Water as a Compass: Hydrostimulation‐Triggered Aerial Root Growth in Phalaenopsis aphrodite

ABSTRACT

Epiphytic orchids have evolved specialized adaptive strategies, such as aerial roots with water‐absorbing velamen tissues, to cope with water‐scarce and nutrient‐deficient habitats. Our previous study revealed that the aerial roots of the epiphytic orchid Phalaenopsis aphrodite lack a gravitropic response, raising the possibility that alternative tropic mechanisms may contribute to their adaptation. In this study, we examined the effects of light and moisture on aerial root growth in P. aphrodite . Surprisingly, we found that light had no effect on root growth orientation. In contrast, localized moisture gradients consistently directed the growth of young aerial roots, indicating a hydrotropic response. To explore the underlying regulatory mechanisms, we performed hormone profiling of hydrostimulated root tissues. Our data showed that indole‐3‐acetic acid (IAA), salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) levels did not differ significantly between the water‐facing and air‐facing sides of the roots, suggesting other mechanisms may regulate this hydrotropism. In summary, our findings demonstrate that hydrotropism, rather than phototropism or gravitropism, guides aerial root growth in P. aphrodite . This hydrotropic response may represent a key adaptation that enables epiphytic orchids to effectively acquire water in the forest canopy.

1. Introduction

Epiphytic orchids typically inhabit the forest canopy in tropical and subtropical regions. Rather than rooting in soil, they grow non‐parasitically on host plants or other structures such as rocks (Zotz et al. 2021). To survive the intermittent availability of water, nutrients, and light in canopy environments, epiphytic orchids have evolved aerial roots with specialized morphological and physiological adaptations. These include the development of the velamen radicum, a multilayered tissue composed of dead epidermal cells, and the use of crassulacean acid metabolism (CAM), a water‐conserving photosynthetic pathway (Roth‐Nebelsick et al. 2017). The velamen radicum, due to its spongy structure, facilitates rapid water absorption within seconds and provides a protective barrier that minimizes water loss from internal tissues (Zotz et al. 2021). Additionally, many epiphytic orchids employ CAM photosynthesis as an adaptive response to water scarcity (Benzing 1989; Lüttge 2004). Stable carbon isotope ¹³C analyses of orchid leaves indicate that CAM photosynthesis has evolved multiple times independently within the Orchidaceae, likely contributing to the diversification and speciation of epiphytic lineages (Silvera et al. 2009). Some C3 orchids are also capable of switching to CAM photosynthesis during periods of drought, highlighting a flexible photosynthetic strategy (Lüttge 2004; Rodrigues et al. 2013; Tay et al. 2019). Together, these morphological and metabolic innovations have enabled epiphytic orchids to thrive across a broad range of ecological habitats.

Root cells in many epiphytic orchids contain chloroplasts and are capable of performing photosynthesis (Martin et al. 2010; Brunello et al. 2024). This root‐based photosynthesis has been shown to alleviate hypoxic stress in the aerial roots (Brunello et al. 2024). Although photosynthesis in aerial roots is well documented, it remains unclear whether light signals influence the directional growth of these roots.

Roots play a crucial role in acquiring water and nutrients from their surrounding environments. Hydrotropism refers to the ability of roots to sense and grow toward sources of moisture (Miyazawa and Takahashi 2020). It has long been proposed that moisture gradients in the soil near roots trigger the hydrotropic response (Gardner 1965). Similar to gravitropic responses, hydrotropism involves moisture sensing, signal transduction, and directional growth (Voothuluru et al. 2024). Recent studies have begun to uncover the molecular links between signal perception and growth reorientation. The root caps of maize and pea are particularly sensitive to hydrostimulation (Takahashi and Scott 1993; Takano et al. 1995). In contrast, Arabidopsis thaliana roots retain their hydrotropic response even after the removal of the root cap and meristem, suggesting that the root cap is not essential for moisture sensing in this species (Dietrich et al. 2017). Thus, the role of root cap cells in hydrotropic sensing remains controversial. Hormonal signaling pathways including auxin (indole‐3‐acetic acid, IAA), abscisic acid (ABA), cytokinins, and brassinosteroids have been implicated in regulating hydrotropism (Miao et al. 2018, 2021; Li et al. 2024; Fu et al. 2025; Zhang et al. 2025). Additional modulators include calcium (Takano et al. 1997; Shkolnik et al. 2018; Ju et al. 2024), reactive oxygen species (Krieger et al. 2016), and H⁺‐ATPases (Miao et al. 2021). Genetic screens conducted in Arabidopsis have identified several key molecular components involved in the regulation of root hydrotropism (Miyazawa and Takahashi 2020). Despite these advances, most research has focused on terrestrial plants with gravitropic roots. How epiphytic species perceive and respond to moisture gradients—and whether aerial roots exhibit hydrotropic growth to cope with dry environments—remains largely unexplored.

Root gravitropism, the directional growth of a root toward gravity, is essential for plant adaptation to arid terrestrial environments (Sato et al. 2015). Root gravitropism allows plants to anchor to the ground and enables efficient water and nutrient uptake from the soil. Similarly, the aerial roots of epiphytic plants provide structural support and absorb water and nutrients, playing a critical role in their survival. However, it remains unclear whether these aerial roots respond to environmental cues. Recent findings indicate that the aerial roots of Phalaenopsis aphrodite do not exhibit gravitropic responses (Chen et al. 2024). This absence of gravitropism is likely due to the absence of the gravity‐sensing mechanism (amyloplast sedimentation) and the auxin efflux transporter PIN2 gene, which regulates the gravity‐dependent asymmetrical auxin distribution. This finding raises the question of which tropic responses aerial roots acquire to adapt to canopy environments. Given that orchid aerial roots contain chloroplasts for photosynthesis (Martin et al. 2010) and are capable of absorbing water from the atmosphere (Roth‐Nebelsick et al. 2017; Zotz et al. 2021), we hypothesized that light and/or water may serve as environmental cues guiding their directional growth. In this study, we investigated the effects of light and hydrostimulants on the growth orientation of aerial roots in the epiphytic orchid P. aphrodite . Our results show that root growth was unresponsive to light but exhibited a response to a hydrostimulant. These findings provide evidence supporting a hydrotropic response in orchid aerial roots, a tropic behavior that may be conserved across other vascular epiphytes.

2. Materials and Methods

2.1. Plant Materials

P. aphrodite subsp. formosana (m1663 seedlings, tetraploid) was purchased from Yard Tale Horticulture (Ping Tung, Taiwan). The plants were grown in a temperature‐controlled greenhouse at 25°C at the Academia Sinica Southern Campus, Tainan.

2.2. Seed Germination and Young Seedlings

Mature seeds were allowed to germinate on 1/4 strength Murashige and Skoog basal medium supplemented with 0.1% (w/v) tryptone, 1.8% (w/v) sucrose, 2% (w/v) potato homogenate, 0.01% (w/v) myoinositol, 0.0001% (w/v) nicotinic acid, 0.00001% (w/v) pyridoxine‐HCl, 0.00001% (w/v) thiamine‐HCl, and 1% (w/v) agar, adjusted to pH 5.7. Germinated protocorms were grown in a temperature‐controlled growth chamber maintained at 24°C in 16 h light/8 h dark cycles under illumination at 45–55 μmol photons m⁻² s⁻¹. The light‐related experiments were conducted in a growth chamber maintained at 24°C in 16 h light/8 h dark cycles under illumination at 45–55 μmol photons m⁻² s⁻¹. P. aphrodite seedlings of approximately 3–5 months old grown from germinated protocorms were used for all the experiments described.

For the light response assays, the seedlings were maintained on T1 medium (0.2% [w/v] Hyponex No. 1, 0.1% [w/v] tryptone, 2% [w/v] sucrose, 0.01% [w/v] citric acid, 0.1% [w/v] charcoal, 1% [w/v] agar, pH 5.4).

For the hydrotropic experiments, the seedlings were grown on sphagnum moss and kept in a seedling germination tray to maintain relatively high humidity (RH = 80%–100%, depending on the time of the day and weather conditions). All plants were grown in a temperature‐controlled (~25°C) greenhouse. The experimental setup is shown in Figure 2A. For the hydrotropic test, 150 mL water was added to the tip‐boxes. A no‐water control was used as a comparison. Water‐containing and control tip‐boxes were kept in the same seedling germination tray. Sphagnum moss was wetted once every week to keep the moisture content stable. The growth of aerial roots was recorded after 69–105 days of growth in the tip‐box chambers.

FIGURE 2.

The effect of water on root growth of Phalaenopsis aprhodite. (A) Schematic illustrating the experimental setup for hydrotropism test. (B) Response of aerial roots to the water‐treated and control conditions. Scale bar = 1 cm. (C) The percentage of plants with at least one root bending downward was compared between control and water‐treated seedlings. Only aerial roots that had grown long enough to reach the tip box holes were considered in the analysis. The plants were allowed to grow for 69–105 days. This experiment was repeated seven times for statistical analysis, with total 30 plants per treatment. The standard error is represented by error bars. *, A significant increase in the percentage of roots that grew downward (p < 0.01). (D) Some aerial roots of P. aphrodite clung to the surface of the tip box. Scale bar = 1 cm. The yellow arrow indicates a root adhering to the tip box. (E) Comparison of the percentage of downward‐growing aerial roots that grew into the tip boxes versus those that adhered to their inner surfaces in both control and water‐treated seedlings. Only downward‐growing aerial roots were included in this analysis. The plants were allowed to grow for 69–105 days. This experiment was repeated seven times for statistical analysis. The standard error is represented by error bars. *, A significant increase in the percentage of roots into the tip boxes (p < 0.05).

2.3. Plant Hormone Analysis

Approximately 100 mg of root tissue (from 4 to 6 roots) was snap‐frozen in liquid nitrogen and homogenized using a tissue homogenizer (Kurabo SH‐100) with a 5 mm stainless steel grinding ball at 15,871g for 30 s, followed by cooling with liquid nitrogen. The homogenization cycle was repeated six times. Metabolites were extracted using 0.95 mL of extraction buffer (2‐propanol/H₂O/HCl, 2:1:0.002) and 0.05 mL of 100% MeOH, supplemented with 1 mM ascorbic acid, 1 mM butylated hydroxytoluene (BHT), in the presence of isotope standards (2 ng ¹³C₆‐IAA, 5 ng d₅‐jasmonic acid (JA), 10 ng ¹³C₆‐salicylic acid (SA), and 1 ng d₆‐ABA). The extract was mixed at 4°C for approximately 2.5 h. One milliliter of dichloromethane was added to the extract, and the mixture was shaken at 4°C for 30 min. Dichloromethane was used to remove polysaccharides from the extract and was essential for obtaining consistent data. Centrifugation was performed at 15,871g at 4°C for 5 min. The bottom phase of the extract was transferred to a new tube and concentrated using a CentriVap Vacuum Concentrator (Labconco 7310021) at room temperature.

Hormones were analyzed and quantified by an ultra‐performance liquid chromatography (UPLC) system (Acquity, Waters) coupled to a Xevo TQ‐XS triple quadrupole mass spectrometer (Waters). Briefly, a 5 μL sample was injected and separated using an Acquity UPLC HSS T3 column (Waters) at 30°C. The Mobile Phase A consisted of 0.1% acetic acid in water, while Mobile Phase B was 0.1% acetic acid in methanol. Characteristic MS transitions were monitored using negative multiple reaction monitoring (MRM) mode for SA (m/z, 137 > 93), ¹³C₆‐SA (m/z, 143 > 99), JA (m/z, 209 > 59), and D5‐JA (m/z, 214 > 62), ABA (m/z, 263 > 153), and d₆‐ABA (m/z, 269 > 159). Positive MRM mode was used for endogenous (IAA, m/z, 176 > 130) and ¹³C₆‐IAA (182 > 136). Data acquisition and processing were carried out using MassLynx (v.4.2) and TargetLynx software (Waters). This experiment was repeated eight times to generate data for statistical analysis (Table S1A and S1B).

During the hormone profiling experiment, substantial variability in the absolute hormone quantities was observed among root samples from different plants. To normalize this variation, the Up/Down ratio of absolute values was used to assess differences between the upper root halves (facing the air) and the lower root halves (facing downward).

2.4. Statistical Analysis

All experiments were performed multiple times for statistical analysis. For the water treatment experiment presented in Figure 2C, this experiment was repeated seven times. A total of 30 plants per treatment were used. For hormone analysis presented in Figure 4B, this experiment was repeated eight times. The data are presented as means and standard deviations obtained from replicates of a single experiment. The significant difference between the treatments was analyzed by running a Student's two‐tailed t test (α = 0.05).

FIGURE 4.

Relative hormone levels in the upper (Up) and lower (Down) parts of aerial roots of water‐treated and control Phalaenopsis aphrodite seedlings. (A) Roots were harvested 4 days after hydrotropic treatment but before conspicuous root bending. Scale bar = 1 cm. (B) The ratios of hormone content between the upper (Up) and lower (Down) parts of root tips in response to hydrostimulant or control treatment (see Section 2). Data are presented as means ± SD. Standard deviations were derived from eight independent experiments. A Student's two‐tailed t test (α = 0.05) was applied to all the data. There was no significant increase (p > 0.05) in hormone content compared with control group. (C) The aerial roots of Phalaenopsis orchids lack phototropism but exhibit a modest yet significant hydrotropic response. Part of this artwork was assisted by ChatGPT.

3. Results and Discussion

3.1. Light Does Not Influence Aerial Root Growth in P. Aphrodite

Given the photosynthetic capability of P. aphrodite aerial roots, we investigated whether light influences their growth direction. In the first experiment (Phototropism Test 1), orchid seedlings were grown in a chamber with light directed from the side (Figure 1A). Over an approximately 4‐week period, aerial root growth appeared unaffected by the direction of light illumination (Figure 1B).

FIGURE 1.

The effect of light on the root growth of Phalaenopsis aprhodite. (A) Schematics illustrating Phototropism Condition Test 1. (B) Response of aerial roots under Phototropism Test Condition 1. (C) Schematics illustrating Phototropism Condition Test 2–1 and 2–2. (D) Response of aerial roots under Phototropism Test Condition 2–1 and 2–2. The plants were allowed to grow for 59–63 days. Scale bar = 1 cm. (E) Quantification analysis of the light effect on aerial root growth using Phototropism Test Condition 2–1 and 2–2. The average percentage was derived from five independent experiments. The standard error is represented by error bars. Red arrow, shoot growing toward the light. Yellow dot, light source. *, A significant proportion of aerial roots failed to bend toward the light (p < 0.005).

To confirm this observation, seedlings were grown in foil‐covered petri dishes with a single hole allowing directional light exposure from a light shelf for approximately eight weeks. In one setup (Phototropism Test 2–1), roots were positioned perpendicular to the light source; in another (Phototropism Test 2–2), roots were oriented away from the light (Figure 1C). Aerial roots were classified as “bending toward light” if they grew at an angle ≥ 40° toward the light source. Roots that did not meet this criterion were categorized as “not bending toward light” and considered light‐independent. Due to variability in growth rates among protocorm‐derived seedlings, only roots that grew more than 0.5 cm during the 8‐week period were included in the analysis. In Phototropism Test 2–1, 94.7% of roots did not bend toward the light, and in Test 2–2, 93.4% also failed to exhibit light‐directed growth (Figure 1D,E). These results indicate that aerial root growth in P. aphrodite is not directed by light, regardless of seedling orientation. Notably, shoots grew toward the light source in both experiments (Figure 1D), confirming a functional phototropic response in the aerial parts of the plant.

Interestingly, P. aphrodite aerial roots did not grow in response to light despite their ability to conduct photosynthesis. This suggests that the aerial roots are not actively seeking a light source, aligning with previous observations that their photosynthetic activity only plays a subsidiary role in carbon fixation (Goh et al. 1983; Hew et al. 1984; Brunello et al. 2024). It is also noteworthy that the aerial roots of P. aphrodite did not exhibit a defined negative phototropic response (Muthert et al. 2019). In our phototropism assays (Figure 1E), a proportion of aerial roots bent toward the light under Test Condition 2–1 (7.9%) and Condition 2–2 (5.3%), suggesting that light does not serve as a tropic cue for aerial roots.

3.2. Hydrotropic Response of Aerial Roots in P. Aphrodite

To determine whether a moisture gradient influences the growth direction of P. aphrodite aerial roots, orchid seedlings were grown on moisturized peat moss placed in tip boxes within a temperature‐controlled greenhouse (Figure 2A). The seedlings were maintained in a closed microenvironment with high humidity (see Section 2), which proved essential for aerial root development. A localized moisture gradient was established by adding 150 mL of water to the tip boxes (water‐treated group), while no water was added to the control group. Aerial root growth was monitored over a period of approximately 69–105 days. In the control group, aerial roots grew in various directions, displaying a seemingly random growth pattern. In contrast, seedlings exposed to the water gradient exhibited a greater tendency for newly emerging roots to grow downward toward the water source (Figure 2B). To validate this observation, the experiment was repeated seven times using a total of 30 seedlings. For comparisons between control and water‐treated seedlings, only aerial roots that had grown long enough to reach the tip box holes were included in the analysis. In the water‐treated group, approximately 76.4% of seedlings developed at least one root growing downward toward the water, compared to 47.9% in the control group (Figure 2C), where downward growth was likely due to random orientation. These results demonstrated that the aerial roots tended to grow toward the source of high moisture. Interestingly, some of the downward‐growing aerial roots adhered to the inner surface of the tip boxes (Figure 2D), suggesting that moisture retained by the sphagnum moss may serve as a source of hydrotropic stimulus. To distinguish the potential effect of moisture from sphagnum moss from water‐induced hydrostimulation, we quantified and compared the downward‐growing aerial roots that either grew into the tip boxes or adhered to their inner surfaces, in both control and water‐treated seedlings. Our results show that 67.4% of the downward‐growing roots in water‐treated plants grew into the tip boxes, whereas only 41.6% of such roots in control plants did so—presumably due to random growth (Figure 2E). In contrast, the proportion of downward‐growing roots adhering to the tip box surface was similar between control and water‐treated seedlings (Figure 2E). Together, these data indicate that aerial roots of water‐treated seedlings have a greater tendency to grow toward the water‐induced hydrostimulant.

Even though the hydrotropic response was detected, it was only observed for relatively young roots, and not all of the roots responded to the hydrostimulant. The basis for this differential response remains unclear. One possibility is that aerial roots prioritize anchorage over directional growth toward water. To attach securely to tree trunks or bark, epiphytic orchids flatten their aerial roots to maximize surface contact, forming strong adhesions that are difficult to detach (Zotz et al. 2021; Figure 3). A strong hydrotropic response, particularly during seasonal rainfall, could disrupt this attachment and compromise the plant's stability. Whether epiphytic orchids have genetically adapted to balance hydrotropism with surface attachment remains to be determined.

FIGURE 3.

Aerial roots of Phalaenopsis orchid clung tightly to the tree trunk.

3.3. Lack of Differential Accumulation of IAA, SA, JA, and ABA Preceding Hydrotropic Growth of Aerial Roots

Cell elongation and expansion are key processes underlying the differential growth of roots in response to tropic stimuli. Hormones such as auxin, cytokinin, and ABA have been implicated in regulating hydrostimulant‐induced root bending (Dietrich 2018; Miyazawa and Takahashi 2020). To investigate the potential role of hormones in the hydrotropic response of P. aphrodite, we quantified the levels of IAA, SA, JA, and ABA in aerial roots. Because cytokinins, including trans‐zeatin, cis‐zeatin, and N ⁶‐(∆ ²‐isopentenyl) adenine, were below the detection threshold, they were excluded from further analysis. Since hormone redistribution, particularly auxin, precedes root curvature in maize (Wang et al. 2020), we focused on analyzing water‐treated roots before the onset of conspicuous tip curvature. In our system, root tip curvature became evident around Days 4–5 following water treatment. We therefore selected Day 4 as the sampling time point to capture early molecular events preceding visible root bending. This experiment was designed as follows: root tips that had not yet developed velamen tissues were harvested after four days of water treatment and then longitudinally divided into two halves (Figure 4A)—the “Water/Down” side, facing the hydrostimulant, and the “Water/Up” side, exposed to air. For comparison, root tips grown in an empty (water‐free) tip box were similarly divided into “Control/Down” (facing downward) and “Control/Up” (facing the air). Our data showed that the levels of IAA, SA, JA, and ABA did not differ significantly between the “UP” and “DOWN” sides in either control or hydrostimulated roots (Figure 4B), suggesting mechanisms other than IAA, SA, JA, and ABA may regulate hydrotropism in P. aphrodite aerial roots.

Unexpectedly, none of the hormones tested showed differential accumulation in aerial roots treated with the hydrostimulant. Auxin, in particular, has been shown to play a key role in regulating root hydrotropism in several plant species, including cucumber, rice, pea, and maize (Morohashi et al. 2017; Nakajima et al. 2017; Fujii et al. 2018; Wang et al. 2020). Particularly, differential auxin dynamics has been reported to be associated with the moistened (concave) side and the opposite (convex) side of the hydrotropically responding roots in cucumber and maize (Morohashi et al. 2017; Fujii et al. 2018; Wang et al. 2020). However, in our study, auxin did not display differential accumulation in hydrostimulant‐triggered aerial roots, suggesting that mechanisms other than auxin may regulate hydrotropism‐associated root bending in Phalaenopsis. Intriguingly, the root hydrotropism regulator MIZU‐KUSSEI1 (MIZ1) in Arabidopsis has been reported to function upstream of the cytokinin signaling pathway (Chang et al. 2019). Specifically, the preferential accumulation of type‐A response regulators, ARR16 and ARR17, on the lower water potential side of the root apical meristem enhances cell division, resulting in root curvature. Whether orchid MIZ1‐like genes and/or cytokinin signaling are involved in the hydrotropic response of orchid aerial roots remains to be determined.

Vascular epiphytes comprise more than 30,000 species, representing approximately 10% of the vascular plant diversity (Zotz et al. 2021). Most vascular epiphytes thrive in humid forest environments characterized by frequent light rainfall, persistent mist, and low evaporation rates. In such habitats, it is plausible that epiphytic aerial roots have evolved hydrotropic mechanisms to efficiently locate and acquire water. Unlike most vascular plants, where the gravitropic response of roots often confounds the study of hydrotropism (Miyazawa and Takahashi 2020), the absence of root gravitropism in the epiphytic orchid P. aphrodite (Chen et al. 2024) presents a unique opportunity to investigate hydrotropic behavior directly. Furthermore, recent advances in molecular tools for Phalaenopsis orchids (Lin et al. 2018; Fang et al. 2022; Chen et al. 2024) provide a valuable foundation for elucidating the molecular pathways underlying hydrotropic responses in epiphytic orchids.

4. Conclusions

Using the epiphytic orchid P. aphrodite, we demonstrate that its aerial roots lack phototropic responses but exhibit a modest yet significant hydrotropic response (Figure 4C). To our knowledge, this represents the first report of hydrotropism in an epiphytic orchid. Notably, the levels of key phytohormones, including IAA, SA, JA, and ABA, did not differ between the two sides of hydrostimulated roots, suggesting that hydrotropism may be regulated by alternative mechanisms.

Author Contributions

S.‐C.F. conceived the idea and coordinated this study. S.‐C.F. and Y.‐J.H. supervised the project. H.‐C.C., I.‐C.C., J.‐C.C., Y.‐J.H., and S.‐C.F. designed the experiments. H.‐C.C., I.‐C.C., and J.‐C.C. conducted the experiments. S.‐C.F. and Y.‐J.H. wrote the manuscript. All authors have read and approved the final manuscript.

Supporting information

Table S1A: Plant hormone analysis—hormone measurement. IAA, SA, JA, and ABA levels extracted from 1 mg of the upper (Up) and lower (Down) parts of aerial root tissues from water‐ and control‐treated Phalaenopsis aphrodite seedlings (pg/mg FW; mean ± SD).

Table S1B: Plant hormone analysis—ratio of the indicated hormone. Relative hormone levels in the upper (Up) and lower (Down) parts of aerial root tips of water‐ and control‐treated P. aphrodite seedlings. Ratio = mean ± SD.

Chang, H.‐C. , Chen I.‐C., Chen J.‐C., Hou Y.‐J., and Fang S.‐C.. 2025. “Water as a Compass: Hydrostimulation‐Triggered Aerial Root Growth in Phalaenopsis aphrodite .” Physiologia Plantarum 177, no. 5: e70505. 10.1111/ppl.70505.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.