under the controlled environment in this study aligns with previous reports. Therefore, in future investigations it will be necessary to clarify the timing at which flower bud differentiation begins in seedlings undergoing phase transition in this environment.
In conclusion, our results indicate that employing hydroponics in a controlled setting is beneficial for accelerating the phase transition of M. hupehensis. Our examination of endogenous plant hormones in the leaves at the tip of the main stem during growth revealed that sustained low ABA levels may encourage phase transition, whereas the concentrations of GA4, Z, ZR, iP, and iPA in the leaves did not significantly impact the phase transition of M. hupehensis.
