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. 2026 Jun 2;28(5):1614–1627. doi: 10.1111/plb.70235

Effects of cotyledon removal on the root growth and physiology properties during early seedling stage of Ricinus communis under salt stress

X Deng 1, R Lin 1, H Liu 1, L Pan 1, Y Li 1, S Mao 1, S Yu 1, J Lin 1,✉
PMCID: PMC13358673  PMID: 42228838

Abstract

  • Castor (Ricinus communis L.) is an important oilseed crop species that demonstrates strong adaptability to saline‐alkaline conditions. Early seedling development is the most vulnerable period to salinity stress during plant establishment. Cotyledons, as the earliest emerging photosynthetic organs, play a crucial role in supporting seedling establishment and contributing to salt stress tolerance. However, the effects of cotyledon removal (CR) on root adaptive strategies and physiological regulation under salt stress remain unclear.

  • Here, the growth, root ultrastructure, ion homeostasis, antioxidant defences, and carbon–nitrogen metabolism of castor roots with the degree of CR (no cotyledon removed, NR; one cotyledon removed, OR; both cotyledons removed, TR) were measured under both salt and non‐salt treatments.

  • The results showed that CR inhibited biomass accumulation and damaged root architecture, and the scanning electron microscopy (SEM) analysis revealed that TR induced loss of root hairs and severe epidermal damage. Under saline conditions, CR had a more significant effect, leading to elevated Na+ concentrations and reduced K+ content in castor roots. In addition, TR treatment decreased MDA content and antioxidant enzyme activity under 150 mM NaCl, compared with NR seedlings. Moreover, CR adversely affected root carbon and nitrogen metabolism. Principal component analysis (PCA) further highlighted distinct physiological profiles, with TR150 samples clustering separately due to severe ionic and oxidative imbalances.

  • Together, these findings provide new insight into the physiological functions of cotyledons and the above and below ground interactions of castor plants under salt conditions.

Keywords: cotyledon removal, physiological response, Ricinus communis, root morphogenesis, salt stress

Cotyledon removal aggravates salt‐induced damage to castor roots by disrupting ion homeostasis, antioxidant capacity, and carbon–nitrogen metabolism, revealing that cotyledons play an essential role in supporting early seedling salt tolerance.

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INTRODUCTION

Soil salinization is a global issue, and affects 1.381 billion hectares of land, which is exacerbated by climate change and water scarcity (FAQ 2025). Unlike halophytes that can survive in highly saline conditions, most crop species are glycophytes and display significant physiological damage even under moderate salinity levels (Flowers & Colmer 2008; Cheeseman 2014). Salt stress affects plants via various physiological pathways. In the saline‐alkaline soils, plants experience osmotic stress due to reduced soil water potential, which limits water uptake (Awlia et al. 2021). Subsequently, the accumulation of Na+ and Cl− disrupts ion homeostasis and induces ionic toxicity, typically occurring after toxic levels of salt are reached in root and shoot tissues (Munns & Tester 2008). Collectively, these stresses impair plants' physiology and hinder their growth.

Castor (Ricinus communis L.), a member of the Euphorbiaceae family, is one of the top 10 oilseed crops. Castor seeds contain over 50% oil and are widely utilized in the production of various industrial chemicals such as lubricants, coatings, biodiesel, and surfactant (Tomar et al. 2017; Shi et al. 2024). Owing to its renewable nature, castor oil is often referred to as ‘green petroleum’. Additionally, numerous studies have focused on the potential of castor plants for rehabilitating the salt‐alkali soils (Zhang et al. 2018). It is widely cultivated in many countries due to its high economic and ecological values, particularly in India, Brazil, and China (Ribeiro et al. 2014). Hence, investigating the salt tolerance mechanisms of this species holds significant value for advancing basic plant stress physiology and for improving crop tolerance under saline conditions.

Early seedling stage is the most vulnerable period in the plant life cycle (Zhou et al. 2013). Cotyledons are the first photosynthetic organs that provide photoassimilates for the subsequent seedling growth until the first pair of true leaves become fully functional (Marshall & Kozlowski 2010). Crucially, during the cotyledon stage, sugars derived from cotyledons act as both an energy source and systemic signals, transported via the phloem to drive root morphogenesis and physiological development (Kircher & Schopfer 2012). Moreover, cotyledons exhibit protective functions during early seedling development under adverse environmental conditions (Wang et al. 2019; Hu et al. 2024). However, cotyledon damage or loss is common in natural environments due to abiotic stress such as extreme weather and soil stressors, as well as biotic factors including mechanical injury. In general, cotyledon removal (CR) in epigeal dicotyledonous plants negatively affects growth and survival, with impacts that may persist into later developmental stages. For example, CR significantly reduces biomass and survival rate in legume seedlings (Hu et al. 2017). The early‐stage disruptions may further affect reproductive development and reduce the yield potential. Previous studies have shown that CR delays flowering in several grass species (Hanley & Fegan 2007), and as observed in Medicago lupulina, reduces seed production by limiting final plant size (Zhang et al. 2010). However, the contributions of cotyledon to the salt tolerance of early‐stage castor seedling have rarely been reported.

In fact, roots are essential for absorbing water and mineral nutrients, serving as a foundation for plant growth and development (Libault et al. 2010). As the primary sensors of salinity, roots' responses influence the overall plant adaptation to salt stress (Bazihizina et al. 2022). Previous research has largely focused on above ground traits, such as biomass accumulation, survival, flowering, and seed production. Consequently, the understanding of how CR influences root adaptive strategies and physiological adjustments under salt stress remains limited. In the present study, we hypothesized that cotyledons play a critical role in the salt tolerance of castor seedlings, and their removal alters root growth and physiological responses. To test this hypothesis, we examined how CR (no cotyledon removed, NR; one cotyledon removed, OR; both cotyledons removed, TR) influences root growth, ultrastructural alterations, antioxidant defence mechanisms, ion homeostasis, and carbon–nitrogen metabolism under salinity stress (0, 75, and 150 mM NaCl).

MATERIALS AND METHODS

Experimental design

The castor seeds (provided by the Institute of Economic Crops, Shanxi Academy of Agricultural Sciences) employed in this study were sourced from the early maturing hybrid ‘Fen Bi 10’. This cultivar completes its developmental cycle in roughly 96 days for the main raceme and 112 days for the primary branches. Owing to its requirement for a frost‐free period of at least 120 days, ‘Fen Bi 10’ is primarily cultivated in several regions across China, including Shanxi, Jilin, Inner Mongolia, Shaanxi, and Xinjiang. Surface‐sterilized castor seeds were sowed in plastic pots (15 × 12.5 × 10.5 cm) containing 2 kg soil and grown in the greenhouse (25 ± 1 °C; 12 h light/12 h dark). Five seeds were planted in each pot as one replicate, with four replicates per group. Two factors were set in this experiment: the degree of CR (NR, removal of no cotyledon; OR, removal of one cotyledon; TR, removal of two cotyledons); salt concentrations (0, 75, and 150 mM NaCl). Method for CR treatment and salt stress test: Select castor seedlings in the fully extended stage of cotyledons (two cotyledons fully expanded and true leaves exposed but not fully expanded) for CR treatment. The salt stress treatment began 3 days after CR. Before applying the NaCl solutions, soil moisture in all pots was adjusted to approximately 70% of field capacity using the gravimetric method. Plants were then irrigated with 150 ml of the designated NaCl solution once daily for 5 days. At the end of the treatment period, root tips were excised and fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.2) and stored at 4 °C for the subsequent ultrastructural analysis. A portion of fresh root tissue was used for root architectural measurements, while the remaining root samples were immediately frozen in liquid nitrogen and stored at −80 °C for physiological analyses.

Growth, root architecture, and ultrastructure

Roots were thoroughly rinsed with deionized water to remove residual soil, blotted to remove surface moisture, and then weighed to determine fresh weight (FW). Subsequently, samples were then dried at 105 °C for 15 min to inactivate enzymes, followed by drying at 80 °C until a constant dry weight (DW) was obtained.

The WinRHIZO (Pro.2013e) root analysis system software (Regent Instruments Inc., Canada) was utilized to measure and analyse parameters including root length, surface area, average diameter, volume, and forks. Root tips (5 mm) from lateral roots were collected from castor, and root surface ultrastructure was observed using SEM (Hitachi S‐3400 N, Japan).

Na+ and K+ content measurements

The Na+ and K+ content in the roots of castor were determined using an atomic absorption spectrophotometer (TAS‐990, Purkinje General, Beijing, China). Briefly, 0.1 g of dried root was extracted with 10 ml of deionized water at 100 °C for 20 min. The extract was centrifuged at 10,000×g for 10 min, and the supernatant was filtered through a 0.22 μm aqueous microporous membrane prior to analysis.

Malondialdehyde contents and activities of antioxidant enzymes measurements

Fresh root samples (0.2 g) were homogenized with 2 ml Tris–HCl buffer (0.05 mM, pH 8.0) and two steel balls in an ice bath, then centrifuged at 4,000×g for 10 min at 4 °C. The supernatant was used to determine soluble protein content and antioxidant enzyme activity. The activity of SOD was measured with nitro blue tetrazolium photoreduction method at 560 nm (Modesti et al. 2018). The activity of POD was measured with guaiacol method at 470 nm (Polle & Seifert 1994). The activity of CAT was measured as described by Knorzer et al. (1996). The activity of APX was measured as described by Nakano & Asada (1980). The MDA content was determined using the thiobarbituric acid colorimetric method as described by Shi et al. (2024).

Measurement of activities of enzymes involved in carbon and nitrogen metabolism

The activity of sucrose synthase (SS) and sucrose phosphate synthase (SPS) was determined according to the method described by Li et al. (2023), with minor modifications. Briefly, 0.5 g of fresh root samples was homogenized with 3 ml Hepes–NaOH buffer (50 mM, pH 7.5, containing 1 mM EDTA, 1 mM MgCl2, and 10 mM 2‐Mercaptoethanol). The homogenate was centrifuged at 1,000×g for 10 min at 4 °C, and the supernatant was collected and used for enzyme activity assays of SS and SPS.

The nitrate reductase (NR) activity was measured following the protocol of Zou (2000), with slight modifications. About 2 g of fresh root samples were homogenized with 100 mM phosphate buffer (pH 7.5). The homogenate was centrifuged at 4,000×g for 6 min at 4 °C. The supernatant was then collected and used for determining NR activity at 540 nm.

To measure the activities of glutamine synthetase (GS) and glutamate synthetase (GOGAT), 0.4 g fresh roots were homogenized with Tris–HCl buffer (pH 7.6, containing 1 mM EDTA, 1 mM MgCl2, and 10 mM 2‐Mercaptoethanol), and the mixture was centrifuged at 13,000×g for 20 min at 4 °C, and the supernatant was taken as enzyme extract.

Measurement of carbohydrate, total nitrogen, and nitrate nitrogen content

The sucrose, fructose, glucose, and starch contents were determined as previously described (Li et al. 2023). Total nitrogen content was determined using the Kjeldahl method (Lu & Li 2012). Briefly, 0.1 g of ground dry root was transferred into a digestion tube, followed by the addition of 0.2 g catalyst mixture (cupric sulfate: potassium sulfate = 1:5, w/w) and 5 ml sulfuric acid. The mixture was digested in a digestion furnace at 400 °C until the solution became clear and transparent. After cooling, the digest was transferred to a 50 ml volumetric flask and diluted to volume with distilled water. Total nitrogen content was subsequently measured using a Kjeldahl nitrogen analyser. Nitrate nitrogen content was determined using the salicylic acid method (Zou 2000). Fresh root samples (0.5 g) were extracted with 2 ml deionized water at 100 °C for 30 min, then centrifuged and diluted to 4 ml. About 0.1 ml supernatant was mixed with 0.4 ml 5% salicylic–H2SO4 at 25 °C for 20 min, and then added 9.5 ml 8% NaOH at 25 °C. The absorbance was recorded at 410 nm.

Data analysis

Statistical analyses were conducted using SPSS 26.0, and graphs were generated with SigmaPlot 14.0. Data are presented as the mean ± SE of three biological replicates. A two‐way ANOVA was performed to evaluate the effects of CR, salinity, and their interaction on physiological traits in castor roots. Tukey's test was used to identify significant differences among means at P < 0.05.

RESULTS

Growth, root architecture, and ultrastructure

The FW and DW of NR, OR, and TR roots decreased significantly with increasing salt concentration (Table 1, P < 0.05). Furthermore, the FW and DW of TR roots were consistently lower than those of NR seedling roots at the same salt concentration level (0, 75, and 150 mM NaCl). Specifically, the FW of TR seedling roots was 72.13%, 86.70%, and 80.00% lower at 0, 75, and 150 mM NaCl, respectively, compared with NR roots. A similar trend was also found for the DW. Two‐way ANOVA revealed that salinity, CR and their interaction had a significant impact on root architecture of castor plants, including root length, surface area, volume, and root forks. Whereas root average diameter was affected by salinity and CR, with no significant interaction between those two main factors (Table S1). In this study, salt stress significantly inhibited root development (Fig. 1A, P < 0.05). In detail, root length, surface area, average diameter, volume, and forks of NR seedlings decreased by 61.62%, 48.17%, 23.08%, 57.83%, and 63.19% under 150 mM NaCl, respectively, in comparison to the salt‐free condition. Moreover, CR (especially TR treatment) further damaged the root architecture of the castor root system under salt stress. For instance, TR treatment reduced root length, surface area, average diameter, volume, and forks by 76.03%, 67.23%, 12.5%, 60.00%, and 81.96% under 150 mM NaCl, respectively, compared with NR treatment.

Table 1.

The FW and DW of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl.

0 mM NaCl 75 mM NaCl 150 mM NaCl
NR OR TR NR OR TR NR OR TR
FW 3.48 ± 0.16Cc 2.47 ± 0.20Bc 0.97 ± 0.07Ab 1.88 ± 0.10Cb 1.30 ± 0.20Bb 0.25 ± 0.06Aa 1.35 ± 0.17Ba 0.61 ± 0.04Aa 0.27 ± 0.04Aa
DW 0.22 ± 0.01Bb 0.19 ± 0.02Bc 0.08 ± 0.01Ab 0.17 ± 0.02Ba 0.12 ± 0.02Bb 0.03 ± 0.01Aa 0.13 ± 0.02Ba 0.06 ± 0.01Aa 0.04 ± 0.01Aa
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Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

Fig. 1.

Fig. 1

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Effects of cotyledon removal on the root growth. (A) Root morphology; (B) Scanning electron microscopy (SEM) images of root hairs in R. communis under different treatments: (a, e) removal of no cotyledon (NR), (b, f) removal of two cotyledons (TR), (c, g) NR at 150 mM NaCl, and (d, h) TR at 150 mM NaCl. Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

As specialized epidermal extensions critical for water and nutrient absorption, root hair in the mature zone was measured through SEM with a magnification of 13.0 mm × 70 SE. In this study, root hair of NR seedlings maintained robustness and was characterized by high density coverage along root epidermal surfaces (Fig. 1a). Root hair development was inhibited significantly under both salt stress and TR treatment (Fig. 1b, c). In addition, TR treatment significantly reduced root hair density under 150 mM NaCl, with complete disappearance observed in localized areas (Fig. 1d). The root surface was observed using SEM with a magnification of 13.0 mm × 800 SE. The root surface of control plants exhibited a relatively smooth morphology with uniform epidermal cells (Fig. 1e). In contrast, the epidermal cells displayed structural irregularities and compromised integrity under either salt stress alone or TR treatment individually (Fig. 1f, g). Notably, TR treatment exacerbated the extent of root surface damage under salt stress (Fig. 1h). These results indicate that CR significantly influences root growth, spatial distribution, and ultrastructure in R. communis, especially under salt stress.

Na+ and K+ contents

Two‐way ANOVA revealed significant effects of both salinity and CR and their interactive influence upon Na+ and K+ contents (Table S1). The Na+ contents of the NR, OR, and TR roots significantly increased with increasing salinity (Fig. 2A, P < 0.05). At the highest salt level of 150 mM NaCl, the Na+ content in the roots of NR, OR, and TR seedlings was elevated by 5.33‐fold, 5.30‐fold, and 10.27‐fold, respectively, compared with without salt treatment. Under non‐saline conditions, CR had no significant impact on the Na+ content. However, CR (OR and TR) resulted in a significant increase in Na+ content under salt stress compared with NR seedlings. Notably, TR treatment increased the Na+ content by 2.18‐fold and 1.96‐fold under 75 mM and 150 mM NaCl, respectively. In contrast, the K+ content declined significantly in NR, OR, and TR roots with increasing salinity (Fig. 2B, P < 0.05). For TR seedlings, the K+ content decreased significantly by 70.9% under 150 mM NaCl in comparison to the control treatment. Moreover, CR significantly reduced the K+ content in the roots of OR and TR seedlings under the same salt level. Specifically, the K+ content decreased by 18.43% and 31.85% in OR and TR roots under 150 mM NaCl, respectively, compared with NR roots. In conclusion, under non‐saline conditions, root Na+ uptake is intrinsically low; thus, CR has no effect on root Na+ levels. Under salt stress, however, CR deprives the roots of the energy and nutrients needed to mitigate ionic stress, thereby weakening their capacity for Na+ efflux and intracellular compartmentalization and consequently exacerbating Na+ accumulation and disrupting K+ homeostasis in the roots.

Fig. 2.

Fig. 2

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The Na+ content (A) and K+ content (B) in the root of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl. Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

Antioxidant enzyme system

Two‐way ANOVA revealed that the MDA content in the roots was significantly affected by salinity, CR, and their interaction (Table S1). The MDA content in the roots of TR seedlings significantly reduced following CR, showing a 57.80% decrease compared with NR seedlings under 150 mM NaCl (Fig. 3A, P < 0.05). For NR seedlings, the MDA content in the roots peaked at a salt level of 75 mM NaCl, subsequently declining at higher salt levels. In contrast, the MDA content in the roots of TR seedlings remained stable across different salt treatments (Fig. 3A).

Fig. 3.

Fig. 3

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The MDA content (A), APX activity (B), CAT activity (C), SOD activity (D), and POD activity (E) in the root of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl. Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

As for antioxidant enzyme activities, two‐way ANOVA revealed that the APX and CAT activities were significantly affected by salinity, CR, and their interaction. Whereas SOD and POD activities were affected by salinity and their interaction (Table S1). The APX activity in the roots of NR and OR seedlings decreased significantly under 75 mM NaCl but increased markedly under 150 mM NaCl, compared with salt‐free condition (Fig. 3B, P < 0.05). Conversely, the APX activity in the roots of TR seedlings showed a positive correlation with salinity level. CR enhanced the APX activity of OR and TR roots by 40.22% and 98.22% under 75 mM NaCl, respectively, compared with NR roots. However, the APX activity in the roots significantly decreased with CR under 150 mM NaCl (Fig. 3B, P < 0.05). The results indicate that the effect of CR on APX activity exhibits a significant dependence on salinity levels. The CAT activity of NR and OR roots showed a peak at 75 mM NaCl followed by a decline at 150 mM NaCl, while that in TR roots exhibited a gradual decrease with increasing salinity (Fig. 3C). CR reduced CAT activity in the roots under all salinity levels. These results indicate that CR weakens the ROS scavenging capacity of CAT, particularly at elevated salinity levels. In NR, OR, and TR roots, the SOD activity was not significantly affected by the increasing salinity. However, CR significantly reduced SOD activity in the roots of TR seedlings by 38.06%, 49.83%, and 12.13% at 0, 75, and 150 mM NaCl, respectively, while OR seedlings showed no significant changes compared with NR seedlings (Fig. 3D). These results suggest a specific sensitivity of the SOD activity in TR roots, which may impair their oxidative stress response. Finally, the POD activity of NR roots increased significantly with salinity level, whereas both OR and TR roots exhibited a gradual decline (Fig. 3E, P < 0.05). The POD activity decreased by 19.38% and 26.39% in OR and TR roots under 150 mM NaCl, respectively, compared with the salt‐free condition. Moreover, the POD activity of the TR roots was lower than that of the NR roots at each salinity level. This trend highlights a reduced capacity for ROS detoxification via POD in seedlings with CR, particularly under salinity stress.

Carbon metabolism

Two‐way ANOVA revealed that the total carbon content was significantly affected by salinity, CR, and their interaction (Table S1). The total carbon content in the roots of NR and TR seedlings decreased significantly with increasing salinity (Fig. 4A, P < 0.05). For OR seedlings, the total carbon content in the roots peaked at 75 mM NaCl and then fell significantly at 150 mM NaCl. In addition, CR significantly reduced the total carbon content in the roots at each salt level (Fig. 4A, P < 0.05). In detail, the total carbon content of TR roots reduced by 4.25%, 5.15%, and 5.33% under 0, 75, and 150 mM NaCl, respectively, compared with NR roots.

Fig. 4.

Fig. 4

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Total carbon (A), glucose content (B), sucrose content (C), fructose content (D), and starch content (E) in the root of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl. Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

According to two‐way ANOVA, the glucose and sucrose contents of castor seedlings were significantly affected by salt concentration, CR, and their interaction. The fructose and starch contents were significantly influenced by salt concentration and CR, with no significant interaction between those two main factors (Table S1). CR significantly increased the glucose content by 48.72%, 15.39%, and 16.84% in the OR roots under each salt stress (0, 40, and 80 mM NaCl), respectively, compared with the NR roots. In contrast, the glucose content of TR roots exhibited a substantial reduction. In addition, salinity significantly elevated the glucose content of NR roots (Fig. 4B, P < 0.05). However, the glucose levels in both OR and TR roots remained unchanged with increasing salt level (Fig. 4B). The sucrose content in the roots of NR, OR, and TR seedlings decreased by 18.97%, 20.41%, and 80.69% under 150 mM NaCl, respectively, compared with salt‐free treatment. In addition, CR increased the sucrose content in the roots under both 0 and 150 mM NaCl; however, under 75 mM NaCl, the sucrose content of OR and TR roots decreased by 7.75% and 12.81%, respectively (Fig. 4C). The fructose contents in the roots of NR, OR, and TR seedlings decreased by 19.71%, 21.47%, and 28.28% under 150 mM NaCl, respectively, compared with salt‐free treatment. Moreover, CR reduced the fructose content, which was much lower in the TR seedling roots than in the other seedlings under salt stress. For example, compared with that in the NR roots under 150 mM salinity, the fructose content of TR roots decreased by 33.41% (Fig. 4D). The starch contents in the roots of NR, OR, and TR seedlings peaked at 75 mM NaCl and then declined at 150 mM NaCl. In addition, under each salt level, OR treatment increased the starch content, whereas TR treatment decreased it (Fig. 4E).

Two‐way ANOVA revealed that the SS and SPS activities were significantly affected by salinity, CR, and their interaction (Table S1). The SS activity in the roots of NR, OR, and TR seedlings declined with increasing salinity (Fig. 5A). For example, the SS activity of NR, OR, and TR roots significantly decreased by 64.42%, 67.66%, and 64.61% under 150 mM NaCl, respectively, compared with salt‐free treatment (P < 0.05). Moreover, CR significantly decreased the SS activity of OR and TR roots compared with the NR roots under 150 mM NaCl (P < 0.05). Similarly, the SPS activity in the roots decreased significantly with the degree of CR (Fig. 5B, P < 0.05). For instance, the SPS activity of TR roots declined by 63.32%, 18.32%, and 42.09% under 0, 75, and 150 mM NaCl, respectively, compared with NR roots. In addition, the SPS activity in the roots of NR seedlings decreased with salinity (by 48.32% and 42.45% under 75 and 150 mM, respectively); however, salinity had no significant effect on SPS activity of OR and TR roots (Fig. 5B).

Fig. 5.

Fig. 5

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SS activity (A) and SPS activity (B) in the root of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl. Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

Nitrogen metabolism

Two‐way ANOVA revealed that total nitrogen and nitrate nitrogen contents were significantly affected by salinity, CR, and their interaction (Table S1). CR significantly decreased the total nitrogen content in the roots under salt‐free condition (Fig. 6A, P < 0.05). For example, compared with NR roots, the total nitrogen content of TR roots declined by 50.20%. Under salt conditions (75 and 150 mM NaCl), the total nitrogen content in the roots of OR seedlings increased and then decreased in TR seedlings. Moreover, the total nitrogen content of NR roots declined with increasing salt concentration, while that of OR roots peaked under 75 mM NaCl and then declined under 150 mM NaCl. In contrast, the total nitrogen of TR roots maintained relatively stable with increasing salt level (Fig. 6A). Similarly, CR significantly decreased the nitrate nitrogen content in the roots under salt‐free condition (Fig. 6B, P < 0.05). In detail, compared with NR roots, the nitrate nitrogen content of TR roots declined by 33.90%. Under salt conditions (75 and 150 mM NaCl), the nitrate nitrogen content of OR roots increased and then decreased in TR roots. Additionally, the nitrate nitrogen content of NR roots decreased and then increased with salt level. For OR seedlings, the nitrate nitrogen content in the roots increased with salt level, while that of TR seedlings showed an initial increase followed by a decline (Fig. 6B).

Fig. 6.

Fig. 6

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Total nitrogen (A), nitrate nitrogen (B), GS activity (C), GOGAT activity (D), and NR activity (E) in the root of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl. Different lowercase letters represent significant differences among different salt levels within NR, OR, and TR seedlings, respectively (P < 0.05, ANOVA with LSD). Different uppercase letters above lines represent significant differences between NR, OR, and TR seedlings within one salt level (P < 0.05, t‐test).

Two‐way ANOVA revealed that the GS activity of castor seedlings was significantly affected by salt concentration, CR, and their interaction. The GOGAT activity was significantly influenced by salt concentration and CR, but was not affected by the interaction of these two factors. The NR activity was significantly affected by salt concentration and the interaction between salt concentration and CR, but was not influenced by CR (Table S1). The GS activity of NR, OR, and TR roots significantly decreased with increasing salinity (Fig. 6C, P < 0.05). Specifically, the GS activity in the roots of the NR, OR, and TR seedlings decreased by 23.19%, 16.51%, and 11.76% under 150 mM NaCl, respectively, compared with the salt‐free condition. Moreover, the GS activity of the TR roots was much lower than those of the NR roots at each salt concentration (0, 75, and 150 mM NaCl). For example, compared with NR roots, the GS activity of TR roots was decreased by 23.68%, 10.53%, and 12.32% under 0, 75, and 150 mM NaCl, respectively (Fig. 6C).

The GOGAT activity in the roots of NR, OR, and TR seedlings peaked at 75 mM NaCl and then fell at 150 mM NaCl. In addition, the GOGAT activity of TR roots was significantly lower than NR roots under 0 and 75 mM NaCl, with reductions of 14.10% and 17.79%, respectively (Fig. 6D, P < 0.05). Under the highest concentration of salt stress, the GOGAT activity of OR and TR roots also showed a decreasing trend compared with TR roots (Fig. 6D).

The NR activity in the roots of NR and OR seedlings peaked at 75 mM NaCl and then fell at 150 mM NaCl, while that of TR seedlings increased with salt concentration (Fig. 6E). In detail, the NR activity of TR roots increased significantly by 48.20% under 150 mM NaCl (P < 0.05). In addition, the NR activity in the roots of TR seedlings was lower than that in NR seedlings under 0 and 75 mM NaCl but increased significantly by 83.13% under 150 mM NaCl.

Principal component analysis (PCA) analysis

To comprehensively assess the integrated physiological responses of castor roots to CR and salt stress, PCA was performed using a set of physiological parameters. The first two principal components (PC1 and PC2) accounted for 47.1% and 15.4% of the total variance, respectively (Fig. 7). Clear separation among the treatment groups was observed along PC1, which reflected both the salt level (0, 75, and 150 mM NaCl) and the degree of CR (NR, OR, and TR). The NR0, OR0, and TR0 clusters were relatively close. However, the degree of CR diverged significantly with increasing salt concentration, with TR150 showing the most distinct separation along PC1. The APX and Na+ exhibited relatively high positive loadings on PC1, suggesting that they have a significant impact on the inter‐group separation along PC1, particularly with removal of two cotyledons and the 150 mM salt stress group, which are clustered on the right side of PC1. In contrast, other physiological parameters (such as nitrate nitrogen, total nitrogen, MDA, SOD, POD, CAT, GS, GOGAT, NR, sucrose, total carbon, fructose, glucose, starch, SS, SPS, DW, and root morphology) displayed higher negative loadings on PC1, indicating that these variables primarily drove the separation pattern along the left side of PC1.

Fig. 7.

Fig. 7

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Integrated physiological shifts in castor roots revealed by principal component analysis (PCA) under varying cotyledon removal and salt concentrations.

Variable loading vectors revealed that traits such as Na+ content, MDA, APX activity, and nitrate nitrogen were strongly associated with TR150. In contrast, traits such as total carbon, total nitrogen, root surface area, root volume, sucrose, and antioxidant enzymes (CAT and POD) were more closely associated with NR75 and OR75, highlighting their potential roles in mitigating salt stress when cotyledons were retained.

Collectively, the PCA results suggest that cotyledons play a critical role in the salt tolerance of castor roots via regulating ion homeostasis, antioxidant defence, and carbon–nitrogen metabolism.

DISCUSSION

The early seedling stage represents the most critical and vulnerable phase in plant development, particularly under saline condition. During this period, the root system functions as the primary organ for environmental perception and physiological adaptation. Our previous study has established that cotyledons are indispensable for the salt tolerance of the aerial parts in castor bean. Specifically, the presence of cotyledons was found to maintain the structural integrity of chloroplasts and alleviate the reduction in photosynthetic capability caused by stomatal closure under salt stress. Furthermore, cotyledons significantly counteracted Na+ toxicity and relieved osmotic stress in the emerging true leaves (Wang et al. 2019a, 2019b). However, previous studies have focused on the responses of intact plant or the application of exogenous substances (such as gibberellins and arbuscular mycorrhizal fungi) to salt stress (Zhang et al. 2018; Jiao et al. 2019; Wang et al. 2019a, 2019b). In contrast, the specific functional role of cotyledons which persist for up to 1 month during the seedling stage in regulating root development and salt stress responses remains poorly understood. Therefore, we conducted a CR experiment to investigate the differential root responses of NR, OR, and TR seedlings under varying salinity levels.

MDA, as a membrane lipid peroxidation product, is a widely used marker of oxidative damage and stress tolerance. Here, the MDA content in the roots of NR seedlings were positively correlated with salt level. Meanwhile, OR seedlings exhibited a response characterized by low concentration promotion and high concentration inhibition, whereas the MDA content in TR seedlings did not show a continuous increase with elevating salt concentrations. Salinity is known to disturb the balance between reactive oxygen species (ROS) production and antioxidant defence mechanisms, leading to oxidative stress. The antioxidant defence system scavenges excess ROS through enzymatic and non‐enzymatic mechanisms, thereby mitigating oxidative damage (Hasanuzzaman et al. 2021). For instance, Attia et al. (2021) reported that salt significantly increased the total phenol content by 60% and ascorbic acid (AsA) levels by 55% in Solanum lycopersicum plants, along with enhanced activities of antioxidant enzymes such as SOD, CAT, POD, and polyphenol oxidase (PPO). In this study, CR altered the antioxidant defence response of castor roots under salt stress, showing a trend similar to that of MDA content. This phenomenon may be attributed to the collapse of the antioxidant system under high salt concentrations and complete CR, where excessive ROS exceed the plant capacity for detoxification. As a result, there is no further increase in MDA content, and a marked decline in antioxidant enzyme activity is observed. PCA revealed that APX and MDA were closely associated with severe stress such as TR treatment, suggesting that complete CR increased oxidative pressure and disrupted redox homeostasis. In contrast, NR and OR roots maintained higher CAT and POD activities under salt condition, supporting more effective ROS scavenging. These results revealed the pivotal role of cotyledons in sustaining antioxidant capacity of castor roots during early seedling stage under salt stress, consistent with prior reports emphasizing cotyledon senescence in Phaseolus vulgaris is associated with increased oxidative stress (Procházková & Wilhelmová 2007).

Excessive Na+ accumulation poses a critical constraint on plant growth under salt stress. High Na+ content competitively inhibits K+ uptake through ionic antagonism, disrupting Na+/K+ homeostasis (Zeng et al. 2017). This ionic imbalance induces physiological dysfunction, including the displacement of essential divalent cations such as Ca2+ and Mg2+. Previous studies showed that salt addition increased Na+ concentration of castor plants (Zheng et al. 2021). Here, CR significantly impaired Na+/K+ homeostasis in castor roots. Specifically, the Na+ content in the roots of TR seedlings was elevated by 10.27‐fold, while the K+ content decreased by 70.9% under 150 mM NaCl, compared with salt‐free condition. The PCA further supported that CR aggravates ionic imbalance under salinity. In addition, the previous study highlights the role of cotyledons in mitigating ion toxicity induced by salt stress by ion compartmentalization during early seedling stage (Wang et al. 2019a, 2019b). The distinct separation of TR150 in PCA suggests that cotyledons are essential for preserving ionic homeostasis, thereby enhancing stress resilience during seedling establishment.

Carbon metabolism is fundamental metabolic processes that are essential for plant growth and development (Marino et al. 2006). SPS is a key regulatory enzyme in sucrose biosynthesis, whereas SS primarily catalyses sucrose degradation (Chen et al. 2018). Under stress conditions, plants activate adaptive regulatory mechanisms by modulating the activities of SPS and SS to adjust sucrose levels and facilitate carbohydrate redistribution in response to environmental challenges (Whittaker et al. 2007). Previous studies have shown that the SS and SPS activities of Tartary buckwheat were enhanced under low salinity stress, thereby promoting sucrose and soluble sugar accumulation to mitigate osmotic stress and maintain growth (Zhang et al. 2023). However, sustained or high salinity typically suppresses these enzyme activities, disrupting carbohydrate biosynthesis and transport, and ultimately impairing plant development (Wu et al. 2019). Consistent with these findings, our results showed that the SS and SPS activities in the roots were reduced under salinity condition, while CR further augmented this decline. Interestingly, the glucose content increased significantly in OR roots but decreased in TR roots. This change was consistent with the variation in root starch content, suggesting an accumulation of glucose that functions as a dynamic intermediate in starch synthesis and degradation. In addition, correlation analysis reveals that the activities of SS and SPS were both positively correlated with fructose levels in the roots (Fig. S1). The strong positive correlation between SS activity and fructose content directly supports the role of SS as a key enzyme responsible for sucrose degradation in sink tissues, with its activity largely determining the rate of fructose production. In contrast, the positive association between SPS activity and fructose may reflect an indirect effect, given that fructose‐6‐phosphate (the phosphorylated derivative of fructose) serves as a substrate for SPS. This suggests that cotyledons sustain key metabolic activities of castor roots under unfavourable conditions.

Nitrogen is an essential nutrient for plant growth, involved in the synthesis of various compounds such as proteins, nucleic acids, enzymes, and alkaloids (Arghavani et al. 2017). Adverse environmental conditions limit nitrate (NO3 −) absorption and movement, thereby suppressing assimilation and translocation processes. Here, the total nitrogen and nitrate nitrogen contents in the roots gradually decreased with increasing degrees of CR under salt‐free condition. However, under 75 mM and 150 mM NaCl, these nitrogen contents initially increased and then declined as CR intensified. This phenomenon may be attributed to the interactive effects between CR and salt stress on nitrogen metabolism in castor roots. In NR seedlings, total nitrogen and nitrate nitrogen levels generally decreased with increasing salt concentration, indicating that salt stress directly suppressed the root capacity for nitrogen uptake and assimilation. In contrast, the OR treatment exhibited higher total nitrogen and nitrate nitrogen contents under 75 mM NaCl, likely reflecting a compensatory mechanism triggered by moderate CR. However, this compensatory response was abolished under severe stress at 150 mM NaCl, resulting in a pronounced inhibition of nitrogen metabolism. Furthermore, in TR seedlings, total nitrogen levels remained consistently low across salt concentrations and showed no further change with increasing salinity, suggesting that the complete removal of cotyledons severely impaired the roots to sustain normal nitrogen metabolism. These results indicate that cotyledons play a positive role in promoting the accumulation of the total and nitrate nitrogen in the roots of castor seedlings. Additionally, environmental stress inhibits the activity of key enzymes involved in nitrogen assimilation, thereby impairing plant growth and development (Hossain et al. 2012; Park Williams et al. 2012). The GS/GOGAT pathway is responsible for catalysing NH4 + assimilation and serves as the primary route for nitrogen assimilation in most plants (Lopez‐Delacalle et al. 2020). Here, the result observed the GS activity of castor roots were decreased under salt stress, which aligns with previous findings in soybean (Zilli et al. 2008), cucumber (Shao et al. 2015), and Glycyrrhiza uralensis (Ma et al. 2023). These results indicate that the GS/GOGAT pathway of castor roots is significantly inhibited under salt stress conditions. Moreover, CR further intensified the inhibition of this pathway under salt stress. As the initial and rate limiting enzyme for nitrate reduction, NR activity exhibited a significance increase in TR roots under 150 mM NaCl, although the nitrate content was lower than at 75 mM NaCl. This suggests that the upregulation of NR activity under high salinity aimed to accelerate the reduction of limited nitrate into available forms to cope with the salt stress. However, CR restricted the activities of GS and GOGAT, thereby impairing the nitrogen assimilation pathway. Collectively, under salt stress, these findings suggest that cotyledons may regulate root nitrogen metabolism by sustaining GS/GOGAT pathway activity and enhancing nitrate uptake and assimilation efficiency, thereby contributing to nitrogen homeostasis of castor seedlings.

In conclusion, cotyledons enhance seedling root adaptation to salt stress in the early stage by maintaining antioxidant defence, stabilizing carbon and nitrogen metabolism, and ensuring ionic balance in the roots. However, this study also has certain limitations. The experiment was conducted in a greenhouse under controlled conditions, and whether the results can be extended to field conditions needs to be verified. In addition, only the early seedling stage of castor was studied, and the effects of CR on the growth and development of castor in the later stages (e.g., flowering and fruiting stages) are still unknown.

The critical dependency of root physiological resilience on cotyledon integrity has significant implications for castor cultivation, particularly in saline‐alkali lands. In field conditions, cotyledons are highly susceptible to damage from soil crusting (mechanical impedance during emergence), early‐stage pests, and diseases. Our findings suggest that loss of cotyledons during the first month of growth can significantly impair the adaptive capacity of the root system, resulting in disturbed ionic homeostasis and attenuated root metabolic activity. To minimize the impact of cotyledon loss in practical production, several agronomic strategies should be emphasized. Optimized sowing techniques, such as precision sowing and maintaining appropriate soil moisture, can effectively reduce soil surface crusting, thereby protecting the large, succulent cotyledons of castor from mechanical injury. Furthermore, in cases where cotyledon function is compromised, the application of exogenous substances, including signalling molecules, mineral nutrients, and plant growth regulators, can serve as an effective strategy to mitigate the physiological damage induced by cotyledon deficiency.

CONCLUSION

This study demonstrates that the root salt tolerance of castor seedlings stage is critically dependent on cotyledon integrity. CR altered root architecture and epidermal cells of castor seedlings under salt condition; specifically, under 150 mM NaCl, the TR treatment significantly reduced root length, surface area, volume, and forks by 76.03%, 67.23%, 60.00%, and 81.96%, respectively, accompanied by a marked decrease in root hair density and structural damage to epidermal cells. Furthermore, complete CR compromised the antioxidant defence system, as evidenced by a 57.80% reduction in MDA content and the suppression of key antioxidant enzyme activities, including APX and CAT, under 150 mM NaCl stress. In addition, complete CR significantly disrupted carbon and nitrogen metabolism in the castor roots (Fig. 8). In contrast, the preservation of cotyledons conferred salt tolerance in seedlings, mitigating growth retardation and metabolic imbalance under salt stress. These findings highlight the pivotal role of cotyledons in supporting root adaptation and seedling establishment under saline conditions.

Fig. 8.

Fig. 8

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Physiological roles of cotyledons in root adaptation to salt stress in castor seedlings. Radar plots show normalized responses of root morphological, ion homeostasis, antioxidant, and carbon–nitrogen metabolism indicators under different levels of cotyledon removal (NR, OR, TR) and salt stress (0, 75, and 150 mM NaCl). All parameters were standardized to the NR (removal of no cotyledon) treatment, and each reference variable was standardized by receiving a numerical value of 1.

AUTHOR CONTRIBUTIONS

XD wrote the manuscript. XD, RL, HL, LP, and YL did the experiments. SM analysed the data. SY and JL revised the grammar, and JL conceived the manuscript.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

Supporting information

Fig. S1. Correlation analysis of carbon metabolites and key enzymes in the roots of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl.

PLB-28-1614-s002.docx (159.2KB, docx)

Table S1. Two‐way analysis of variance (ANOVA) of the effects of salinity (S), cotyledon removal (CR) and their interactions on the growth, Na+ and K+ contents, and carbon and nitrogen metabolism of Ricinus communis seedlings.

PLB-28-1614-s001.docx (19.2KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the Natural Science Foundation of Heilongjiang Province (LH2024C036).

X. Deng and R. Lin contributed equally to this work.

Editor: B. Hu

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Correlation analysis of carbon metabolites and key enzymes in the roots of Ricinus communis under different treatments: removal of no cotyledon (NR) at 0, 75, and 150 mM NaCl; removal of one cotyledon (OR) at 0, 75, and 150 mM NaCl; removal of two cotyledons (TR) at 0, 75, and 150 mM NaCl.

PLB-28-1614-s002.docx (159.2KB, docx)

Table S1. Two‐way analysis of variance (ANOVA) of the effects of salinity (S), cotyledon removal (CR) and their interactions on the growth, Na+ and K+ contents, and carbon and nitrogen metabolism of Ricinus communis seedlings.

PLB-28-1614-s001.docx (19.2KB, docx)

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