Mechanisms underlying rhizosheath dynamics in Kengyilia hirsuta in response to alternating drought and rewatering

Mechanisms underlying rhizosheath dynamics in Kengyilia hirsuta in response to alternating drought and rewatering | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanisms underlying rhizosheath dynamics in Kengyilia hirsuta in response to alternating drought and rewatering Yutao Yuan, Li Wu, Chen Chen, Rong Li, Qingping Zhou, Youjun Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8055104/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Under increasing extreme climate events, plants must adapt to alternating drought–rewatering stress. Kengyilia hirsuta , a pioneer forage grass in alpine desert regions, depends on its rhizosheath for drought resistance. This study simulated alternating and continuous drought–rewatering treatments (10%–40% field capacity) to investigate root architecture, biomass allocation, arbuscular mycorrhizal fungi (AMF) symbiosis, and rhizosheath formation. Results showed that rhizosheath accumulation responded dynamically to water regimes: continuous 25% moisture stress (W 5 ) significantly promoted rhizosheath biomass, whereas 40% stress (W 6 ) enhanced early-stage development and 10% rewatering (W 1 ) boosted later-stage formation. AMF colonization increased progressively, with total colonization rising from 41.51% (T 1 ) to 61.40% (T 3 ). The W 5 treatment consistently showed the highest vesicle, arbuscule, and hyphal colonization, alongside increased soil spore density and hyphal density by T3. Root morphological traits—including surface area, volume, tip number, hair length, and hair density—peaked under W 5 . Structural equation modelling identified AMF colonization (total effect: − 0.90) and root hair traits (total effect: +0.80) as pivotal regulators of rhizosheath formation, interacting through biomass allocation, root architecture, and soil microenvironment to form a multidimensional adaptive network. These findings elucidate the ecophysiological mechanisms underlying plant–AMF collaboration in rhizosheath formation under water fluctuation, supporting selection of stress-tolerant grasses for restoring desertified grasslands. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Plant sciences drought- rehydration Kengyilia hirsuta rhizosheath root architecture AM fungi Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Kengyilia hirsuta is a perennial hexaploid species within the Poaceae family, characterised by long, yellow-brown, stiff hairs on the lemmas (Sun et al., 1995). As a pioneer forage grass and a source of stress-tolerant germplasm, it plays a vital role in combating desertification and in alpine forage breeding on the Qinghai–Tibet Plateau (Cai et al., 1999; Chen, 2020). This species forms a highly specialised rhizosheath—a sheath-like structure in which root exudates bind soil particles (Mo et al., 2020). Functioning as a critical interface between roots and soil, the rhizosheath offers physical protection to roots, regulates water dynamics, and facilitates microbial interactions (Aslam et al., 2021; Brown et al., 2017). Under drought stress, it enhances plant tolerance by improving water uptake (Brown et al., 2017; Young, 1995), promoting the growth of beneficial microorganisms (Rabbi et al., 2018), and strengthening soil aggregation (Othman et al., 2004). Consistent with these roles, our field observations revealed pronounced rhizosheath development in K. hirsuta under arid conditions, supporting the hypothesis that this trait has evolved in response to long-term water scarcity. Drought stress significantly inhibits root development and biomass accumulation in plants, largely due to disruptions in water metabolism, photosynthetic efficiency, and cellular homeostasis (Yang et al., 2023). To adapt to arid environments, plants have evolved diverse strategies, among which rhizosheath formation represents a key mechanism for drought tolerance. In particular, roots promote rhizosheath development through architectural modifications and the secretion of mucilaginous substances (Rahim et al., 2024). This enhances water-use efficiency—a phenomenon documented in crops such as rice ( Oryza sativa ) and chickpeas ( Cicer arietinum ) (Rabbi et al., 2018; Lei et al., 2023). Moreover, the rhizosheath serves as an important ecological niche for functional microorganisms, selectively enriching drought-resistant taxa such as Massilia , Nocardioides , and arbuscular mycorrhizal fungi (AMF) (Lei et al., 2023; Gao et al., 2022; Chen et al., 2014). AMF, in particular, exhibit synergistic effects with their host plants by improving water uptake and nutrient translocation (Gao et al., 2022; Chen et al., 2014). Although rehydration following drought has been shown to stimulate compensatory growth in plants (Acevedo et al., 1971; Schimel, 2018), its influence on the formation and restructuring of rhizosheaths and their associated microbial communities remains unclear. Current understanding of the mechanisms by which K. hirsuta responds to drought remains limited. Several key knowledge gaps persist: first, the dynamics of rhizosheath formation under alternating drought–rehydration conditions and its feedback relationship with AMF; second, the interplay between root architecture, biomass allocation, and rhizosheath development; and third, the mechanisms underlying rhizosheath-mediated plant–microbe synergism in drought resistance. This study employs graded drought and rehydration treatments to systematically analyse the relationships among rhizosheath formation, AMF colonisation, root phenotype, and biomass allocation, with the aim of addressing these research gaps. The ultimate objectives are to clarify the functional role of the rhizosheath in drought adaptation and to establish a theoretical foundation for the restoration of desertified grasslands. MATERIALS AND METHODS Plant Materials and Growth Conditions Seeds of K. hirsuta were provided by the College of Grassland Resources, Southwest Minzu University. These seeds were collected from WaQie Town, Hongyuan County (33.18° N, 102.62° E, altitude 3490 m), Aba Prefecture, Sichuan Province, China in September 2022, with permission granted by the Forestry and Grassland Bureau of Hongyuan County. The plant species was authoritatively identified by Professor Junliang Yang from the College of Life Sciences, Sichuan Agricultural University. A voucher specimen (voucher number: SAUT 201402232) has been deposited in the Herbarium of the Triticeae Research Institute, Sichuan Agricultural University (SAUT). The growth substrate consisted of sandy soil with the following physicochemical properties: pH 7.69, organic carbon 6.3 g/kg, total phosphorus 0.35 g/kg, nitrate nitrogen 6.68 mg/kg, ammonium nitrogen 6.69 mg/kg, and a maximum field capacity of 23%. Before use, the soil was air-dried naturally and sieved through a 4 mm mesh. Experimental Design At the three-leaf stage, plants were assigned to two water regimes over 21 days: A drought–rehydration group, subjected to a 7-day cycle of drought followed by rehydration to 10% (W 1 ), 25% (W 2 ), or 40% (W 3 ) of field capacity. A sustained drought group, maintained at 10% (W 4 ), 25% (W 5 ), or 40% (W 6 ) of field capacity. Soil moisture was carefully regulated daily using the weighing method to maintain target levels within ±2% of the set values. Each treatment included four biological replicates. Determination Indicators and Methods Quantitative of Rhizosheath Rhizosheath biomass was assessed using a combined mechanical vibration–ultrasonic separation approach. The whole plant was carefully excavated, and loose soil was removed by gentle shaking while preserving the intact rhizosheath. After root excision, the fresh weight of the root–rhizosheath complex (X1) was recorded. Roots were then ultrasonicated (40 kHz, 30 min) in deionised water to detach the rhizosheath completely. The roots were blot-dried and reweighed (X2), and rhizosheath biomass was calculated as the difference: Rhizosheath weight (mg/cm) = (X1 – X2) / total root length. Total root length was measured using a root scanner (EPSON Expression 12000XL, 600 dpi) and analysed with WinRHIZO Pro 2017a software. Analysis of AM Fungal Colonization Colonization Rate Root segments (1 cm) were cleared in 10% KOH at 90 °C for 90 min, acidified in 2% HCl, and stained with 0.05% Trypan blue in a lactic acid–glycerol solution at 90 °C for 30 min. After destaining in lactic acid–glycerol for 72 h, segments were mounted on slides and examined under an OLYMPUS BX53 microscope. Colonisation by vesicles, arbuscules, and hyphae was assessed using the grid intersection method across 400 fields per treatment. Colonisation rates were calculated as follows: Vesicle colonisation rate = (Number of intersections with vesicles / Total intersections) × 100% Arbuscule colonisation rate = (Number of intersections with arbuscules / Total intersections) × 100% Hyphal colonisation rate = (Number of intersections with hyphae / Total intersections) × 100% Total colonisation rate = [(Total intersections – No-colonised intersections) / Total intersections] × 100% Hyphal Density A soil suspension was prepared from 5 g of air-dried soil (sieved to 1 mm) mixed with 50 mL deionised water, followed by sequential fractionation on 20/400-mesh sieves. Material retained on the 400-mesh sieve was collected, centrifuged (1000 × g, 30 s), and 5 mL of supernatant was vacuum-filtered through a 0.45 μm membrane. After Trypan blue staining, hyphal density was determined microscopically by counting hyphal–grid intersections across 25 random fields under an OLYMPUS CX23 microscope. Hyphal length and density were calculated as: Hyphal length (m) = (11/14 × Number of intersections × Grid cell length × Membrane area × Dilution factor) / (25 × Grid area) Hyphal density (m/g) = Hyphal length (m) / Soil mass (g) Spore Density Spores were extracted from 10 g soil by wet sieving and enriched via sucrose gradient centrifugation (45% solution, 3,000 × g, 3 min). Collected spores were transferred to a Petri dish and counted under a Nikon SMZ25 stereomicroscope. Results are expressed as spores per gram of dry soil (spores/g). Root Phenotype Analysis Overall root architecture parameters—including total root length, surface area, volume, diameter, tip count, and branch number—were analysed using washed roots scanned at 600 dpi (Epson Expression 12000XL) and processed with WinRHIZO Pro 2017a. For root hair characterisation, segments taken 3–5 cm behind the root tip were mounted on slides, and the length of ten fully expanded root hairs per segment was measured using a Nikon Eclipse Ni phase-contrast microscope. Root hair density per unit length (root hairs/mm) was quantified with ImageJ 1.53s. Data Processing Data were standardised and organised in Excel 2021, with outliers removed by Grubbs’ test. One-way ANOVA was performed in R 4.2.1, followed by LSD multiple comparisons (α = 0.05). Principal component analysis (PCA) was conducted using the vegan package, and Mantel tests (ape package) were used to assess associations between environmental factors and phenotypic traits. Figures were prepared with Origin 2021. RESULTS AND ANALYSIS Dynamic Response Characteristics of Rhizosheath Formation Rhizosheath formation responds non-linearly to water stress As shown in Figure 1A, rhizosheath biomass responded non-linearly to increasing water stress. Under sustained 25% water stress (W 5 ), rhizosheath dry weight reached its maximum at T 1 and T 2 (19.49 mg/cm and 24.60 mg/cm, respectively)—6.7 and 3.04 times greater, respectively, than under the W1 treatment (drought–rehydration at 10%; P < 0.05 ). At T 3 , the W 5 treatment maintained the highest rhizosheath accumulation (15.13 mg/cm), whereas values under W 2 , W 3 , and W 6 were significantly lower than in other groups ( P < 0.05 ). Temporal Trajectory of Rhizosheath Development The applied water treatments induced clear temporal shifts in rhizosheath development (Figure 1B). Under W 1 , rhizosheath weight increased continuously across growth stages, reaching 11.18 mg/cm at T 3 —a 341.9% increase relative to T 1 ( P < 0.05 ). In contrast, under W 5 , rhizosheath weight peaked at T 2 (24.60 mg/cm) and decreased by 38.5% by T 3 , yet remained significantly higher than all other treatments at that stage ( P < 0.05 ). Under W 6 , rhizosheath weight declined linearly over time, falling by 57.3% at T 3 compared to T1 ( P < 0.05) . Overall, the W 5 treatment markedly promoted rhizosheath accumulation, likely by enhancing root exudation and stimulating microbial activity. Notably, rehydration to 10% (W 1 ) activated rhizosheath formation in later growth (T 3 ) through a compensatory growth response. Conversely, higher initial soil moisture (W 6 ) appeared to reduce root stress perception, thereby delaying rhizosheath development. Variation Characteristics of AM Fungal Colonization in K. hirsuta Characteristics of colonization changes of AM fungi The colonisation dynamics of arbuscular mycorrhizal (AM) fungi showed significant spatiotemporal variation (Fig. 2). Total colonisation rate increased progressively across growth stages, from 41.51% at T 1 to 61.40% at T 3 (Fig. 2d), and was highest under sustained 25% water stress (W 5 ; P < 0.05 ). Vesicle colonisation rates (ranging from 1.18% to 33.10%) were significantly higher under W 5 and W 6 than in other treatments ( P < 0.05 ; Fig. 2a), suggesting that relatively high moisture conditions (≥25%) favour the formation of these storage structures. The arbuscule colonisation rate showed greater fluctuation (Fig. 2b), with W 5 maintaining the highest values throughout T 1 –T 3 (1.56–1.81%). The synchronisation of arbuscule development with rhizosheath formation points to a potential role in rhizosheath construction through enhanced carbon–water exchange. Hyphal colonisation under W 5 exhibited a stress-adaptation pattern (Fig. 2c), increasing by 30.08–40.89% at T 3 compared to T 1 /T 2 . This supports the view that hyphal network expansion is a key mechanism in AM–host interaction during mid-to-late drought stages. Notably, total colonisation under W 5 exceeded 90% at T 3 , significantly surpassing all other treatments ( P < 0.05 ). This indicates that 25% water stress effectively promotes a functional AM–plant symbiosis by improving hyphal colonisation efficiency and host resource allocation. Spatial Distribution Characteristics of AM Fungal Spores and Hyphae The spatial distribution of AM fungal propagules and hyphal networks was significantly influenced by soil water regime (Fig. 3). Sustained 25% water stress (W 5 ) markedly enhanced both spore and hyphal densities in the rhizosheath (WR) and rhizosphere (WS) soils. By T 3 , under W 5 , spore density reached 29 spores/10 g and hyphal density 1.82 m/g in the WR zone, while values in the WS zone were 14.5 spores/10 g and 1.17 m/g. These represent increases of 3.83‑fold, 0.68‑fold, 3.14‑fold, and 0.49‑fold, respectively, compared to the W 1 treatment ( P < 0.05 ). Over time, AM fungal biomass in both WR and WS zones accumulated progressively. Under W 5 , WR spore density increased by 45% from T 1 to T 3 , and hyphal density by 26.39% ( P < 0.05 )—significantly greater than under other treatments. Biomass was consistently higher in the WR than in the WS zone, indicating that the rhizosheath acts as a hotspot for AM fungal enrichment, likely due to root exudates and physical retention. These results suggest that 25% water stress promotes spore germination and hyphal network expansion by optimising soil moisture and host carbon supply. This functional coupling is reflected in the total colonisation rate of 90.88% at T 3 (Fig. 2d), supporting a coordinated rhizosheath–AMF drought adaptation strategy. Adaptive Responses of Root Architecture and Root Hair Traits to Water Stress Graded water stress induced coordinated plasticity in both root architecture and root hair phenotypes of K. hirsuta (Figs. 4, 5). Under sustained 25% water stress (W 5 ), plants developed a coordinated drought-resistance strategy, characterised as “spatially efficient exploration and precise resource capture”, through optimisation of root topology and root hair morphology. At the architectural level, total root length increased linearly with moisture deficit. The W 5 treatment promoted a highly branched root system, with root tip and branch numbers at T 3 increasing by 5.48‑fold and 6.93‑fold, respectively, compared to W 1 . Root surface area under W 5 reached 27.87 cm² at T 3 —an 8.02‑fold increase over W 1 ( P < 0.05 )—indicating enhanced soil exploration via modified branching angles. Root volume increased initially before declining, peaking at T 3 (0.18 cm³), reflecting a shift in resource allocation toward absorptive fine roots during mid‑to‑late drought. In contrast, the W 6 treatment (sustained high moisture) suppressed such functional root differentiation. Root hairs, key structures for moisture perception and uptake, exhibited strong spatiotemporal plasticity. Under W 5 , root hair length increased continuously from T 1 to T3 (650.29 → 871.03 μm), and by T 3 was 34.78% longer than under W 6 ( P < 0.05 ). The highest root hair density (66–68.5 root hairs/mm) during T 2 –T 3 facilitated an expanded root–soil interface, improving water capture. These morphological adjustments correlated positively with AM fungal hyphal colonisation and rhizosheath biomass, suggesting that root hairs contribute to rhizosheath formation through synergistic interaction with hyphal networks. In contrast, sustained high moisture (W 6 ) reduced root hair density by 34.90–36.74% ( P < 0.05 ), confirming that moderate water deficit stimulates root hair development. In summary, 25% water stress shifted root architecture from wide-ranging exploration to localised efficiency, likely through reprogrammed auxin transport, while regulating root hair elongation via ABA signalling. This integrated morphological and functional adaptation, together with expanded AM fungal symbiosis, forms a dual-engine mechanism driving rhizosheath development—providing structural and functional support for plant ecological adaptation in sandy habitats. Principal component analysis reveals multidimensional root adaptation to water stress Principal component analysis (PCA) of root phenotypic traits indicated that K. hirsuta responds to water stress through multidimensional morphological strategies (Fig. 6). The first two principal components (PC1 and PC2) together explained 83.93% of the total phenotypic variation, with PC1 accounting for 70.02% (λ = 5.60) and PC2 for 13.91% (λ = 1.11). PC1 was strongly associated with total root length (loading: 0.404), total root surface area (0.406), total root volume (0.406), and number of branches (0.408), reflecting the spatial expansion capacity of the root system. PC2 was closely linked to root hair density (0.80) and average root diameter (0.49), representing adaptation to heterogeneous soil microenvironments. Notably, the W 5 treatment exhibited a pronounced distribution along both PC1 and PC2 axes. Under this regime, plants synergistically enhanced branching complexity and root hair density, achieving a functional integration of wide-ranging exploration and local optimisation. These findings support the hypothesis that 25% water stress reshapes both root topological structure and fine-scale phenotypic traits, forming a hierarchical drought adaptation network. Together with the rhizosheath–AMF interaction system, this network elucidates the integrated adaptive strategy employed by plants in sandy habitats. Biomass allocation shifts in response to water stress gradient Gradient water stress significantly influenced biomass accumulation patterns in K. hirsuta , regulating the allocation of photosynthetic products (Fig. 7). Under sustained high moisture (W 6 ), aboveground growth was accelerated, with aboveground dry weight reaching 50.83 mg at T 3 —3.86 times that under W 1 ( P < 0.05) . This supports the hypothesis that photosynthates are preferentially allocated to stem and leaf expansion under ample water supply. In contrast, under 25% water stress (W 5 ), root fresh weight increased substantially to 304.88 mg at T 3 , exceeding the W 6 treatment by 33.4% ( P < 0.05 ), indicating a shift in assimilate partitioning toward drought-resistant structures such as the rhizosheath. Although root dry weight generally increased with moisture availability—peaking at 29.45 mg under W 6 at T 3 —analysis of the root–shoot ratio (Fig. 7E) revealed that the W 5 treatment enhanced root functionality through dynamic source–sink modulation. At T 1 , the root–shoot ratio peaked (0.57) under W 1 (10% rehydration), reflecting compensatory root investment after drought. By T 3 , the ratio under W 5 (0.71) significantly exceeded those under W 1 and W 2 ( P < 0.05 ), indicating that sustained moderate stress balances growth and resistance by maintaining root activity. This spatiotemporally specific regulation forms a synergistic network with AMF-mediated carbon–phosphorus exchange and root hair phenotypic plasticity, collectively enhancing plant ecological fitness in fluctuating soil moisture environments. Functional Analysis of the AMF–Plant Interaction Network in Drought Resistance Mantel tests revealed a highly significant correlation ( P < 0.01 ) between soil-borne arbuscular mycorrhizal fungi (S-AMF) and multiple biomass traits in K. hirsuta (Fig. 8). S-AMF abundance was significantly associated with aboveground fresh weight (FWAG), root fresh weight (FRW), aboveground dry weight (DWAG), root dry weight (DWR), root–shoot ratio (RSR), and key root morphological traits—including total root length (TRL), root surface area (RSA), root volume (RV), number of root tips (NRT), and number of forks (NF). Rhizosheath-related traits such as rhizosheath weight (RW), root hair length (RHL), and root hair density (RHD) were also significantly correlated with S-AMF. These results suggest that S-AMF enhance host drought resistance by expanding the root–soil interface and facilitating efficient carbon–phosphorus exchange. In comparison, root-colonising AMF (R-AMF) showed significant positive correlations ( P < 0.05 ) with all traits except RSR. Aboveground and root fresh weights (FWAG and FRW) exhibited highly consistent variation, displaying strong positive correlations ( P < 0.01 ) with most biomass, architectural, and root hair traits, and a significant correlation with RSR ( P < 0.05 ). Dry weight traits (DWAG and DWR) were significantly correlated ( P < 0.05 ) with TRL, RSA, RV, NRT, NF, and RHL, and showed a highly significant positive correlation ( P < 0.01 ) with RW. Within the root system, strong interdependencies ( P < 0.01 ) were observed among architectural traits (TRL, RSA, RV, NRT, NF). Root volume was also significantly correlated with rhizosheath weight ( P < 0.05 ), and tightly coupled relationships ( P < 0.01 ) linked RW, RHL, and RHD.Collectively, these findings indicate that AMF colonisation—both in soil and roots—enhances drought adaptation in K. hirsuta through synergistic regulation of biomass allocation, root architecture, and root hair development. This functional integration highlights the key role of AMF in strengthening plant resilience in arid environments. Structural equation modelling reveals hierarchical regulation of rhizosheath formation A segmented structural equation model (SEM) was used to elucidate how drought stress and growth stage interact to regulate rhizosheath formation, revealing a hierarchical regulatory pathway (Fig. 9). The model demonstrated good fit (Fisher's C = 28.97, P = 0.42). Root hair traits (path coefficient = 0.80) and AM fungal colonisation rate (–0.90) exerted the strongest direct effects on rhizosheath formation, while biomass allocation (indirect effect = 1.26), root architectural plasticity (1.55), and AM fungal activity in rhizosheath soil (1.14) functioned through indirect pathways. Growth stage progression significantly enhanced the positive effects of root architecture (0.86) and root hair plasticity (1.40) on rhizosheath development, but suppressed soil AM fungal activity (–0.25), indicating a stage-dependent resource trade-off in plant–microbe interactions. Sustained 25% water stress (W 5 ) strengthened the synergistic contribution of root hair traits (–2.92) and AM fungal colonisation (1.17) to rhizosheath formation via treatment–stage interaction, highlighting its role in modulating development through the mycorrhizal network. AM fungal colonisation rate (total effect: –0.90) balanced root carbon expenditure via negative regulation, whereas root hair traits (total effect: 0.80) served as physical structures expanding the root–fungus interface. Together, these form a complementary mechanism of “hyphal synergy and morphological adaptation.” The model indicates that rhizosheath formation arises from combined contributions of plant phenotypic plasticity (54.3%) and microbial interaction networks (45.7%). Under experimental conditions, the W 5 treatment promoted functional assembly of rhizosheath modules in sandy habitats by optimising dynamic equilibrium among root hairs, AMF, and the soil environment. These findings offer a theoretical framework for understanding ecological and evolutionary mechanisms in plant–microbe drought adaptation. DISCUSSION Adaptive Mechanism of Root Morphological Plasticity to Alternate Drought–Rehydration Root architectural plasticity enables plants to efficiently explore and acquire soil resources, a trait of considerable ecological importance in arid habitats (Fig. 4 ). Unlike the inhibitory effect of drought on root phenotypes in tartary buckwheat (Fagopyrum tataricum) (Lu et al., 2018 ), this study found that K. hirsuta significantly increased total root length (5.48-fold at T 3 vs. W 1 ), root surface area (8.02-fold), and branch number (6.93-fold) under 25–40% moisture gradients, forming a dual adaptation strategy of "wide-range branching and local optimisation" (Fig. 6 ). This difference may reflect evolutionary adaptations of K. hirsuta as a pioneer species in desertified grasslands: sandy soils maintain adequate pore oxygen even at 25–40% moisture, satisfying root aerobic metabolism. As shown in prior studies, K. hirsuta enhances deep soil water acquisition while optimising resource capture in surface microzones by increasing root branching angles and root hair density. Notably, under W 5 treatment, periodic fluctuation in root volume (peak at T 3 : 0.18 cm³) and synchronous growth in root tip number (1875.25 at T 3 ) reflect functional complementarity, confirming a "carbon investment–phenotypic benefit" trade-off mechanism (Song et al., 2015 ). This allows dynamic biomass allocation between storage and absorptive roots, balancing immediate survival and long-term adaptation. In contrast to the root expansion of perennial ryegrass ( Lolium perenne ) under ample moisture (Grzesiak et al., 2013 ), K. hirsuta under sustained 40% moisture (W 6 ) showed increased total root length (389.06 cm at T 3 ) but a decreased root–shoot ratio (from 0.71 to 0.58) and lower AM fungal colonization. This "moisture paradox" suggests that moderate drought (25% field capacity) may activate ABA signal-mediated architectural reprogramming, shifting branching patterns from quantitative expansion to qualitative optimisation. This establishes a positive feedback loop with the mycorrhizal network, collectively supporting rhizosheath ecological function. These findings provide new theoretical insight into the ecological evolution of root plasticity in psammophytes. Root hairs, as frontline sensors of soil heterogeneity, exhibit phenotypic plasticity in length and density (Fig. 5 ), a key strategy for adapting to moisture variation. Under 25% water stress (W 5 ), root hair length increased significantly (871.03 µm at T 3 , 34.78% longer than W 6 , P < 0.05 ) while maintaining high density (66 root hairs/mm), forming a "super surface area" absorption mode similar to Japanese brome (Bromus japonicus) (Zhang et al., 2022 ). However, K. hirsuta shows unique spatiotemporal regulation: root hair length increased continuously through growth stages (T 1 →T 3 : 650.29→871.03 µm), while density peaked at T 2 then declined, suggesting that mature plants extend the lifespan of existing root hairs rather than producing new ones. This contrasts with the "quantity–quality" trade-off in typical dicot root hair development (Liu et al., 2019 ). The 40% moisture treatment (W 6 ) inhibited root hair density, and drought–rehydration (W 1 ) impeded both length and density, indicating that moderate sustained drought may activate ethylene-mediated root hair elongation, while high or fluctuating moisture disrupts calcium oscillation signalling, leading to phenotypic imbalance. The strong synergy among root hair length, AM fungal hyphal colonization, and rhizosheath biomass (Fig. 9 ) suggests root hairs act not only as physical absorption organs but also as chemical signalling hubs for microbial interaction. Flavonoids and other signals secreted from root hair tips may guide hyphal growth via chemotaxis. These results support the "root hair–hyphal synergy" theory and indicate that psammophytes have evolved a unique rhizosheath construction strategy integrating phenotypic plasticity and microbial cooperation, offering biomimetic inspiration for drought-resistant crop root design. Regulatory Mechanism of Moisture Threshold on the AMF–Plant Symbiotic Relationship The intensity of AM fungal symbiosis with host plants is non-linearly regulated by moisture and exhibits species and habitat specificity (Fig. 2 ). AM fungi form symbiotic associations with most terrestrial plants (Marulanda et al., 2003 ) and enhance host stress resistance, competitive ability, and survival, thereby influencing plant community structure and ecosystem productivity (Dong et al., 2021 ; He et al., 2018 ). In this study, the total AM fungal colonization rate of K. hirsuta peaked (90.88% at T 3 ) under sustained 25% water stress (W 5 ), showing a strong positive correlation with soil moisture—contrasting with the "drought-promoted" symbiosis observed in grasses like crested wheatgrass ( Agropyron cristatum ) (Monz et al., 1994 ). This may reflect an evolutionary adaptation of psammophytes: sandy soils retain adequate pore oxygen even at 25–40% moisture, meeting AM fungal aerobic requirements (Hattori et al., 2014 ), whereas loamy soils under similar moisture can develop anaerobic microsites. The synchronous increase in root–shoot ratio and hyphal colonization under W 5 suggests the host allocates photosynthetic carbon to AM fungi in exchange for phosphorus and other scarce resources, a strategy particularly advantageous in infertile sandy soils (Lv et al., 2019). Moderate drought (25%) may activate JA signalling and strigolactone secretion, specifically attracting AM fungal hyphae. Although the 40% moisture treatment (W 6 ) maintained high colonization (78.99%), its hyphal network density (1.46 m/g) was significantly lower than under W 5 ( P < 0.05 ), indicating excessive moisture may inhibit functional hyphal differentiation. Rehydration treatments (W 1 –W 3 ) reduced AM fungal colonization by 21.8–38.5% compared to sustained drought ( P < 0.05 ), aligning with the "moisture fluctuation inhibition hypothesis" (Hattori et al., 2014 ), where rapid changes in soil oxygen after rehydration disrupt hyphal network stability. In contrast, prolonged drought enriches drought-tolerant AM fungal communities through selection. This supports the idea that the psammophyte–AM fungal symbiotic system achieves dynamic equilibrium via a "stress memory" mechanism, offering a theoretical basis for mycorrhizal-assisted restoration of degraded grasslands. Analysis of Rhizosheath Formation in K. hirsuta Under Drought and Rehydration Conditions The rhizosheath in K. hirsuta seedlings is a structure formed by the entanglement of roots, soil microorganisms, and root exudates, which adhere soil particles (Chen, 2020 ). This adaptation is key to its success in alpine arid environments. Although rhizosheath research has largely focused on Poaceae species—e.g., Bailey et al. observed rhizosheaths in most wild grasses—it is not exclusive to this family (Basirat et al., 2019 ). Soil moisture critically influences aggregation: the rhizosheath retains water by suction, maintaining higher moisture than bulk soil, and is more evident in dry sandy soil (Haling et al., 2010 ). In this study, rhizosheath weight under the 25% moisture treatment was significantly higher than under other moisture levels, and its response varied with culture duration, indicating optimal development under sustained moderate drought. However, rhizosheath formation is multifactorial (North & Nobel, 1997 ). This study identified root hair traits and AM fungal colonization as having direct, significant effects. Other essential factors include root mucilage concentration, root metabolites, and rhizosheath microbial composition, which warrant further investigation. CONCLUSION This study demonstrates that K. hirsuta adapts to water fluctuations by dynamically modulating root system architecture, biomass allocation, and arbuscular mycorrhizal fungi (AMF) symbiosis, which act together to enhance rhizosheath formation. In conditions of soil moisture equivalent to 25% of field capacity (W 5 ), significant stimulation was observed in the accumulation of rhizosheath biomass, the development of root morphology, and the colonisation of arbuscular mycorrhizal (AMF) fungi. Furthermore, AMF colonisation extent and root hair traits were identified as key regulators of rhizosheath formation. These factors interact through coordinated effects on biomass allocation, root architecture, and the soil microenvironment, constituting a multidimensional adaptive network. The findings elucidate the ecophysiological basis of plant–microbial collaboration under varying moisture conditions, thus providing a theoretical foundation for the selection of stress-tolerant grasses with a view to restoring degraded grassland ecosystems. Declarations Declaration of funding. This work was supported by the National Natural Science Foundation of China (Grant No. 31802123). Acknowledgements I would like to thank the National Natural Science Foundation of China and the “College of Grassland Resources, Southwest University for Nationalities” for their support and funding of this research. Author contributions In this collaborative effort, Dr. Yuan Yutao mainly contributed by focusing on his writing and manuscript. 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1","display":"","copyAsset":false,"role":"figure","size":429832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic response of rhizosheath biomass in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. hirsuta\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seedlings to gradient water stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: In Figure 1-A, W\u003csub\u003e1\u003c/sub\u003e, W\u003csub\u003e2\u003c/sub\u003e, and W\u003csub\u003e3\u003c/sub\u003e represent the 10%, 25%, and 40% moisture gradient treatments for rehydration, respectively. W\u003csub\u003e4\u003c/sub\u003e, W\u003csub\u003e5\u003c/sub\u003e, and W\u003csub\u003e6\u003c/sub\u003e represent the 10%, 25%, and 40% moisture gradient treatments, respectively; The different lowercase letters in Figure 1-B indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) in different moisture gradients during the same period; Different capital letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) at different stages under the same moisture gradient, with T\u003csub\u003e1\u003c/sub\u003e, T\u003csub\u003e2\u003c/sub\u003e, and T\u003csub\u003e3\u003c/sub\u003e representing stages 1, 2, and 3, respectively. The same below.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/431caa0b3042bcd95fe94f41.png"},{"id":96757923,"identity":"cfa455e9-2194-4048-9771-44d60b7c82c8","added_by":"auto","created_at":"2025-11-25 18:21:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal heterogeneity of AMF colonization: Synergistic regulation by water thresholds and growth stages\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/10f2f7444b88432b1a6ae893.png"},{"id":96757926,"identity":"5d26ad2c-8755-4f1c-aeed-226066ee0a56","added_by":"auto","created_at":"2025-11-25 18:21:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial differentiation of AMF propagules in rhizosheath microzones driven by soil water availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: The light-colored areas in the diagram represent rhizosheath soil, while the dark-colored areas indicate root zone soil.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/9587e3069e46789d345acbbc.png"},{"id":96757927,"identity":"225f1663-de5a-46e0-b395-c98685001dab","added_by":"auto","created_at":"2025-11-25 18:21:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":281910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdaptive strategies of root morphological plasticity to drought rehydration alternation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/b08621e04ebca9bba28fc318.png"},{"id":96915569,"identity":"56b7dfdd-7123-49d8-bb86-701d3a86a31a","added_by":"auto","created_at":"2025-11-27 14:07:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":157645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological responses of root hair plasticity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. hirsuta\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seedlings to gradient water stress\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/8c8371fdd4fdb5d8db04b70f.png"},{"id":96757929,"identity":"67827d44-df15-4f00-a279-67d607a8d1af","added_by":"auto","created_at":"2025-11-25 18:21:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2113682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultidimensional characterization of root architectural traits: Drought adaptation strategies revealed by principal component analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: X1~X8 in the figure represent the total root length, total root surface area, average root diameter, total root volume, number of root tips, number of branches, root hair length, and root hair density of the root system, respectively.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/e60c89da9891684e57792e2e.png"},{"id":96914521,"identity":"855a7c5f-25ce-4496-8823-5c000fd53012","added_by":"auto","created_at":"2025-11-27 14:06:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":347434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiomass allocation and dynamic regulation of root cap\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/bc8f3bb6d9aface26e4d3bab.png"},{"id":96914925,"identity":"93bded5f-5cc1-46db-b08b-0ff3f27c6924","added_by":"auto","created_at":"2025-11-27 14:06:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":290269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional integration of AMF-plant phenotypic interaction networks via Mantel test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: S-AMF in the figure represents soil-borne arbuscular mycorrhizal fungi, R-AMF represents root-colonising arbuscular mycorrhizal fungi, FWAG represents aboveground fresh weight, FRW represents root fresh weight, DWAG represents aboveground dry weight, DWR represents root dry weight, RSR represents root–shoot ratio, TRL represents total root length, RSA represents root surface area, RV represents root volume, NRT represents number of root tips , NF represents number of forks, RW represents rhizosheath weight, RHL represents root hair length, RHD represents root hair density.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/61d9bce93c69baf807e11395.png"},{"id":96757950,"identity":"83abd3c6-3c7a-44f6-a8fd-8df8f2e6afff","added_by":"auto","created_at":"2025-11-25 18:21:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":180069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultifactorial pathways driving rhizosheath formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: CL in the figure represents different water treatments, SQ represents different periods, CL: SQ represents the interaction between treatments and periods, and Biomass represents biomass (aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, root shoot ratio); RA represents root morphology (total root length, root surface area, average root diameter, root volume, number of root tips, number of bifurcations); RH represents root hair characteristics (root hair length, root hair density); AMF represents the infection rate of arbuscular mycorrhizal fungi (vesicle infection rate, arbuscular infection rate, hyphal infection rate, and total infection rate); SAMF represents the infection rate of arbuscular mycorrhizal fungi (AMF hyphae, spores) in rhizosheath soil; RSW represents the weight of rhizosheath soil.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/bfb38052d4b96c1a15324c51.png"},{"id":107928181,"identity":"0c66b460-f835-4aca-a019-9465a5b71ee7","added_by":"auto","created_at":"2026-04-27 16:08:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4445186,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8055104/v1/ae7a11be-a927-48bc-84fa-b6a463f09cc2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanisms underlying rhizosheath dynamics in Kengyilia hirsuta in response to alternating drought and rewatering","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e\u003cem\u003eKengyilia hirsuta\u003c/em\u003e is a perennial hexaploid species within the Poaceae family, characterised by long, yellow-brown, stiff hairs on the lemmas (Sun et al., 1995). As a pioneer forage grass and a source of stress-tolerant germplasm, it plays a vital role in combating desertification and in alpine forage breeding on the Qinghai\u0026ndash;Tibet Plateau (Cai et al., 1999; Chen, 2020). This species forms a highly specialised rhizosheath\u0026mdash;a sheath-like structure in which root exudates bind soil particles (Mo et al., 2020). Functioning as a critical interface between roots and soil, the rhizosheath offers physical protection to roots, regulates water dynamics, and facilitates microbial interactions (Aslam et al., 2021; Brown et al., 2017). Under drought stress, it enhances plant tolerance by improving water uptake (Brown et al., 2017; Young, 1995), promoting the growth of beneficial microorganisms (Rabbi et al., 2018), and strengthening soil aggregation (Othman et al., 2004). Consistent with these roles, our field observations revealed pronounced rhizosheath development in \u003cem\u003eK. hirsuta\u003c/em\u003e under arid conditions, supporting the hypothesis that this trait has evolved in response to long-term water scarcity.\u003c/p\u003e\n\u003cp\u003eDrought stress significantly inhibits root development and biomass accumulation in plants, largely due to disruptions in water metabolism, photosynthetic efficiency, and cellular homeostasis (Yang et al., 2023). To adapt to arid environments, plants have evolved diverse strategies, among which rhizosheath formation represents a key mechanism for drought tolerance. In particular, roots promote rhizosheath development through architectural modifications and the secretion of mucilaginous substances (Rahim et al., 2024). This enhances water-use efficiency\u0026mdash;a phenomenon documented in crops such as rice (\u003cem\u003eOryza sativa\u003c/em\u003e) and chickpeas (\u003cem\u003eCicer arietinum\u003c/em\u003e) (Rabbi et al., 2018; Lei et al., 2023). Moreover, the rhizosheath serves as an important ecological niche for functional microorganisms, selectively enriching drought-resistant taxa such as \u003cem\u003eMassilia\u003c/em\u003e, \u003cem\u003eNocardioides\u003c/em\u003e, and arbuscular mycorrhizal fungi (AMF) (Lei et al., 2023; Gao et al., 2022; Chen et al., 2014). AMF, in particular, exhibit synergistic effects with their host plants by improving water uptake and nutrient translocation (Gao et al., 2022; Chen et al., 2014). Although rehydration following drought has been shown to stimulate compensatory growth in plants (Acevedo et al., 1971; Schimel, 2018), its influence on the formation and restructuring of rhizosheaths and their associated microbial communities remains unclear.\u003c/p\u003e\n\u003cp\u003eCurrent understanding of the mechanisms by which \u003cem\u003eK. hirsuta\u003c/em\u003e responds to drought remains limited. Several key knowledge gaps persist: first, the dynamics of rhizosheath formation under alternating drought\u0026ndash;rehydration conditions and its feedback relationship with AMF; second, the interplay between root architecture, biomass allocation, and rhizosheath development; and third, the mechanisms underlying rhizosheath-mediated plant\u0026ndash;microbe synergism in drought resistance. This study employs graded drought and rehydration treatments to systematically analyse the relationships among rhizosheath formation, AMF colonisation, root phenotype, and biomass allocation, with the aim of addressing these research gaps. The ultimate objectives are to clarify the functional role of the rhizosheath in drought adaptation and to establish a theoretical foundation for the restoration of desertified grasslands.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePlant Materials and Growth Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeeds of \u003cem\u003eK. hirsuta\u003c/em\u003e were provided by the College of Grassland Resources, Southwest Minzu University. These seeds were collected from WaQie Town, Hongyuan County (33.18° N, 102.62° E, altitude 3490 m), Aba Prefecture, Sichuan Province, China in September 2022, with permission granted by the Forestry and Grassland Bureau of Hongyuan County. The plant species was authoritatively identified by Professor Junliang Yang from the College of Life Sciences, Sichuan Agricultural University. A voucher specimen (voucher number: SAUT 201402232) has been deposited in the Herbarium of the Triticeae Research Institute, Sichuan Agricultural University (SAUT).\u003c/p\u003e\n\u003cp\u003eThe growth substrate consisted of sandy soil with the following physicochemical properties: pH 7.69, organic carbon 6.3 g/kg, total phosphorus 0.35 g/kg, nitrate nitrogen 6.68 mg/kg, ammonium nitrogen 6.69 mg/kg, and a maximum field capacity of 23%. Before use, the soil was air-dried naturally and sieved through a 4 mm mesh.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the three-leaf stage, plants were assigned to two water regimes over 21 days: A drought–rehydration group, subjected to a 7-day cycle of drought followed by rehydration to 10% (W\u003csub\u003e1\u003c/sub\u003e), 25% (W\u003csub\u003e2\u003c/sub\u003e), or 40% (W\u003csub\u003e3\u003c/sub\u003e) of field capacity. A sustained drought group, maintained at 10% (W\u003csub\u003e4\u003c/sub\u003e), 25% (W\u003csub\u003e5\u003c/sub\u003e), or 40% (W\u003csub\u003e6\u003c/sub\u003e) of field capacity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSoil moisture was carefully regulated daily using the weighing method to maintain target levels within ±2% of the set values. Each treatment included four biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination Indicators and Methods \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative of Rhizosheath \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRhizosheath biomass was assessed using a combined mechanical vibration–ultrasonic separation approach. The whole plant was carefully excavated, and loose soil was removed by gentle shaking while preserving the intact rhizosheath. After root excision, the fresh weight of the root–rhizosheath complex (X1) was recorded. Roots were then ultrasonicated (40 kHz, 30 min) in deionised water to detach the rhizosheath completely. The roots were blot-dried and reweighed (X2), and rhizosheath biomass was calculated as the difference:\u003c/p\u003e\n\u003cp\u003eRhizosheath weight (mg/cm) = (X1 – X2) / total root length.\u003c/p\u003e\n\u003cp\u003eTotal root length was measured using a root scanner (EPSON Expression 12000XL, 600 dpi) and analysed with WinRHIZO Pro 2017a software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of AM Fungal Colonization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColonization Rate\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRoot segments (1 cm) were cleared in 10% KOH at 90 °C for 90 min, acidified in 2% HCl, and stained with 0.05% Trypan blue in a lactic acid–glycerol solution at 90 °C for 30 min. After destaining in lactic acid–glycerol for 72 h, segments were mounted on slides and examined under an OLYMPUS BX53 microscope. Colonisation by vesicles, arbuscules, and hyphae was assessed using the grid intersection method across 400 fields per treatment. Colonisation rates were calculated as follows:\u003c/p\u003e\n\u003cp\u003eVesicle colonisation rate = (Number of intersections with vesicles / Total intersections) × 100%\u003c/p\u003e\n\u003cp\u003eArbuscule colonisation rate = (Number of intersections with arbuscules / Total intersections) × 100%\u003c/p\u003e\n\u003cp\u003eHyphal colonisation rate = (Number of intersections with hyphae / Total intersections) × 100%\u003c/p\u003e\n\u003cp\u003eTotal colonisation rate = [(Total intersections – No-colonised intersections) / Total intersections] × 100%\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHyphal Density \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA soil suspension was prepared from 5 g of air-dried soil (sieved to 1 mm) mixed with 50 mL deionised water, followed by sequential fractionation on 20/400-mesh sieves. Material retained on the 400-mesh sieve was collected, centrifuged (1000 × g, 30 s), and 5 mL of supernatant was vacuum-filtered through a 0.45 μm membrane. After Trypan blue staining, hyphal density was determined microscopically by counting hyphal–grid intersections across 25 random fields under an OLYMPUS CX23 microscope. Hyphal length and density were calculated as:\u003c/p\u003e\n\u003cp\u003eHyphal length (m) = (11/14 × Number of intersections × Grid cell length × Membrane area × Dilution factor) / (25 × Grid area)\u003c/p\u003e\n\u003cp\u003eHyphal density (m/g) = Hyphal length (m) / Soil mass (g)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpore Density \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpores were extracted from 10 g soil by wet sieving and enriched via sucrose gradient centrifugation (45% solution, 3,000 × g, 3 min). Collected spores were transferred to a Petri dish and counted under a Nikon SMZ25 stereomicroscope. Results are expressed as spores per gram of dry soil (spores/g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRoot Phenotype Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall root architecture parameters—including total root length, surface area, volume, diameter, tip count, and branch number—were analysed using washed roots scanned at 600 dpi (Epson Expression 12000XL) and processed with WinRHIZO Pro 2017a. For root hair characterisation, segments taken 3–5 cm behind the root tip were mounted on slides, and the length of ten fully expanded root hairs per segment was measured using a Nikon Eclipse Ni phase-contrast microscope. Root hair density per unit length (root hairs/mm) was quantified with ImageJ 1.53s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Processing \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were standardised and organised in Excel 2021, with outliers removed by Grubbs’ test. One-way ANOVA was performed in R 4.2.1, followed by LSD multiple comparisons (α = 0.05). Principal component analysis (PCA) was conducted using the vegan package, and Mantel tests (ape package) were used to assess associations between environmental factors and phenotypic traits. Figures were prepared with Origin 2021.\u003c/p\u003e"},{"header":"RESULTS AND ANALYSIS ","content":"\u003cp\u003e\u003cstrong\u003eDynamic Response Characteristics of Rhizosheath Formation \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRhizosheath formation responds non-linearly to water stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1A, rhizosheath biomass responded non-linearly to increasing water stress. Under sustained 25% water stress (W\u003csub\u003e5\u003c/sub\u003e), rhizosheath dry weight reached its maximum at T\u003csub\u003e1\u0026nbsp;\u003c/sub\u003eand T\u003csub\u003e2\u003c/sub\u003e (19.49 mg/cm and 24.60 mg/cm, respectively)\u0026mdash;6.7 and 3.04 times greater, respectively, than under the W1 treatment (drought\u0026ndash;rehydration at 10%; \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). At T\u003csub\u003e3\u003c/sub\u003e, the W\u003csub\u003e5\u003c/sub\u003e treatment maintained the highest rhizosheath accumulation (15.13 mg/cm), whereas values under W\u003csub\u003e2\u003c/sub\u003e, W\u003csub\u003e3\u003c/sub\u003e, and W\u003csub\u003e6\u003c/sub\u003e were significantly lower than in other groups (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemporal Trajectory of Rhizosheath Development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe applied water treatments induced clear temporal shifts in rhizosheath development (Figure 1B). Under W\u003csub\u003e1\u003c/sub\u003e, rhizosheath weight increased continuously across growth stages, reaching 11.18 mg/cm at T\u003csub\u003e3\u003c/sub\u003e\u0026mdash;a 341.9% increase relative to T\u003csub\u003e1\u003c/sub\u003e (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). In contrast, under W\u003csub\u003e5\u003c/sub\u003e, rhizosheath weight peaked at T\u003csub\u003e2\u003c/sub\u003e (24.60 mg/cm) and decreased by 38.5% by T\u003csub\u003e3\u003c/sub\u003e, yet remained significantly higher than all other treatments at that stage (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). Under W\u003csub\u003e6\u003c/sub\u003e, rhizosheath weight declined linearly over time, falling by 57.3% at T\u003csub\u003e3\u003c/sub\u003e compared to T1 (\u003cem\u003eP \u0026lt; 0.05)\u003c/em\u003e. Overall, the W\u003csub\u003e5\u003c/sub\u003e treatment markedly promoted rhizosheath accumulation, likely by enhancing root exudation and stimulating microbial activity. Notably, rehydration to 10% (W\u003csub\u003e1\u003c/sub\u003e) activated rhizosheath formation in later growth (T\u003csub\u003e3\u003c/sub\u003e) through a compensatory growth response. Conversely, higher initial soil moisture (W\u003csub\u003e6\u003c/sub\u003e) appeared to reduce root stress perception, thereby delaying rhizosheath development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVariation Characteristics of AM Fungal Colonization in\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. hirsuta\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristics of colonization changes of AM fungi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colonisation dynamics of arbuscular mycorrhizal (AM) fungi showed significant spatiotemporal variation (Fig. 2). Total colonisation rate increased progressively across growth stages, from 41.51% at T\u003csub\u003e1\u003c/sub\u003e to 61.40% at T\u003csub\u003e3\u003c/sub\u003e (Fig. 2d), and was highest under sustained 25% water stress (W\u003csub\u003e5\u003c/sub\u003e; \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). Vesicle colonisation rates (ranging from 1.18% to 33.10%) were significantly higher under W\u003csub\u003e5\u0026nbsp;\u003c/sub\u003eand W\u003csub\u003e6\u003c/sub\u003e than in other treatments (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e; Fig. 2a), suggesting that relatively high moisture conditions (\u0026ge;25%) favour the formation of these storage structures. The arbuscule colonisation rate showed greater fluctuation (Fig. 2b), with W\u003csub\u003e5\u003c/sub\u003e maintaining the highest values throughout T\u003csub\u003e1\u003c/sub\u003e\u0026ndash;T\u003csub\u003e3\u003c/sub\u003e (1.56\u0026ndash;1.81%). The synchronisation of arbuscule development with rhizosheath formation points to a potential role in rhizosheath construction through enhanced carbon\u0026ndash;water exchange. Hyphal colonisation under W\u003csub\u003e5\u003c/sub\u003e exhibited a stress-adaptation pattern (Fig. 2c), increasing by 30.08\u0026ndash;40.89% at T\u003csub\u003e3\u003c/sub\u003e compared to T\u003csub\u003e1\u003c/sub\u003e/T\u003csub\u003e2\u003c/sub\u003e. This supports the view that hyphal network expansion is a key mechanism in AM\u0026ndash;host interaction during mid-to-late drought stages. Notably, total colonisation under W\u003csub\u003e5\u003c/sub\u003e exceeded 90% at T\u003csub\u003e3\u003c/sub\u003e, significantly surpassing all other treatments (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). This indicates that 25% water stress effectively promotes a functional AM\u0026ndash;plant symbiosis by improving hyphal colonisation efficiency and host resource allocation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatial Distribution Characteristics of AM Fungal Spores and Hyphae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe spatial distribution of AM fungal propagules and hyphal networks was significantly influenced by soil water regime (Fig. 3). Sustained 25% water stress (W\u003csub\u003e5\u003c/sub\u003e) markedly enhanced both spore and hyphal densities in the rhizosheath (WR) and rhizosphere (WS) soils. By T\u003csub\u003e3\u003c/sub\u003e, under W\u003csub\u003e5\u003c/sub\u003e, spore density reached 29 spores/10 g and hyphal density 1.82 m/g in the WR zone, while values in the WS zone were 14.5 spores/10 g and 1.17 m/g. These represent increases of 3.83‑fold, 0.68‑fold, 3.14‑fold, and 0.49‑fold, respectively, compared to the W\u003csub\u003e1\u003c/sub\u003e treatment (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). Over time, AM fungal biomass in both WR and WS zones accumulated progressively. Under W\u003csub\u003e5\u003c/sub\u003e, WR spore density increased by 45% from T\u003csub\u003e1\u003c/sub\u003e to T\u003csub\u003e3\u003c/sub\u003e, and hyphal density by 26.39% (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e)\u0026mdash;significantly greater than under other treatments. Biomass was consistently higher in the WR than in the WS zone, indicating that the rhizosheath acts as a hotspot for AM fungal enrichment, likely due to root exudates and physical retention. These results suggest that 25% water stress promotes spore germination and hyphal network expansion by optimising soil moisture and host carbon supply. This functional coupling is reflected in the total colonisation rate of 90.88% at T\u003csub\u003e3\u003c/sub\u003e (Fig. 2d), supporting a coordinated rhizosheath\u0026ndash;AMF drought adaptation strategy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdaptive Responses of Root Architecture and Root Hair Traits to Water Stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraded water stress induced coordinated plasticity in both root architecture and root hair phenotypes of\u003cem\u003e\u0026nbsp;K. hirsuta\u003c/em\u003e (Figs. 4, 5). Under sustained 25% water stress (W\u003csub\u003e5\u003c/sub\u003e), plants developed a coordinated drought-resistance strategy, characterised as \u0026ldquo;spatially efficient exploration and precise resource capture\u0026rdquo;, through optimisation of root topology and root hair morphology. At the architectural level, total root length increased linearly with moisture deficit. The W\u003csub\u003e5\u003c/sub\u003e treatment promoted a highly branched root system, with root tip and branch numbers at T\u003csub\u003e3\u003c/sub\u003e increasing by 5.48‑fold and 6.93‑fold, respectively, compared to W\u003csub\u003e1\u003c/sub\u003e. Root surface area under W\u003csub\u003e5\u003c/sub\u003e reached 27.87 cm\u0026sup2; at T\u003csub\u003e3\u003c/sub\u003e\u0026mdash;an 8.02‑fold increase over W\u003csub\u003e1\u003c/sub\u003e (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e)\u0026mdash;indicating enhanced soil exploration via modified branching angles. Root volume increased initially before declining, peaking at T\u003csub\u003e3\u003c/sub\u003e (0.18 cm\u0026sup3;), reflecting a shift in resource allocation toward absorptive fine roots during mid‑to‑late drought. In contrast, the W\u003csub\u003e6\u003c/sub\u003e treatment (sustained high moisture) suppressed such functional root differentiation.\u003c/p\u003e\n\u003cp\u003eRoot hairs, key structures for moisture perception and uptake, exhibited strong spatiotemporal plasticity. Under W\u003csub\u003e5\u003c/sub\u003e, root hair length increased continuously from T\u003csub\u003e1\u003c/sub\u003e to T3 (650.29 \u0026rarr; 871.03 \u0026mu;m), and by T\u003csub\u003e3\u003c/sub\u003e was 34.78% longer than under W\u003csub\u003e6\u003c/sub\u003e (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). The highest root hair density (66\u0026ndash;68.5 root hairs/mm) during T\u003csub\u003e2\u003c/sub\u003e\u0026ndash;T\u003csub\u003e3\u003c/sub\u003e facilitated an expanded root\u0026ndash;soil interface, improving water capture. These morphological adjustments correlated positively with AM fungal hyphal colonisation and rhizosheath biomass, suggesting that root hairs contribute to rhizosheath formation through synergistic interaction with hyphal networks. In contrast, sustained high moisture (W\u003csub\u003e6\u003c/sub\u003e) reduced root hair density by 34.90\u0026ndash;36.74% (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e), confirming that moderate water deficit stimulates root hair development. In summary, 25% water stress shifted root architecture from wide-ranging exploration to localised efficiency, likely through reprogrammed auxin transport, while regulating root hair elongation via ABA signalling. This integrated morphological and functional adaptation, together with expanded AM fungal symbiosis, forms a dual-engine mechanism driving rhizosheath development\u0026mdash;providing structural and functional support for plant ecological adaptation in sandy habitats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrincipal component analysis reveals multidimensional root adaptation to water stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) of root phenotypic traits indicated that \u003cem\u003eK. hirsuta\u003c/em\u003e responds to water stress through multidimensional morphological strategies (Fig. 6). The first two principal components (PC1 and PC2) together explained 83.93% of the total phenotypic variation, with PC1 accounting for 70.02% (\u0026lambda; = 5.60) and PC2 for 13.91% (\u0026lambda; = 1.11). PC1 was strongly associated with total root length (loading: 0.404), total root surface area (0.406), total root volume (0.406), and number of branches (0.408), reflecting the spatial expansion capacity of the root system. PC2 was closely linked to root hair density (0.80) and average root diameter (0.49), representing adaptation to heterogeneous soil microenvironments. Notably, the W\u003csub\u003e5\u003c/sub\u003e treatment exhibited a pronounced distribution along both PC1 and PC2 axes. Under this regime, plants synergistically enhanced branching complexity and root hair density, achieving a functional integration of wide-ranging exploration and local optimisation. These findings support the hypothesis that 25% water stress reshapes both root topological structure and fine-scale phenotypic traits, forming a hierarchical drought adaptation network. Together with the rhizosheath\u0026ndash;AMF interaction system, this network elucidates the integrated adaptive strategy employed by plants in sandy habitats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiomass allocation shifts in response to water stress gradient\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGradient water stress significantly influenced biomass accumulation patterns in \u003cem\u003eK. hirsuta\u003c/em\u003e, regulating the allocation of photosynthetic products (Fig. 7). Under sustained high moisture (W\u003csub\u003e6\u003c/sub\u003e), aboveground growth was accelerated, with aboveground dry weight reaching 50.83 mg at T\u003csub\u003e3\u003c/sub\u003e\u0026mdash;3.86 times that under W\u003csub\u003e1\u003c/sub\u003e (\u003cem\u003eP \u0026lt; 0.05)\u003c/em\u003e. This supports the hypothesis that photosynthates are preferentially allocated to stem and leaf expansion under ample water supply. In contrast, under 25% water stress (W\u003csub\u003e5\u003c/sub\u003e), root fresh weight increased substantially to 304.88 mg at T\u003csub\u003e3\u003c/sub\u003e, exceeding the W\u003csub\u003e6\u003c/sub\u003e treatment by 33.4% (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e), indicating a shift in assimilate partitioning toward drought-resistant structures such as the rhizosheath. Although root dry weight generally increased with moisture availability\u0026mdash;peaking at 29.45 mg under W\u003csub\u003e6\u003c/sub\u003e at T\u003csub\u003e3\u003c/sub\u003e\u0026mdash;analysis of the root\u0026ndash;shoot ratio (Fig. 7E) revealed that the W\u003csub\u003e5\u003c/sub\u003e treatment enhanced root functionality through dynamic source\u0026ndash;sink modulation. At T\u003csub\u003e1\u003c/sub\u003e, the root\u0026ndash;shoot ratio peaked (0.57) under W\u003csub\u003e1\u003c/sub\u003e (10% rehydration), reflecting compensatory root investment after drought. By T\u003csub\u003e3\u003c/sub\u003e, the ratio under W\u003csub\u003e5\u003c/sub\u003e (0.71) significantly exceeded those under W\u003csub\u003e1\u0026nbsp;\u003c/sub\u003eand W\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e), indicating that sustained moderate stress balances growth and resistance by maintaining root activity. This spatiotemporally specific regulation forms a synergistic network with AMF-mediated carbon\u0026ndash;phosphorus exchange and root hair phenotypic plasticity, collectively enhancing plant ecological fitness in fluctuating soil moisture environments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Analysis of the AMF\u0026ndash;Plant Interaction Network in Drought Resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMantel tests revealed a highly significant correlation (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) between soil-borne arbuscular mycorrhizal fungi (S-AMF) and multiple biomass traits in K. hirsuta (Fig. 8). S-AMF abundance was significantly associated with aboveground fresh weight (FWAG), root fresh weight (FRW), aboveground dry weight (DWAG), root dry weight (DWR), root\u0026ndash;shoot ratio (RSR), and key root morphological traits\u0026mdash;including total root length (TRL), root surface area (RSA), root volume (RV), number of root tips (NRT), and number of forks (NF). Rhizosheath-related traits such as rhizosheath weight (RW), root hair length (RHL), and root hair density (RHD) were also significantly correlated with S-AMF. These results suggest that S-AMF enhance host drought resistance by expanding the root\u0026ndash;soil interface and facilitating efficient carbon\u0026ndash;phosphorus exchange. In comparison, root-colonising AMF (R-AMF) showed significant positive correlations (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e) with all traits except RSR. Aboveground and root fresh weights (FWAG and FRW) exhibited highly consistent variation, displaying strong positive correlations (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) with most biomass, architectural, and root hair traits, and a significant correlation with RSR (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e). Dry weight traits (DWAG and DWR) were significantly correlated (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e) with TRL, RSA, RV, NRT, NF, and RHL, and showed a highly significant positive correlation (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) with RW. Within the root system, strong interdependencies (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) were observed among architectural traits (TRL, RSA, RV, NRT, NF). Root volume was also significantly correlated with rhizosheath weight (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e), and tightly coupled relationships (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e) linked RW, RHL, and RHD.Collectively, these findings indicate that AMF colonisation\u0026mdash;both in soil and roots\u0026mdash;enhances drought adaptation in K. hirsuta through synergistic regulation of biomass allocation, root architecture, and root hair development. This functional integration highlights the key role of AMF in strengthening plant resilience in arid environments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural equation modelling reveals hierarchical regulation of rhizosheath formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA segmented structural equation model (SEM) was used to elucidate how drought stress and growth stage interact to regulate rhizosheath formation, revealing a hierarchical regulatory pathway (Fig. 9). The model demonstrated good fit (Fisher\u0026apos;s C = 28.97, P = 0.42). Root hair traits (path coefficient = 0.80) and AM fungal colonisation rate (\u0026ndash;0.90) exerted the strongest direct effects on rhizosheath formation, while biomass allocation (indirect effect = 1.26), root architectural plasticity (1.55), and AM fungal activity in rhizosheath soil (1.14) functioned through indirect pathways. Growth stage progression significantly enhanced the positive effects of root architecture (0.86) and root hair plasticity (1.40) on rhizosheath development, but suppressed soil AM fungal activity (\u0026ndash;0.25), indicating a stage-dependent resource trade-off in plant\u0026ndash;microbe interactions. Sustained 25% water stress (W\u003csub\u003e5\u003c/sub\u003e) strengthened the synergistic contribution of root hair traits (\u0026ndash;2.92) and AM fungal colonisation (1.17) to rhizosheath formation via treatment\u0026ndash;stage interaction, highlighting its role in modulating development through the mycorrhizal network. AM fungal colonisation rate (total effect: \u0026ndash;0.90) balanced root carbon expenditure via negative regulation, whereas root hair traits (total effect: 0.80) served as physical structures expanding the root\u0026ndash;fungus interface. Together, these form a complementary mechanism of \u0026ldquo;hyphal synergy and morphological adaptation.\u0026rdquo; The model indicates that rhizosheath formation arises from combined contributions of plant phenotypic plasticity (54.3%) and microbial interaction networks (45.7%). Under experimental conditions, the W\u003csub\u003e5\u003c/sub\u003e treatment promoted functional assembly of rhizosheath modules in sandy habitats by optimising dynamic equilibrium among root hairs, AMF, and the soil environment. These findings offer a theoretical framework for understanding ecological and evolutionary mechanisms in plant\u0026ndash;microbe drought adaptation.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003eAdaptive Mechanism of Root Morphological Plasticity to Alternate Drought\u0026ndash;Rehydration\u003c/h2\u003e\u003cp\u003eRoot architectural plasticity enables plants to efficiently explore and acquire soil resources, a trait of considerable ecological importance in arid habitats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Unlike the inhibitory effect of drought on root phenotypes in tartary buckwheat (Fagopyrum tataricum) (Lu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), this study found that \u003cem\u003eK. hirsuta\u003c/em\u003e significantly increased total root length (5.48-fold at T\u003csub\u003e3\u003c/sub\u003e vs. W\u003csub\u003e1\u003c/sub\u003e), root surface area (8.02-fold), and branch number (6.93-fold) under 25\u0026ndash;40% moisture gradients, forming a dual adaptation strategy of \"wide-range branching and local optimisation\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This difference may reflect evolutionary adaptations of \u003cem\u003eK. hirsuta\u003c/em\u003e as a pioneer species in desertified grasslands: sandy soils maintain adequate pore oxygen even at 25\u0026ndash;40% moisture, satisfying root aerobic metabolism. As shown in prior studies, \u003cem\u003eK. hirsuta\u003c/em\u003e enhances deep soil water acquisition while optimising resource capture in surface microzones by increasing root branching angles and root hair density. Notably, under W\u003csub\u003e5\u003c/sub\u003e treatment, periodic fluctuation in root volume (peak at T\u003csub\u003e3\u003c/sub\u003e: 0.18 cm\u0026sup3;) and synchronous growth in root tip number (1875.25 at T\u003csub\u003e3\u003c/sub\u003e) reflect functional complementarity, confirming a \"carbon investment\u0026ndash;phenotypic benefit\" trade-off mechanism (Song et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This allows dynamic biomass allocation between storage and absorptive roots, balancing immediate survival and long-term adaptation. In contrast to the root expansion of perennial ryegrass (\u003cem\u003eLolium perenne\u003c/em\u003e) under ample moisture (Grzesiak et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eK. hirsuta\u003c/em\u003e under sustained 40% moisture (W\u003csub\u003e6\u003c/sub\u003e) showed increased total root length (389.06 cm at T\u003csub\u003e3\u003c/sub\u003e) but a decreased root\u0026ndash;shoot ratio (from 0.71 to 0.58) and lower AM fungal colonization. This \"moisture paradox\" suggests that moderate drought (25% field capacity) may activate ABA signal-mediated architectural reprogramming, shifting branching patterns from quantitative expansion to qualitative optimisation. This establishes a positive feedback loop with the mycorrhizal network, collectively supporting rhizosheath ecological function. These findings provide new theoretical insight into the ecological evolution of root plasticity in psammophytes.\u003c/p\u003e\u003cp\u003eRoot hairs, as frontline sensors of soil heterogeneity, exhibit phenotypic plasticity in length and density (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), a key strategy for adapting to moisture variation. Under 25% water stress (W\u003csub\u003e5\u003c/sub\u003e), root hair length increased significantly (871.03 \u0026micro;m at T\u003csub\u003e3\u003c/sub\u003e, 34.78% longer than W\u003csub\u003e6\u003c/sub\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) while maintaining high density (66 root hairs/mm), forming a \"super surface area\" absorption mode similar to Japanese brome (Bromus japonicus) (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, \u003cem\u003eK. hirsuta\u003c/em\u003e shows unique spatiotemporal regulation: root hair length increased continuously through growth stages (T\u003csub\u003e1\u003c/sub\u003e\u0026rarr;T\u003csub\u003e3\u003c/sub\u003e: 650.29\u0026rarr;871.03 \u0026micro;m), while density peaked at T\u003csub\u003e2\u003c/sub\u003e then declined, suggesting that mature plants extend the lifespan of existing root hairs rather than producing new ones. This contrasts with the \"quantity\u0026ndash;quality\" trade-off in typical dicot root hair development (Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The 40% moisture treatment (W\u003csub\u003e6\u003c/sub\u003e) inhibited root hair density, and drought\u0026ndash;rehydration (W\u003csub\u003e1\u003c/sub\u003e) impeded both length and density, indicating that moderate sustained drought may activate ethylene-mediated root hair elongation, while high or fluctuating moisture disrupts calcium oscillation signalling, leading to phenotypic imbalance. The strong synergy among root hair length, AM fungal hyphal colonization, and rhizosheath biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) suggests root hairs act not only as physical absorption organs but also as chemical signalling hubs for microbial interaction. Flavonoids and other signals secreted from root hair tips may guide hyphal growth via chemotaxis. These results support the \"root hair\u0026ndash;hyphal synergy\" theory and indicate that psammophytes have evolved a unique rhizosheath construction strategy integrating phenotypic plasticity and microbial cooperation, offering biomimetic inspiration for drought-resistant crop root design.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eRegulatory Mechanism of Moisture Threshold on the AMF\u0026ndash;Plant Symbiotic Relationship\u003c/h2\u003e\u003cp\u003eThe intensity of AM fungal symbiosis with host plants is non-linearly regulated by moisture and exhibits species and habitat specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). AM fungi form symbiotic associations with most terrestrial plants (Marulanda et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and enhance host stress resistance, competitive ability, and survival, thereby influencing plant community structure and ecosystem productivity (Dong et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; He et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, the total AM fungal colonization rate of \u003cem\u003eK. hirsuta\u003c/em\u003e peaked (90.88% at T\u003csub\u003e3\u003c/sub\u003e) under sustained 25% water stress (W\u003csub\u003e5\u003c/sub\u003e), showing a strong positive correlation with soil moisture\u0026mdash;contrasting with the \"drought-promoted\" symbiosis observed in grasses like crested wheatgrass (\u003cem\u003eAgropyron cristatum\u003c/em\u003e) (Monz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). This may reflect an evolutionary adaptation of psammophytes: sandy soils retain adequate pore oxygen even at 25\u0026ndash;40% moisture, meeting AM fungal aerobic requirements (Hattori et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), whereas loamy soils under similar moisture can develop anaerobic microsites. The synchronous increase in root\u0026ndash;shoot ratio and hyphal colonization under W\u003csub\u003e5\u003c/sub\u003e suggests the host allocates photosynthetic carbon to AM fungi in exchange for phosphorus and other scarce resources, a strategy particularly advantageous in infertile sandy soils (Lv et al., 2019). Moderate drought (25%) may activate JA signalling and strigolactone secretion, specifically attracting AM fungal hyphae. Although the 40% moisture treatment (W\u003csub\u003e6\u003c/sub\u003e) maintained high colonization (78.99%), its hyphal network density (1.46 m/g) was significantly lower than under W\u003csub\u003e5\u003c/sub\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), indicating excessive moisture may inhibit functional hyphal differentiation. Rehydration treatments (W\u003csub\u003e1\u003c/sub\u003e\u0026ndash;W\u003csub\u003e3\u003c/sub\u003e) reduced AM fungal colonization by 21.8\u0026ndash;38.5% compared to sustained drought (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), aligning with the \"moisture fluctuation inhibition hypothesis\" (Hattori et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), where rapid changes in soil oxygen after rehydration disrupt hyphal network stability. In contrast, prolonged drought enriches drought-tolerant AM fungal communities through selection. This supports the idea that the psammophyte\u0026ndash;AM fungal symbiotic system achieves dynamic equilibrium via a \"stress memory\" mechanism, offering a theoretical basis for mycorrhizal-assisted restoration of degraded grasslands.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of Rhizosheath Formation in\u003c/b\u003e \u003cb\u003eK. hirsuta\u003c/b\u003e \u003cb\u003eUnder Drought and Rehydration Conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rhizosheath in \u003cem\u003eK. hirsuta\u003c/em\u003e seedlings is a structure formed by the entanglement of roots, soil microorganisms, and root exudates, which adhere soil particles (Chen, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This adaptation is key to its success in alpine arid environments. Although rhizosheath research has largely focused on Poaceae species\u0026mdash;e.g., Bailey et al. observed rhizosheaths in most wild grasses\u0026mdash;it is not exclusive to this family (Basirat et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Soil moisture critically influences aggregation: the rhizosheath retains water by suction, maintaining higher moisture than bulk soil, and is more evident in dry sandy soil (Haling et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, rhizosheath weight under the 25% moisture treatment was significantly higher than under other moisture levels, and its response varied with culture duration, indicating optimal development under sustained moderate drought. However, rhizosheath formation is multifactorial (North \u0026amp; Nobel, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This study identified root hair traits and AM fungal colonization as having direct, significant effects. Other essential factors include root mucilage concentration, root metabolites, and rhizosheath microbial composition, which warrant further investigation.\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eK. hirsuta\u003c/em\u003e adapts to water fluctuations by dynamically modulating root system architecture, biomass allocation, and arbuscular mycorrhizal fungi (AMF) symbiosis, which act together to enhance rhizosheath formation. In conditions of soil moisture equivalent to 25% of field capacity (W\u003csub\u003e5\u003c/sub\u003e), significant stimulation was observed in the accumulation of rhizosheath biomass, the development of root morphology, and the colonisation of arbuscular mycorrhizal (AMF) fungi. Furthermore, AMF colonisation extent and root hair traits were identified as key regulators of rhizosheath formation. These factors interact through coordinated effects on biomass allocation, root architecture, and the soil microenvironment, constituting a multidimensional adaptive network. The findings elucidate the ecophysiological basis of plant\u0026ndash;microbial collaboration under varying moisture conditions, thus providing a theoretical foundation for the selection of stress-tolerant grasses with a view to restoring degraded grassland ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of funding.\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 31802123).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to thank the National Natural Science Foundation of China and the \u0026ldquo;College of Grassland Resources, Southwest University for Nationalities\u0026rdquo; for their support and funding of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this collaborative effort, Dr. Yuan Yutao mainly contributed by focusing on his writing and manuscript. Ms. Wu Li, Ms. Chen Chen, Ms. Li Rong, Professor Chen Youjun, and Professor Zhou Qingping have played key roles in the development of methodology, formal analysis, investigation, and paper writing. Professor Chen Youjun provided valuable guidance throughout the entire project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability.\u003c/strong\u003e Data will be available upon request from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest.\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eE. Acevedo, T. C. Hsiao, D. W. Henderson. Immediate and subsequent growth responses of maize leaves to changes in water status [J]. Plant Physiology, 1971, 48(5): 631-636. https://doi.org/10.1104/pp.48.5.631\u003c/li\u003e\n\u003cli\u003eM. M. Aslam, J. K. Karanja, W. Yuan, et al. 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Flavel, et al. Plant roots redesign the rhizosphere to alter the three-dimensional physical architecture and water dynamics [J]. New Phytologist, 2018, 219(2): 542-550. https://doi.org/10.1111/nph.15213\u003c/li\u003e\n\u003cli\u003eJ. P. Schimel. Life in dry soils: effects of drought on soil microbial communities and processes [J]. Annual Review of Ecology, Evolution, and Systematics, 2018, 49: 409-432. https://doi.org/10.1146/annurev-ecolsys-110617-062614\u003c/li\u003e\n\u003cli\u003eQ. H. Song, C. Z. Zhao, Y. C. Shi, et al. Trade-off between root branching number and connection length of Melica przewalskyi on different slope aspects [J]. Chinese Journal of Plant Ecology, 2015, 39 (6): 577-585. https://doi.org/10.17521/cjpe.2015.0055\u003c/li\u003e\n\u003cli\u003eG. L. Sun, J. Yan, J. L. Yang. Studies on the biosystematics between Psathyrostachys huashanica and Roegneria caucasica, \u003cem\u003eKengyilia hirsuta\u003c/em\u003e [J]. 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New Phytologist, 1995, 130(1): 135-139. https://doi.org/10.1111/j.1469-8137.1995.tb01823.x\u003c/li\u003e\n\u003cli\u003eY. M. Zhang, H. Y. Hu, X. M. Bai, et al. Adaptation of root morphology and growth of perennial ryegrass and Japanese brome to soil drought [J]. Chinese Journal of Eco-Agriculture, 2022, 30 (11): 1784-1794. https://doi.org/10.12357/cjea.20220336\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"drought- rehydration, Kengyilia hirsuta, rhizosheath, root architecture, AM fungi","lastPublishedDoi":"10.21203/rs.3.rs-8055104/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8055104/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnder increasing extreme climate events, plants must adapt to alternating drought\u0026ndash;rewatering stress. \u003cem\u003eKengyilia hirsuta\u003c/em\u003e, a pioneer forage grass in alpine desert regions, depends on its rhizosheath for drought resistance. This study simulated alternating and continuous drought\u0026ndash;rewatering treatments (10%\u0026ndash;40% field capacity) to investigate root architecture, biomass allocation, arbuscular mycorrhizal fungi (AMF) symbiosis, and rhizosheath formation. Results showed that rhizosheath accumulation responded dynamically to water regimes: continuous 25% moisture stress (W\u003csub\u003e5\u003c/sub\u003e) significantly promoted rhizosheath biomass, whereas 40% stress (W\u003csub\u003e6\u003c/sub\u003e) enhanced early-stage development and 10% rewatering (W\u003csub\u003e1\u003c/sub\u003e) boosted later-stage formation. AMF colonization increased progressively, with total colonization rising from 41.51% (T\u003csub\u003e1\u003c/sub\u003e) to 61.40% (T\u003csub\u003e3\u003c/sub\u003e). The W\u003csub\u003e5\u003c/sub\u003e treatment consistently showed the highest vesicle, arbuscule, and hyphal colonization, alongside increased soil spore density and hyphal density by T3. Root morphological traits\u0026mdash;including surface area, volume, tip number, hair length, and hair density\u0026mdash;peaked under W\u003csub\u003e5\u003c/sub\u003e. Structural equation modelling identified AMF colonization (total effect: \u0026minus;\u0026thinsp;0.90) and root hair traits (total effect: +0.80) as pivotal regulators of rhizosheath formation, interacting through biomass allocation, root architecture, and soil microenvironment to form a multidimensional adaptive network. These findings elucidate the ecophysiological mechanisms underlying plant\u0026ndash;AMF collaboration in rhizosheath formation under water fluctuation, supporting selection of stress-tolerant grasses for restoring desertified grasslands.\u003c/p\u003e","manuscriptTitle":"Mechanisms underlying rhizosheath dynamics in Kengyilia hirsuta in response to alternating drought and rewatering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 18:21:46","doi":"10.21203/rs.3.rs-8055104/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-16T05:17:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-01T20:15:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131253959654203845812212955883272232007","date":"2025-12-11T18:09:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T22:56:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147960942799648417190996020703938787950","date":"2025-11-19T16:34:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T15:32:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-14T15:29:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-14T08:36:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T09:25:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-12T09:20:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"919e45f1-9505-42a8-83c0-cbd1254e7668","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58521633,"name":"Biological sciences/Ecology"},{"id":58521634,"name":"Earth and environmental sciences/Ecology"},{"id":58521635,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-27T16:06:22+00:00","versionOfRecord":{"articleIdentity":"rs-8055104","link":"https://doi.org/10.1038/s41598-026-49036-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-24 15:58:03","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-11-25 18:21:46","video":"","vorDoi":"10.1038/s41598-026-49036-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-49036-7","workflowStages":[]},"version":"v1","identity":"rs-8055104","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8055104","identity":"rs-8055104","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}