Root cap cuticles confer a transient, penetration-optimized phase during early seedling establishment

Root cap cuticles confer a transient, penetration-optimized phase during early seedling establishment | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 27 January 2026 V1 Latest version Share on Root cap cuticles confer a transient, penetration-optimized phase during early seedling establishment Authors : Woo-Taek Jeon 0009-0003-5984-5489 , Jeong-A Kim , Ahyeon Cheon , Yesol Shin , Shawn S.Y. Lee , and Yuree Lee 0000-0002-4663-6974 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176949232.24395129/v1 185 views 80 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract [1]¿p#1 newcommands Roots emerging from seeds encounter substantial mechanical resistance during soil entry. A transient root cap cuticle (RCC) forms early in development, yet its contribution to root tip function during this mechanically challenging transition remains unclear. Here, we define an early functional window in which the root tip is transiently optimized for establishment under soil-imposed constraints, and show that this window depends on the RCC. Consistent with a protective role, RCC-deficient seedlings were less efficient at traversing dense substrates and accumulated greater mechanical damage at the tip. Notably, RCC function extended beyond protection: when roots were grown on agar surfaces, RCC deficiency accelerated detachment of the outermost root cap cell layer and disrupted its layer integrity, indicating an additional role in maintaining coordinated root cap turnover. This early window faded with seedling age, preceding RCC loss, and coincided with a developmental reduction in gravitropic responsiveness, thereby defining a restricted period for directional control. RCC-defective mutants exhibited impaired gravitropism across stages, whereas hydrotropic responses were largely unaffected, linking RCC function specifically to gravitropic regulation. Together, these results delineate a transient establishment phase in which the RCC integrates mechanical protection, coordinated turnover, and gravitropic control to promote robust root function during early growth. Root cap cuticles confer a transient, penetration-optimized phase during early seedling establishment Woo-Taek Jeon 1* , Jeong-A Kim 1* , Ahyeon Cheon 1,2 , Yesol Shin 1 , Shawn S.Y. Lee 1 , Yuree Lee 1,3,4‡ 1 School of Biological Sciences, Seoul National University; Seoul 08826, Republic of Korea 2 Department of Plant Science, Seoul National University; Seoul 08826, Republic of Korea 3 Research Center for Plant Plasticity, Seoul National University; Seoul 08826, Republic of Korea 4 Plant Genomics and Breeding Institute, Seoul National University; Seoul 08826, Republic of Korea ‡ Author for correspondence: [email protected] , ORCID: 0000-0002-4663-6974 *These authors contributed equally to this work Keywords : Arabidopsis; root cap cuticle; root penetration; mechanical stress Abstract Roots emerging from seeds encounter substantial mechanical resistance during soil entry. A transient root cap cuticle (RCC) forms early in development, yet its contribution to root tip function during this mechanically challenging transition remains unclear. Here, we define an early functional window in which the root tip is transiently optimized for establishment under soil-imposed constraints, and show that this window depends on the RCC. Consistent with a protective role, RCC-deficient seedlings were less efficient at traversing dense substrates and accumulated greater mechanical damage at the tip. Notably, RCC function extended beyond protection: when roots were grown on agar surfaces, RCC deficiency accelerated detachment of the outermost root cap cell layer and disrupted its layer integrity, indicating an additional role in maintaining coordinated root cap turnover. This early window faded with seedling age, preceding RCC loss, and coincided with a developmental reduction in gravitropic responsiveness, thereby defining a restricted period for directional control. RCC-defective mutants exhibited impaired gravitropism across stages, whereas hydrotropic responses were largely unaffected, linking RCC function specifically to gravitropic regulation. Together, these results delineate a transient establishment phase in which the RCC integrates mechanical protection, coordinated turnover, and gravitropic control to promote robust root function during early growth. 1. Introduction Roots grow through soil to acquire water and nutrients, continually exposing the root tip to mechanical stress and potential tissue damage (Potocka and Szymanowska-Pułka, 2018). Therefore, sustained growth depends on mechanisms that preserve tissue integrity while retaining the ability to sense the surrounding environment and adjust growth direction accordingly (Berhin et al., 2019). These competing demands impose an inherent trade-off, whereby a fully rigid protective barrier would compromise environmental sensing, whereas excessive exposure would increase vulnerability to damage. Plants resolve this trade-off through the root cap, a multicellular structure positioned at the forefront of root–soil interactions (Dolan et al., 1993). The root cap covers the root tip and forms the primary interface with the surrounding soil (Dolan et al., 1993). Moreover, the root cap protects meristematic tissues during growth through mechanically challenging substrates while remaining a major site for environmental perception and signal integration that steers directional growth, including gravity and moisture sensing and responses to mechanical cues at the root–soil interface (Juniper et al., 1966; Barlow, 2002; Kobayashi et al., 2007; Zhu et al., 2025). Notably, the root cap maintains a dynamically renewed surface that provides both protection and sensory function, in which the outermost cell layers are shed and replaced in a regulated detachment–regeneration program, enabling the tip to withstand mechanical damage while sustaining growth (Barlow, 2002). This turnover cycle is coordinated by hormonal and redox signaling, including auxin and reactive oxygen species (ROS), to maintain root cap integrity during growth (Shin et al., 2025). Additionally, the secretion of pectic mucilage from the root cap provides local buffering against mechanical stress and facilitates root movement through the substrate (Iijima et al., 2004). Together, this coordinated surface renewal and extracellular secretion provide protection while preserving sensory competence at the growing tip. This strategy contrasts with that of aerial tissues, which do not require direct nutrient or water uptake from the external environment and can, therefore, rely on a stable epidermal cuticle (Yeats and Rose, 2013). In shoots, the cuticle, composed of a cutin matrix with associated waxes, forms a hydrophobic diffusion barrier that limits non-stomatal water loss and restricts pathogen entry, while also contributing to the mechanical robustness of the epidermis (Nawrath et al., 2013; Ingram and Nawrath, 2017). Although changes in wax load and composition across tissues and environmental conditions can tune cuticular properties (Lee et al., 2025; Jeon et al., 2026), the primary function of the shoot cuticle remains protective. Recent studies have revealed that a root cap cuticle (RCC) transiently forms on the outermost root cap cells (Berhin et al., 2019). At first glance, this cuticle-like layer is counterintuitive since a hydrophobic barrier would be expected to compromise the balance between mechanical protection and environmental sensing at the root–soil interface. Therefore, this apparent paradox suggests that the RCC is unlikely to function solely as a mechanical barrier, but may instead serve additional roles specific to early root tip physiology. Indeed, the RCC has been reported to contribute to abiotic stress tolerance, such as protecting the root meristem under salt stress, as well as to facilitate lateral root emergence (Berhin et al., 2019; Uemura et al., 2024; Berhin et al., 2026). However, these reported roles do not readily explain the developmental specificity of the RCC. Unlike shoot cuticles, the RCC exists only briefly and is not regenerated after the first root cap detachment event (Berhin et al., 2019). Moreover, because salt stress can occur throughout root growth, the transient nature of the RCC suggests a function uniquely required during early root development. Hence, this study aimed to address this possibility by examining the challenges faced by emerging seedlings and testing how the RCC contributes to early root penetration. Additionally, we assess whether this transient structure integrates mechanical protection with root cap turnover and directional control during the initial stages of root establishment. Consequently, we show that the RCC supports efficient initial penetration into dense substrates and is linked to orderly root cap detachment dynamics, including under non-penetrating conditions. We further identified that changes in penetration competence along the seedling development track are coupled with gravitropic responsiveness, and that this coupling is disrupted in RCC-deficient roots, whereas hydrotropic responses are largely preserved. These findings position the RCC as a transient, integrative element that coordinates early root cap performance during establishment. 2. Materials and Methods Plant material All Arabidopsis thaliana lines examined in this study were derived from the Columbia-0 (Col-0) ecotype. Detailed descriptions of the Arabidopsis mutant and transgenic lines have been reported previously: gpat4 gpat8 (Li et al., 2007), bdg-1 (Kurdyukov et al., 2006), dcr-2 (Panikashvili et al., 2009), pin2 (Luschnig et al., 1998), DR5::GUS (Ulmasov et al., 1997), pin2 DR5::GUS (Ditengou et al., 2008). Plant growth conditions Seedlings were grown on half-strength Murashige and Skoog (MS) medium supplemented with 1% (w/v) sucrose and solidified with agar. Seeds were surface sterilized by vapor-phase sterilization and stratified at 4 °C for 2 days before sowing. Plants were grown under long-day photoperiods, with light (70 μmol m⁻² s⁻¹) at 22 °C for 16 h, followed by darkness at 18 °C for 8 h. Agar concentrations of either 1.2% or 2.0% (w/v) were used, and seedlings were grown in either vertical or horizontal orientations as indicated. Mechanical impedance and penetration assays Two complementary agar-based assays were performed to assess root penetration capacity. In assay 1, the upper portion of the agar medium was removed, and seeds were placed directly onto the exposed cut surface. Plates were maintained in a vertical orientation to allow root growth toward the agar surface and subsequent penetration into the agar. In assay 2, seeds were sown on intact agar surfaces and initially grown under vertical conditions. After primary root growth was established, plates were repositioned horizontally to promote root penetration into the agar matrix. Penetration efficiency was quantified as indicated. [1]¿p#1 newcommands Root cap phenotyping and treatments For cuticle visualization, roots were stained with Fluorol Yellow 088 (0.01% (w/v) in methanol) at room temperature for 4 days, counterstained with aniline blue (0.5% (w/v) in water) for 1 h, and rinsed before imaging. Root cap mucilage was visualized by staining with ruthenium red (0.005% (w/v)) for 1 min, followed by washes. Cell walls and nonviable cells were visualized by propidium iodide (PI) staining (10 μM, 2 min) followed by rinsing. For enzymatic perturbation, seedlings were grown on medium supplemented with 3.8 units of pectinase from Aspergillus aculeatus ; controls received an equivalent volume of dimethyl sulfoxide (DMSO). Root cap detachment assay Agar blocks containing intact seedlings were excised from the growth medium and transferred onto glass slides. For imaging, a small droplet of water was applied adjacent to the root apex before a cover slip was placed on the agar block. Root tips were imaged in bright-field mode using an Axio Observer 5 microscope (Zeiss, Jena, Germany). Based on these observations, detachment cycles and layered, unlayered, and multilayered detachment events were quantified as described. Gravitropism assay For the phenotypic analysis following gravistimulation, vertically grown seedlings were reoriented by 90°, and images were acquired 3 h after reorientation. To examine auxin distribution, DR5::GUS reporter lines were subjected to gravistimulation and sampled immediately before and 20 min after reorientation. Seedlings were incubated in β-glucuronidase (GUS) staining solution for 1 h and subsequently imaged in bright-field mode using an Axio Observer 5 microscope (Zeiss, Jena, Germany). Hydrotropism assay For the hydrostimulation experiments, a split-agar system was established to generate a moisture gradient. Square plates were first poured with half-strength MS medium containing 1% (w/v) sucrose and 1% (w/v) agar. After solidification, the lower right portion of the agar was removed and replaced with half-strength MS medium supplemented with 1% (w/v) sucrose, 1% (w/v) agar, and 400 mM D-sorbitol. Scanning electron microscopy For scanning electron microscopy (SEM) analysis, plant tissues were prepared for ultrastructural integrity by critical-point drying. Samples were dehydrated in a critical-point dryer (EM CPD300, Leica, Vienna, Austria) and subsequently affixed to stainless steel specimen stubs with conductive carbon tape. To improve surface conductivity, mounted samples were sputter-coated with platinum using a coating system (EM ACE200, Leica, Vienna, Austria). SEM observations were performed under high-vacuum conditions using a JSM-6390LV microscope (JEOL, Tokyo, Japan). Microscopy and imaging Bright-field imaging was performed using an Axio Observer 5 microscope (Zeiss, Jena, Germany). Confocal imaging was performed using an LSM 900/900M laser-scanning confocal microscope (Zeiss, Jena, Germany). PI was excited at 488 nm, and emission was collected at 550–720 nm. Fluorol Yellow 088 was excited at 488 nm and emission collected at 490–555 nm. Quantification and statistical analysis The statistical analyses employed are presented in the figure legends. Data were measured using ImageJ. Measurements for the phenotypic assays were statistically analyzed in GraphPad Prism 10.5.0. Graphs were generated using GraphPad Prism 10.5.0. 3. Results Root cap cuticle facilitates root penetration through mechanical protection Efficient penetration of the surrounding substrate represents a primary challenge for emerging roots during early seedling establishment. Since penetration is governed by the mechanical properties of the growth interface, we first established agar-based conditions that impose distinct constraints on root entry. Therefore, we compared cut agar, where the surface was incised to disrupt continuity and expose the inner porous structure, with intact agar, where the surface remained uninterrupted (Fig. 1A). The wild-type (WT) roots readily penetrated the 1.2% cut agar, whereas penetration into the 1.2% intact agar was markedly reduced and declined further as the agar concentration increased (Fig. 1B, C), thereby supporting the view that surface continuity and overall stiffness impose a major constraint on root entry. Roots that failed to penetrate showed pronounced growth retardation (Fig. 1D). SEM revealed increased surface damage in these roots (Fig. 1E, F), indicating that penetration failure is associated with enhanced mechanical damage at the root tip. In contrast, roots that successfully penetrated exhibited comparable post-penetration growth across conditions (Fig. 1D), indicating that, once penetration occurs, subsequent growth proceeds similarly across substrate conditions. Having established assay conditions that impose distinct mechanical constraints on root entry, we next tested the contribution of the RCC. We analyzed the glycerol-3-phosphate acyltransferase mutant gpat4 gpat8 , which exhibits impaired RCC formation (Berhin et al., 2019). In WT roots, penetration efficiency declined progressively as the mechanical constraint increased, whereas the gpat4 gpat8 mutant roots exhibited a strong reduction in penetration efficiency in the 1.2% cut agar (Fig. 1B, C). SEM revealed a higher frequency of damaged root tips in gpat4 gpat8 than in WT (Fig. 1F), a difference attributable to roots that failed to penetrate; damaged-tip frequencies were comparable among penetrated roots between genotypes (Fig. 1F). Notably, PI staining revealed elevated cellular permeability in gpat4 gpat8 even among penetrated roots (Fig. 1G), indicating that the loss of the RCC broadly compromises root tip cell integrity under mechanically challenging conditions. Enhanced mucilage buffers RCC loss during root penetration To further examine the contribution of the RCC to root penetration, we extended our analysis to additional cuticle-deficient mutants, bodyguard ( bdg) and defective in cuticular ridges ( dcr) . Consistent with previous reports (Berhin et al., 2019), Fluorol Yellow 088 (FY 088) staining revealed more severe defects in RCC formation in the bdg and dcr mutants than in the gpat4 gpat8 mutant , in which a partial columella cuticle remained detectable (Fig. 2A, B). However, penetration performance did not scale with RCC abundance. In both the cut and intact 1.2% agar conditions, the bdg and dcr roots penetrated at efficiencies comparable to WT; a significant reduction was observed in the bdg roots only for the 2% cut agar (Fig. 2C). Notably, this reduction was less severe than that observed for the gpat4 gpat8 mutant (Fig. 1C). These results indicate that penetration efficiency is not determined solely by residual cuticle levels and suggest that additional factors can buffer RCC loss. Therefore, we investigated whether compensatory mechanisms are active in mutants deficient in RCC. Previous immunolabeling with an xylogalacturonan-specific antibody revealed enhanced mucilage accumulation in the bdg and dcr mutant roots (Berhin et al., 2019). In agreement, ruthenium red staining, which labels acidic polysaccharides enriched in pectin-based mucilage, produced strong signals in the bdg and dcr mutant roots but not in WT or gpat4 gpat8 (Fig. 2D). Next, to test whether elevated mucilage contributes functionally to penetration in RCC-null backgrounds, we quantified penetration efficiency following pectinase treatment. Enzymatic removal of pectin significantly reduced penetration in bdg and dcr , with the strongest reduction in the most RCC-deficient backgrounds (Fig. 2E). Together, these results indicate that enhanced mucilage secretion in RCC-null mutants functionally compensates for the absence of the cuticle, improving lubrication and local buffering at the root–substrate interface to support efficient root penetration. RCC coordinates root cap detachment dynamics Penetration is not achieved by a static, shielded tip alone. The root cap surface must be renewed in a coordinated manner, removing compromised cells while preserving an organized, functional interface for continued growth (Fig. 3A) (Shin et al., 2025). Given that the RCC forms on the outermost root cap layers, we evaluated whether the RCC contributes to both mechanical protection and the timing and collective organization of root cap detachment. We first quantified how penetration affects detachment dynamics in WT roots. Under surface growth conditions, most WT roots had progressed to detachment cycle 2—completion of the first detachment event—by 5 to 6 days after germination (DAGs) (Figs. 3B, S1). WT roots progressed further under conditions that impose penetration, with penetration can accelerate progression through the detachment cycle. Next, we analyzed the RCC-defective mutants under the same experimental conditions. The RCC-defective mutants detached more rapidly than WT even on the agar surface, with an increased frequency by cycle 3, indicating that RCC loss accelerates detachment independently of penetration (Figs. 3B, S1). Moreover, acceleration was further enhanced under penetration conditions: the gpat4 gpat8 roots frequently reached cycle 4, whereas the bdg and dcr roots showed elevated frequencies at cycle 3 (Fig. 3B). Thus, penetration and RCC loss act additively to advance the detachment cycle, with the magnitude of acceleration reflecting the functional state of the root cap surface. In addition to altering the timing of root cap turnover, the mechanical challenge imposed by penetration also affected the structural organization of detachment. In WT seedlings grown on the surface, the outermost root cap cells typically detached as a cohesive sheet (Fig. 3C, D). Meanwhile, under penetration conditions, this organized pattern was disrupted from the first cycle onward, with the outermost cells separating individually or as fragmented units rather than forming an intact layer; disorganization became more pronounced during the second cycle, when cuticle regeneration no longer occurs (Fig. 3D). Disorganized detachment was evident in RCC-defective mutants even under surface conditions. This phenotype was particularly pronounced in the gpat4 gpat8 mutant , which lacks mucilage-based compensation, with up to ~38% of roots exhibiting disorganized detachment (Fig. 3D). Notably, the frequency of disorganized detachment increased over successive cycles, suggesting that defects arising during the initial detachment event persist and influence later stages when cuticle regeneration no longer occurs. In contrast, bdg and dcr frequently exhibited multilayered detachment under both surface and penetration conditions, with severity increasing over successive cycles (Fig. 3E, F), potentially reflecting excessive or misregulated mucilage accumulation. Together, these results reveal that the RCC regulates both the timing and the collective organization of detachment of the outermost root cap cells. Notably, the loss of the RCC accelerates and disorganizes detachment even under surface conditions, indicating that RCC status shapes the turnover program ahead of physical entry into the substrate, with defects initiated at the first detachment event persisting across subsequent cycles. RCC supports gravitropic responsiveness during early root penetration While the RCC facilitates penetration into mechanically resistant media, cuticle formation ceases once the first root cap detaches (Fig. 4A). Therefore, to examine how this developmental change affects penetration capacity, seedlings were initially grown on the agar surface for varying durations. Subsequently, the plates were tilted by 90° and the roots were allowed to grow into the medium for three days (Fig. 4B). Under these conditions, penetration efficiency was markedly reduced in 7-day-old seedlings, in which the RCC is no longer present (Fig. 4C), consistent with reduced penetration competence following cuticle loss. However, penetration efficiency was already lower at 3 DAGs than at 0 DAGs, despite the continued presence of the cuticle, indicating that additional age-dependent factors contribute to the progressive decline in penetration capacity. This age-dependent decline was not observed in the gpat4 gpat8 mutant, in which penetration efficiency remained uniformly low across developmental stages (Fig. 4C), consistent with an early and persistent defect associated with impaired RCC formation. To identify age-dependent factors beyond the cuticle, we focused on the gravitropic response. Since penetration into the medium requires rapid reorientation of the root tip in response to gravistimulation, we considered that a developmental weakening of gravitropic responsiveness might underlie the progressive loss of penetration capacity. This proved to be the case: gravitropic curvature declined sharply with age, decreasing from ~80° in freshly germinated roots to This developmental decline in gravitropism was further supported by altered auxin distribution patterns in older roots (Figs. 5D, S2). In contrast, the RCC mutants showed no age-dependent changes in gravitropic response (Fig. 5E). Although the gravitropic response in RCC mutants was less severely impaired than in the pin2 mutant (Fig. 5E), these mutants consistently displayed reduced gravitropism across developmental stages, regardless of mucilage presence, indicating that the RCC contributes to optimal gravitropic responsiveness. Alongside the decline in gravitropism, hydrotropic responses also decreased with age in WT roots (Fig. 6). However, no substantial differences were observed among the WT and gpat4 gpat8 and bdg mutants, with only the dcr mutant showing a tendency toward a reduced response. These observations indicate that the RCC function is less tightly linked to hydrotropism than to gravitropism. Together, these findings suggest that the early root tip represents a developmental state optimized for penetration, supported by a robust gravitropic response and a protective cuticle. 4. Discussion Roots sustain growth in soil by continuously sensing the surrounding environment, coping with mechanical pressure, and adjusting development to optimize water and nutrient acquisition. However, for germination, these longer-term functions depend on an immediate, stage-specific prerequisite: the emerging root must rapidly achieve reliable soil entry. Nonetheless, the mechanism through which the root tip is configured to meet this early priority has previously remained unclear. Notably, this study demonstrates that the RCC defines an early, penetration-optimized root tip state (Fig. 7). In addition to providing mechanical protection, the RCC coordinates the timing and collective organization of root cap detachment while sustaining robust gravitropic responsiveness, thereby promoting effective soil entry at the onset of development. Our penetration assays suggest that the first breach constitutes a mechanically demanding bottleneck for the root tip. Thus, as the mechanical resistance increased, the surface damage rose sharply and was most evident in roots that failed to penetrate, whereas growth after successful entry was comparatively insensitive to these differences in resistance. Together, these patterns indicate that the dominant mechanical challenge is concentrated at the moment of entry. These findings are consistent with a stage-specific division of labor in which plants deploy distinct surface systems before and after soil entry. During the initial breach, a stiffer and more wear-resistant surface layer, such as the RCC, may be required to resist localized compression and abrasion that cannot be fully buffered by mucilage alone. After entry, pectin-rich mucilage may confer greater benefits by dissipating stress through hydration and viscoelastic damping, and by reducing friction at the root–substrate interface (Iijima et al., 2004). In line with this view, enhanced pectin accumulation in the bdg and dcr mutants improved penetration efficiency yet did not fully prevent cellular damage under penetration-imposing conditions. Together, our results provide a functional rationale for why the root tip employs distinct surface barriers across establishment: the RCC protects the tip during the mechanically extreme first breach, whereas pectin-rich mucilage is better suited for buffering and lubrication during subsequent growth through the substrate. In addition to supporting penetration, our results show that the RCC influences root cap detachment dynamics. Indeed, RCC loss accelerated detachment progression even in surface-grown roots before mechanical stress imposed by penetration, indicating that the altered detachment is not simply a passive consequence of increased external pressure. Importantly, this earlier progression persisted beyond the first detachment event, when the newly formed root cap no longer carries a cuticle, suggesting that early RCC status imprints on the detachment program and helps establish the timing of turnover early in development. The accompanying changes in detachment organization further support a role beyond stress buffering. In the RCC mutants, the outermost cells are often separated in a disorganized manner rather than forming a cohesive sheet, suggesting that a continuous cuticular layer influences the distribution of forces across the detaching surface. Mechanical stress applied to a mechanically coupled sheet is expected to dissipate more evenly than stress borne by individual cells detaching independently. Thus, mechanical protection, force distribution, and collective detachment behavior are closely linked, providing a mechanistic basis for the deployment of a cuticle-based surface at the earliest stage of root cap development. Notably, the process through which the RCC status is perceived and translated into detachment timing remains unclear. However, one possibility is that altered mechanics or adhesion at the outermost root cap layer modulate upstream signaling that governs the turnover cycle. In this context, recent work has identified ROS as oscillatory regulators acting upstream of auxin to control root cap detachment dynamics (Dubreuil et al., 2018; Shin et al., 2025). Hence, exploring potential interactions between RCC-dependent surface properties, ROS dynamics, and auxin signaling may provide valuable insight into how detachment timing is established and maintained during early root development. Another defining feature of this early, penetration-optimized root tip state is unusually strong gravitropic responsiveness immediately after germination. We observed that gravitropic curvature declines with seedling age, consistent with a transition from an early, strongly gravitropic mode to a more integrated growth strategy that balances gravity with other heterogeneous environmental cues. The initial high directional bias is likely advantageous for rapid, downward-directed growth, minimizing deviations and promoting efficient soil entry during the narrow window when penetration is critical for seedling survival. Notably, the RCC was essential for this early gravitropic competence: RCC-deficient mutants exhibited persistently reduced gravitropic responses across developmental stages. Given that both gravitropic regulation and root cap detachment rely heavily on auxin signaling (Su et al., 2017; Dubreuil et al., 2018), RCC-dependent modulation of the root cap surface properties may act through a shared upstream framework that coordinates directional growth and turnover dynamics. Therefore, defining how surface properties contribute to auxin-mediated regulation will be important for understanding how this early, penetration-optimized state is established. In contrast to the pronounced effects on gravitropic responsiveness, hydrotropic responses were largely preserved in the RCC-defective mutants, with the gpat4 gpat8 and bdg mutants showing no substantial differences from WT roots. This distinction is consistent with current models of hydrotropism, which emphasize the involvement of abscisic acid (ABA) signaling and growth regulation in the elongation and transition zones rather than auxin-driven polarity changes in the root cap (Shkolnik et al., 2016; Dietrich et al., 2017). A modest reduction was observed in the dcr mutants; however, this effect did not correlate with RCC deficiency or mucilage overproduction, suggesting a dcr -specific contribution independent of RCC function. Overall, these results support a selective link between the RCC and gravity-driven directional growth during early root development, while hydrotropic responses remain largely intact. From an evolutionary perspective, a transient, penetration-optimized root tip may represent an adaptive solution to the high-risk period immediately following germination. Our findings redefine the RCC as a multifunctional, transient surface structure that integrates mechanical protection, tissue organization, and environmental responsiveness during this early window. By prioritizing mechanical robustness and gravity-driven directional growth before transitioning toward broader environmental responsiveness, seedlings may maximize the likelihood of successful establishment in heterogeneous soil environments (Berhin et al., 2019; Zhang et al., 2019). More broadly, our study highlights how transient extracellular structures can exert disproportionate control over developmental robustness and early environmental adaptation, offering a framework for understanding how plants establish growth competence at the onset of soil exploration. Acknowledgments This work was funded by the Suh Kyungbae Foundation (SUHF-19010003), the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT, No. RS-2021-NR060084), and the Mid-Career Bridging Program through Seoul National University, Republic of Korea. W.-T.J. was supported by the Stadelmann–Lee Scholarship Fund at Seoul National University, Republic of Korea. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement Data are available in the supporting material of the article. References Barlow, P.W. (2002). The root cap: cell dynamics, cell differentiation and cap function. Journal of plant growth regulation 21, 261-286. 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Keywords arabidopsis development growth mechanical stress root cap cuticle root penetration Authors Affiliations Woo-Taek Jeon 0009-0003-5984-5489 Seoul National University School of Biological Sciences View all articles by this author Jeong-A Kim Seoul National University School of Biological Sciences View all articles by this author Ahyeon Cheon Seoul National University School of Biological Sciences View all articles by this author Yesol Shin Seoul National University School of Biological Sciences View all articles by this author Shawn S.Y. Lee Seoul National University School of Biological Sciences View all articles by this author Yuree Lee 0000-0002-4663-6974 [email protected] Seoul National University School of Biological Sciences View all articles by this author Metrics & Citations Metrics Article Usage 185 views 80 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Woo-Taek Jeon, Jeong-A Kim, Ahyeon Cheon, et al. 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