Alginate Oligosaccharide (AOS) Promoted the Nutrient Uptake and Growth of Cucumber Seedlings Under Suboptimal Temperature Conditions

Under suboptimal temperature conditions, the application of AOS improved cucumber seedlings’ nutrient absorption and growth more efficiently than merely raising nutrient levels, as it enhanced root surface area, root vitality, and N metabolic enzyme activities.

Introduction

Cucumber (Cucumis sativus L.) is a widely cultivated horticultural crop that thrives at a temperature range of 22-32 ℃/15-18 ℃ (day/night). While winter- and early spring-grown cucumbers command higher market prices due to off-season supply, their cultivation in northern China’s solar greenhouses is severely limited by suboptimal temperature stress, a key limiting factor that impairs plant nutrient uptake. Suboptimal temperatures inhibit the expression of AKT1 (which encodes a K⁺ channel), reducing whole-plant K⁺ content and net K⁺ uptake in tomato. In cucumbers, suboptimal temperatures lead to decreased root growth, reduced root vitality, lower N metabolism enzyme activities, and downregulated expression of nitrate transporter genes, resulting in diminished NO^3--N uptake. An enhanced nutrient supply substantially promotes plant growth. Thus, farmers often overuse chemical fertilizers to increase out-of-season vegetable yields, a practice that is both costly and damaging to the environment.

Biostimulants are natural or synthetic substances which can improve plant growth, stress tolerance, or nutrient use efficiency. The supplementation of biostimulants in fertilization programs can enhance plant growth and crop yields while reducing fertilizer inputs. Alginate oligosaccharides (AOSs), a type of biostimulant, are produced through enzymatic or acid hydrolysis of alginate – a natural polysaccharide extracted from brown algae that consists of mannuronic acid and guluronic acid residues. AOSs are biodegradable and have the advantages of being environmentally friendly. Accumulating evidence has demonstrated the multifaceted physiological roles of AOSs, including the regulation of root and shoot morphogenesis, photosynthetic efficiency, and enhanced abiotic stress (drought, salinity, acid rain, and high temperature) tolerance. Application of AOSs under normal temperatures significantly increased tissue N, P, and K concentrations in multiple crop species. Therefore, it is interesting to investigate whether AOS can promote growth and nutrient acquisition in horticultural crops at suboptimal temperatures, as this could potentially reduce dependence on chemical fertilizers.

Materials and Methods

Experimental Materials

The cucumber seeds of Cv. Jinchun 4 were used in this study. Seeds were soaked in water at 55 ℃ for 15 min. When the water cooled to room temperature, seeds were transferred into a constant-temperature incubator and germinated at a temperature of 28 ℃ for 24 h. Germinated seeds were sowed into 72 holes seedling-raising plates filled with a mixture of coir-coconut, vermiculite, and perlite (2:1:1, v:v:v). Seedlings were watered with 0.5 strength of Hoagland solution and raised under natural temperature and light conditions in a solar greenhouse. Seedlings were then transplanted into pots containing coir-coconut when the first true leaves started to develop. After a 5-day rejuvenation period, the seedlings were transferred and acclimated in the climate chambers for 3 days under conditions of 25 ± 1 ℃/18 ± 1 ℃ (day/night) with a 12 h photoperiod (500 ± 20 μmol·m^-2·s^-1).

Experimental Design

Suboptimal temperatures were set at 15 ± 1 ℃ during the day and 8 ± 1 ℃ at night, with a 12 h photoperiod (400 ± 20 μmol·m^-2·s^-1). Cucumber seedlings were treated with 0.5×, 1.0×, and 1.5× strength of Hoagland solution without AOS (N0.5, N1, and N1.5), or with 30 mg·L^-1 AOS (A0.5, A1, and A1.5). Seedlings were irrigated every 3 days (100 mL per plant) and 5 times during the experiment. Sampling and measurements were conducted on day 18 after treatment. Each treatment consisted of 25 cucumber seedlings with three replicates.

Results

Effects of Different Nutrient Solution Concentrations with AOS on the Growth of Cucumber Seedlings Under Suboptimal Temperatures

Growth parameters were quantified to evaluate the growth response of cucumber seedlings to elevated nutrient supply with or without AOS under suboptimal temperature conditions. Compared to N0.5, only a limited number of measured parameters showed significant increases in N1.0 and N1.5 (Figure 1). When maintaining equivalent nutrient supply levels, AOS application improved all examined parameters to varying degrees compared to those that were non-AOS treated. Two-way ANOVA revealed that nutrient level (NL) alone significantly influenced only plant height, stem thickness, leaf area, and stem fresh weight (Figure 1). Moreover, AOS and the interaction between NL and AOS (NL × AOS) significantly affected all nine plant growth parameters measured.

As shown in Figure 2, elevated nutrient supply increased the measured root architecture parameters, consistent with its effects on plant growth traits. Moreover, AOS application significantly improved these parameters under suboptimal temperature conditions compared to non-AOS treatments. Two-way ANOVA revealed that both the AOS and the NL × AOS interaction significantly influenced all six root traits, while NL alone significantly affected root tip numbers, total root length, root forks, and root surface area (Figure 2).

Effects of AOS and Different Nutrient Solution Levels on the Chlorophyll Content and Pn of Cucumber Seedlings Under Suboptimal Temperatures

The contents of chlorophyll a (Chl. a), chlorophyll b (Chl. b), and Pn all showed increasing trends with elevated nutrient levels, particularly under AOS treatments, where the enhancement was more pronounced (Figure 3). Two-way ANOVA demonstrated that both AOS and the NL × AOS interaction had significant effects on the contents of Chl. a, Chl. b, and Pn, while NL significantly affected Chl. a content and Pn.

Effects of AOS and Different Nutrient Solution Levels on the Root Activity of Cucumber Seedlings Under Suboptimal Temperatures

Plants in A0.5 exhibited significantly higher root activity compared to those in N0.5, N1, and N1.5, while A1.5 provided an additional 19.97% enhancement compared to A0.5 (Figure 4). Statistical analysis revealed that root activity was significantly influenced by NL, AOS, and NL × AOS.

Effects of AOS and Different Nutrient Solution Levels on the N Metabolism Enzymes Activities in Cucumber Seedlings Under Suboptimal Temperatures

The results in Figure 5 showed that while elevated nutrient supply alone exerts only marginal effects on N metabolism enzyme activities, AOS treatment significantly enhances these enzymatic activities in cucumber seedlings. Statistical analysis revealed significant effects of AOS and the NL × AOS interaction on all four N metabolism enzymes in both leaves and roots. However, NL significantly affected the activities of GDH in leaf, as well as GOGAT, GDH, and GS in root.

Effects of AOS and Different Nutrient Solution Levels on N Content, Accumulation, and Distribution in Different Cucumber Organs Under Suboptimal Temperatures

As shown in Figure 6a,b, the N1.5 treatment significantly increased N content and accumulation in all examined tissues (root, stem, leaf) compared to N0.5, i.e., increasing nutrient supply had a positive effect on N content and accumulation in the roots, stems, and leaves of cucumber seedlings under suboptimal temperature conditions. Furthermore, AOS treatments amplified this effect, since the AOS treatments consistently resulted in higher N content and accumulation compared to non-AOS treatments. Two-way ANOVA analysis showed that NL, AOS, and NL × AOS all significantly affected tissue N content and plant N accumulation.

Neither NL and AOS, nor NL × AOS significantly affected distribution of N in cucumber seedlings (Figure 6c). The N distribution ratio in cucumber roots decreased slightly with increasing nutrient solution concentration, while leaf N distribution increased slightly without significance. However, AOS significantly enhanced N allocation to leaves, indicating that AOS application promoted N transport from roots to shoots, enhancing overall plant growth.

Correlation Analysis Between Different Traits

As shown in the correlation heat map (Figure 7), N content in root, stem, and leaf tissues exhibited significant positive correlations with root morphology indices, root activity, and N metabolism enzyme activities.

Discussion

Uptake rates of most essential mineral ions are regulated by specific demand-driven mechanisms that dynamically adjust nutrient acquisition to meet physiological requirements across growth rate. Under suboptimal temperature conditions, plant growth is inhibited and the NO^3- influx rate decreases, while exogenous application of 24-epibrassinolide and gibberellin (GA) promotes plant growth and enhances both NO^3- influx rate and N accumulation. In our study, elevated nutrient solution concentrations have positive effects on N, P, and K content and accumulation in all examined tissues (roots, stems, and leaves) of cucumber seedlings, meanwhile, the co-application of AOS further enhanced these nutrient parameters compared to nutrient elevation alone (Figure 6, Figures S1 and S2). This may be attributed to the suppressed growth and consequently lower nutrient demand of cucumber seedlings under suboptimal temperature stress, where increased nutrient supply shows limited efficacy in enhancing uptake. In contrast, AOS application can stimulate plant growth (Figure 1) and enhance nutrient demand in cucumber seedlings, which may improve nutrients acquisition through coordinated modulation of uptake systems and root growth.

Root architecture is of great importance for the ability of crops to absorb mineral nutrients. Additionally, the modification of root architecture represents an effective strategy to enhance nutrient use efficiency and crop yield. Suboptimal temperatures inhibited root growth in cucumbers by downregulating GA 20-oxidase and GA 3-oxidase genes (involved in active GA biosynthesis) while upregulating GA 2-oxidase genes (responsible for GA deactivation), ultimately decreasing bioactive GA4 levels. Previous studies indicated that AOS could elevate the levels of auxins and gibberellins in roots, stimulate cell division, and promote root elongation, thereby expanding the root absorption area and enhancing N uptake. In this study, the combined application of varying nutrient solution concentrations and AOS enhanced cucumber root architecture, significantly increasing root tip number, average root diameter, total root length, root volume, and root surface area. Additionally, these root morphological characteristics exhibited strong positive correlations with organ-level N content (Figure 7), which align with established theories connecting root system architecture to nutrient uptake capacity. Root activity is closely correlated with mineral nutrient uptake. Previous studies have reported that AOS increased not only the root absorption area but also root activity in Chinese cabbage. Our results also demonstrate that AOS, nutrient solution, and their synergistic interaction significantly enhanced root activity in cucumber seedlings (Figure 4). These findings suggest that AOS improves root vitality and architecture to facilitate more efficient nutrient uptake, thereby promoting plant growth under suboptimal temperature conditions.

Crop N use efficiency (NUE) is governed by four key physiological processes: uptake, transport, assimilation, and remobilization. The assimilation phase involves several critical enzymes, including NR, GS, GOGAT, and GDH. Among these, NR initiates inorganic N utilization, while the GS/GOGAT cycle drives the conversion of inorganic N to organic forms, ultimately determining NUE. Genetic evidence underscores the functional importance of these enzymes in NUE. In Arabidopsis, the nia1 nia2 double mutant retains merely 0.5% of WT NR activity and exhibits severe growth defects under nitrate supply. Similarly, the knockdown of OsNR2 in rice significantly reduces grain yield. GS and GOGAT encoding genes in roots are transcriptionally activated by NO^3- supply. The Arabidopsis gdh1 mutant shows impaired growth when supplemented with inorganic N. Increased N metabolism enzyme activities facilitate rapid N assimilation, promoting growth and development. The application of chitosan and chitosan oligosaccharides significantly enhanced activities of NR, GS, and GOGAT, which enables plants to efficiently convert nitrite into amino acids, facilitating N uptake. Moreover, someone showed that seaweed extract significantly increased the GS and GDH activities in leaves, and promoted the effective absorption and assimilation of N in sorghum plants under cadmium stress. Low-molecular-weight sodium alginate fragments produced by radiation degradation were also reported to enhance N metabolic enzyme activities in various crop species. Studies by others confirmed that AOS stimulates NR enzymatic activity via coordinated gene expression induction and post-transcriptional control mechanisms in wheat roots. They also reported that the Ca²⁺ metabolism inhibitors attenuated the promoting effects of AOS on NR and GS activities, as well as total N concentration in flowering Chinese cabbage, which suggested Ca²⁺ signaling participates in mediating AOS-triggered N metabolic regulation. In this study, AOS combined with nutrient solutions significantly enhanced NR, GOGAT, GDH, and GS activities in plants, and showing a positive correlation with stem N content.

Conclusions

Under suboptimal temperature conditions, increasing nutrient input promoted cucumber seedling growth. However, supplementing with AOS further enhanced root architecture, root activity, and N metabolism enzyme activities, resulting in superior growth promotion and nutrient uptake compared to raising nutrient input alone.

Dora Arouse is a new elicitor product based on AOS/ADO (Alginate-derived Oligosaccharide). This product is produced in full compliance with organic standards and obtained Ecocert in May 2023. (Use as raw material for fertilizer and amendment.) Compared to alginates, AOS exhibit enhanced biological activities due to their lower molecular weight and improved water solubility.

See more details of Plant Immune Elicitors Dora AOS (Alginate Oligosaccharide)