Chiang Mai Journal of Science

Enhanced Cold Hardiness in Pinus koraiensis Seedlings: Physiological Responses to Exogenous Organic Acids

1. INTRODUCTION

Spring cold snaps in the cold-temperate forests of Northeast Asia refer to a rapid temperature drop of 8–15°C within 24h, with the minimum temperature ranging from -5 to 4°C and the cold stress lasting for 3–7d, which are a key environmental stressor for plants, causing physiological and metabolic disorders. At low temperatures, plants undergo cell membrane lipid phase transitions and structural damage, as well as experiencing a rapid accumulation of reactive oxygen species (ROS). This leads to metabolic disruption, growth inhibition and even plant death [1,2]. P. koraiensis, a rare native tree species in Northeast China, plays an irreplaceable role in maintaining forest ecosystem stability and providing timber resources. However, P. koraiensis seedlings and young trees are highly sensitive to diurnal temperature fluctuations and frost events. Cold-induced increases in membrane permeability, oxidative damage and decreased photosynthetic efficiency severely limit the success of artificial afforestation [2,3]. Therefore, it is crucial to clarify the cold resistance regulation mechanisms of P. koraiensis seedlings and develop efficient and practical cold protection techniques for seedling cultivation.

Low-molecular-weight organic acids (LMWOAs) act as key active components in the decomposition of forest litter and the exudation of plant roots. They play a core role in regulating the adaptation of plant species to environmental stress [4,5]. Recent studies confirm that LMWOAs can alleviate cold, drought and heavy metal stress through multiple mechanisms, including chelating soil nutrients, activating antioxidant systems and stabilising cell membranes [6]. For example, applying exogenous citric acid significantly reduces membrane lipid peroxidation in Larix olgensis under cold stress by increasing the activity of antioxidant enzymes and the content of osmolytes [7]. Salicylic acid (SA), a well-studied phenolic organic acid, enhances cold resistance in species such as Fraxinus mandshurica and rice by regulating the expression of genes that respond to the cold [8]. At present, few studies have reported on SA-mediated cold hardiness in P. koraiensis; only a small number of studies have found that exogenous SA can slightly increase the superoxide dismutase (SOD) activity of P. koraiensis seedlings under low temperature, while its regulatory mechanism and optimal concentration remain unclear. However, existing research has significant limitations. Firstly, most studies have focused on angiosperms, with limited investigation into LMWOA regulatory mechanisms in conifers, particularly Pinus species. Secondly, research predominantly centres on SA-type organic acids, while the cold resistance effects of non-SA-type acids, such as oxalic acid, citric acid and malic acid, which are highly abundant in north-east forest litter, remain unclear. Thirdly, although transcriptome studies of P. koraiensis at low temperatures show the enrichment of antioxidant-related differentially expressed genes, the regulatory role of exogenous signalling molecules in these physiological processes remains unknown. Therefore, we hypothesize that exogenous oxalic acid (OA), citric acid (CA), and malic acid (MA) enhance the cold adaptation ability of P. koraiensis seedlings by regulating membrane stability and antioxidant metabolism. To test this, we applied foliar sprays of these acids within their natural concentration range found in cold-temperate forest litter leachates [6], then subjected the seedlings to simulated 4°C cold stress. Subsequently, we measured various core physiological indicators in the seedlings' needles, including cell membrane permeability and the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), as well as the content of malondialdehyde (MDA) and osmoregulatory substances (proline and soluble protein). The “nutrient activation-physiological regulation” synergistic mechanism in this study refers to the fact that exogenous organic acids can not only directly act as signal molecules to regulate physiological processes such as plant cell membrane stability and antioxidant enzyme activity, but also activate intracellular mineral nutrients (such as Fe and Mg) through chelation and acidification, providing a material basis for the synthesis of cold resistance-related functional proteins.

In this study, the foliar spraying method was adopted, as organic acids can be directly absorbed by leaves and participate in intracellular metabolic processes, without relying on the root system to absorb nutrients from the soil; organic acids in leaves can chelate mineral elements stored in cells (for example, Fe²⁺ is a key cofactor of CAT and POD), indirectly achieving 'nutrient activation', which is not necessarily related to fertilization. This study aims to explore the regulatory effects and optimal concentrations of non-SA organic acids on cold resistance of P. koraiensis seedlings, preliminarily verify their physiological regulatory pathways on cold hardiness, explicitly propose the "nutrient activation-physiological regulation" synergistic mechanism as a core scientific hypothesis, provide a theoretical basis for the study of non-SA organic acids regulating cold resistance in conifers, and offer technical references for optimizing cold resistance cultivation practices of P. koraiensis seedlings in northern cold regions.

2. MATERIALS AND METHODS

2.1 Study Material and Location

The experiment was conducted in the P. koraiensis nursery at the Liangshui National Nature Reserve (47°10′N, 128°53′E), which is managed by the Northeast Forestry University in Heilongjiang Province. Healthy, uniformly sized, pest-free, two-year-old P. koraiensis seedlings were selected for the experiment. Experimental soil was collected from the A1 horizon (humus layer) of a typical P. koraiensis plantation within the reserve. This soil was then air-dried for 7 days at room temperature, sieved to remove stones and roots, and thoroughly mixed to an initial moisture content of 18%. Uniform, circular plastic pots with a diameter of 18 cm and a height of 20 cm were filled with a consistent amount of the prepared, dark brown soil (approximately 2.5 L), leaving a headspace of 2–3 cm.

2.2 Experimental Design and Treatment

2.2.1 Seedlings transplantation and acclimation

The seedlings were transplanted on May 5–10, 2024. To avoid inter-individual competition and ensure data from independent individuals, each pot received one seedling. A thin layer of sand was spread over the soil to reduce evaporation. Pots were placed in the reserve's experimental nursery under natural light with a daily average temperature of 22±2℃ and relative humidity of 65±5%, received regular watering (200 mL distilled water per pot per week to maintain soil moisture at 15%-20%) and no fertilization, and were acclimated for two weeks.

2.2.2 Organic acid treatment

Following acclimatisation, the organic acid treatments began. Three LMWOAs were selected for this purpose: oxalic acid (OA), citric acid (CA) and malic acid (MA). Each acid was tested at six different concentrations: 0 mmol·L⁻¹ (CK solvent control), 0.2 mmol·L⁻¹, 1.0 mmol·L⁻¹, 5.0 mmol·L⁻¹, 25.0 mmol·L⁻¹, and 50.0 mmol·L⁻¹. To ensure solubility and pH stability, all acids were dissolved as their sodium salts (sodium oxalate, sodium citrate and sodium malate) in distilled water and the pH was adjusted to 6.0±0.2 using 0.1 mol·L⁻¹ NaOH or HCl. Using a handheld sprayer, the seedling needles were sprayed each evening uniformly (around 18:00) with a spray volume of 10 mL per seedling until runoff. Each concentration was applied daily for three consecutive days. Standard nursery management continued during spraying.

2.2.3 Cold stress treatment

Cold stress commenced approximately 2 h after the final organic acid spray. Seedlings (with pots) were transferred to a programmable artificial climate chamber (model: RXZ-300B, volume: 0.3m³, 10 seedlings were placed in each chamber with a spacing of 10cm between seedlings). The chamber temperature was decreased from 23°C (ambient) to 4°C at 2°C/h and held at 4°C for 12h under dark conditions (simulating night), with relative humidity (RH) controlled at 60-70%.

Control settings:

(1) Constant temperature control (CK): Maintained at 23 ± 2°C in the nursery and sprayed with distilled water only (no organic acid) during the treatment period.

(2) Low-temperature stress control (LT): Received the same 4°C cold stress treatment as the experimental groups and was sprayed with distilled water only (no organic acid) before stress. Low-temperature stress control (LT): Seedlings sprayed with distilled water only (no organic acid) and subjected to the same 4°C cold stress treatment as the experimental groups.

2.2.4 Recovery and sampling

After 12h of cold stress, the seedlings were returned to a room temperature of 23 ± 2°C for a 24h recovery period. Following this, five seedlings per treatment group (including controls) were randomly selected for analysis (n=5 biological replicates). Current-year healthy needles were collected and immediately frozen in liquid nitrogen before being stored at -80°C for subsequent physiological assays. Each treatment included 5 biological replicates, and 3 technical replicates were performed for each physiological indicator measurement.

2.3 Measured Indicators and Methods

The following physiological and biochemical indicators were measured in the needle samples using standard methods and three technical replicates per sample. Mature true needles (from positions 11–20) were used for all assays.

Relative electrolyte leakage (REL) was determined using a DDS-6700 conductivity meter (Shanghai Lei Magnetic, China). Fresh needle discs were soaked in distilled water for 24 h at room temperature to measure initial electrical conductivity, and then autoclaved at 121 °C for 30 min to measure final conductivity. Relative electrolyte leakage was calculated as the percentage ratio of initial conductivity to total conductivity [37].

Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) colorimetric method. Needle samples were homogenized in trichloroacetic acid solution, and the supernatant was mixed with TBA reagent and heated in a boiling water bath. The absorbance values were recorded at 532 nm and 600 nm, and MDA content was calculated using a standard extinction coefficient [38].

Proline (Pro) content was quantified using the acidic ninhydrin colorimetric method. Needle tissues were extracted with sulfosalicylic acid, and the supernatant was reacted with acidic ninhydrin reagent under boiling conditions. The reaction mixture was extracted with toluene, and absorbance was measured at 520 nm for proline quantification [39].

Soluble protein content was determined by the Coomassie brilliant blue G-250 staining method. Plant extracts were mixed with G-250 working solution, and absorbance was measured at 595 nm. Soluble protein concentration was calculated using a bovine serum albumin standard curve [40].

Chlorophyll content was determined by acetone extraction spectrophotometry. Needle samples were immersed in 80% acetone solution under dark conditions for pigment extraction. The absorbance values at 663 nm and 645 nm were recorded, and chlorophyll a, chlorophyll b, and total chlorophyll contents were calculated according to Arnon’s formulas [41].

Superoxide dismutase (SOD) activity was assayed using the nitro blue tetrazolium (NBT) photochemical reduction method. The reaction solution was illuminated, and the absorbance at 560 nm was recorded. One unit of SOD activity was defined as the amount of enzyme inhibiting 50% of NBT photoreduction [42].

Peroxidase (POD) activity was measured using the guaiacol colorimetric method. The absorbance change at 470 nm was continuously monitored to calculate POD activity based on the rate of guaiacol oxidation [43].

Catalase (CAT) activity was determined by ultraviolet absorption spectrophotometry. The decrease in absorbance at 240 nm caused by hydrogen peroxide decomposition was monitored to calculate CAT activity [44].

Ascorbate peroxidase (APX) activity was measured by ultraviolet absorption spectrophotometry. The decrease in absorbance at 290 nm resulting from ascorbic acid oxidation was recorded to evaluate APX activity [45].

Repeat the process 3 times each time.

2.4 Statistical Analysis

All data were statistically analysed using SPSS 26.0 software. One-way analysis of variance (ANOVA) was used to compare the differences among treatment groups, and Duncan's multiple range test was employed to determine the significance of differences at the p<0.05 level. All statistical tests were two-tailed. Data are presented as mean ± standard error (SE). Figures were plotted using Origin 2025 software, with error bars representing SE and lowercase letters (a/b/c) indicating significant differences among groups (p<0.05).

3. RESULTS AND DISCUSSION

3.1 Organic Acids Protect the Cell Membrane System of P. koraiensis Seedlings Under Cold Stress by Mitigating Membrane Lipid Peroxidation

Cold stress significantly induced membrane lipid peroxidation in P. koraiensis seedlings. Malondialdehyde (MDA) content increased by 39.5% to 8.2±0.5 μmol·g⁻¹FW compared to the normal temperature control (CK, 5.9±0.3 μmol·g⁻¹FW) (p<0.05). Concurrently, cell membrane permeability, as indicated by relative electrolyte leakage, increased slightly compared to the CK. The application of exogenous citric acid (CA), oxalic acid (OA) and malic acid (MA) alleviated this membrane damage. Changes in relative electrolyte leakage were highly consistent with MDA levels. As shown in Figure 1a, treatment with 5.0mmol·L⁻¹ CA significantly reduced MDA content by 62.6% to 3.1±0.2 μmol·g⁻¹FW (p<0.05) and relative electrolyte leakage decreased to its lowest value of 12.5% (12.5±1.2%) (p<0.01) compared to the cold stress control group. OA (5.0 mmol·L⁻¹) and MA (1.0 mmol·L⁻¹) exhibited a lesser effect, reducing MDA by 52.7% to 3.9±0.3 μmol·g⁻¹FW and 49.8% to 4.1±0.4 μmol·g⁻¹FW (p<0.05), respectively, and reducing relative electrolyte leakage by 35.8% and 32.1% (p<0.05) compared to the cold control group. Within the low concentration range (0.2–5.0 mmol/L), MDA content decreased significantly with increasing concentration. However, when the concentration exceeded a critical threshold (OA>5.0 mmol/L, CA/MA>1.0 mmol/L), the protective effect weakened. At concentrations ≥25 mmol/L, the protective effect disappeared (Figure 1b). Relative electrical conductivity reflects the integrity of the cell membrane structure, while MDA reflects the degree of membrane lipid peroxidation. Their responses to organic acid concentrations differ. The optimal concentration of oxalic acid is 5.0 mmol·L⁻¹ (at which the cell membrane structure is the most stable), whereas citric acid/malic acid can effectively inhibit membrane lipid peroxidation at 1.0 mmol·L⁻¹. Based on comprehensive membrane stability indicators, 5.0mmol·L⁻¹ is determined as the applicable critical concentration for the three organic acids. Within the effective concentration range (≤5.0 mmol/L), the protective efficacy of the three organic acids differed significantly and was ranked as follows: CA > OA > MA.

3.2 Organic Acids Significantly Enhance Antioxidant Enzyme Activities in P. koraiensis Seedling Needles Under Cold Stress

Under 4 °C cold stress, the activities of four pivotal antioxidant enzymes (SOD, POD, CAT, APX) in the needles of P. koraiensis seedlings exhibited differential responses. Exogenous organic acid application at concentrations ranging from 0.2 to 5.0 mmol·L⁻¹ significantly enhanced antioxidant enzyme activities, with the most significant outcomes observed at concentrations between 2.5% and 72.4%, relative to the low-temperature control (LT) group (p<0.05) (Figure 2). SOD activity showed a unimodal response to organic acid concentration, reaching a maximum at the concentration range of 1.0–5.0 mmol·L⁻¹. POD activity exhibited a marginal increase under low temperatures; however, the induction was found to be constrained. The application of organic acid treatment resulted in a significant enhancement of POD activity (p<0.01), with the 1.0 mmol·L⁻¹ treatment exhibiting the most pronounced increase (1.5-fold higher than the low-temperature control). In contrast, CAT activity exhibited a significant decline of 32.5% to 12.3±1.1 U·mg⁻¹prot under low-temperature conditions (p<0.05). A significant increase in CAT activity was observed in response to the organic acid treatment, as compared with the temperature control maintained at low levels (p<0.05), reaching a maximal level at 1.0 mmol·L⁻¹ (20.5±1.8 U·mg⁻¹prot); CAT activity at 5.0 mmol·L⁻¹ was slightly lower but still significantly higher than the LT group. APX activity exhibited a slight increase under low temperatures; however, the magnitude of this increase was limited. The level of activity in the majority of organic acid treatment groups was significantly higher than that observed in the low-temperature control group (p<0.05), with the 1.0mmol·L⁻¹ CA treatment demonstrating the most substantial increase (45.7% higher than LT group, p<0.01). Based on the calculation of the average activity of four antioxidant enzymes at their optimal concentrations, CA increased the average enzyme activity by 42.3%, which was significantly higher than that of MA (38.5%) and OA (31.2%) (p<0.05). The efficacy of the three organic acids in promoting the activities of these four enzymes followed the order: CA > MA > OA.

3.3 Organic Acids Promote the Accumulation of Osmotic Adjustment Substances in P. koraiensis Seedling Needles Under Cold Stress

After low-temperature treatment, the Proline (Pro) content in P. koraiensis seedling needles increased by 5.2% to 6.2±0.4 μg·g⁻¹FW compared to the CK control (5.9±0.3 μg·g⁻¹FW), indicating that the seedlings initiated an adaptive response involving Pro accumulation to mitigate low-temperature damage. The Pro content in all three organic acid treatments was higher than The LT group, with 5.0mmol·L⁻¹ concentration demonstrating the most significant promoting effect. The increases induced by OA, CA, and MA reached 21.5% to 7.5±0.5 μg·g⁻¹FW, 50.5% to 9.3±0.6 μg·g⁻¹FW, and 34.5% to 8.3±0.5 μg·g⁻¹FW, respectively. This result indicated that appropriate concentrations of organic acids positively regulate low-temperature-induced Pro accumulation, significantly enhancing seedling adaptability to periods of cold stress. In contrast, after 4°C low-temperature treatment, there was a 5.3% decrease in soluble protein (SP) content in the seedlings to 12.5±0.8 mg·g⁻¹FW. Treatment with OA, CA, and MA, within the 0.2-5.0mmol·L⁻¹ concentration range, generally resulted in a linear increase in SP content with increasing concentration, peaking at 5.0mmol·L⁻¹ (CA: 18.3±1.2 mg·g⁻¹FW, MA: 16.5±1.0 mg·g⁻¹FW, OA: 14.2±0.9 mg·g⁻¹FW). At higher concentrations, the SP content decreased but remained above the level observed in the cold control. In the context of low-temperature stress, the impact of the three organic acids on Pro and SP content in P. koraiensis seedling needles manifested a sequential pattern, with the following order observed: CA > MA > OA (Figure 3).

3.4 Organic Acids Effectively Maintain Photosynthetic Pigment Content in P. koraiensis Seedling Needles Under Cold Stress

Exposure to 4°C cold stress significantly damaged the photosynthetic pigment system of P. koraiensis seedlings (p<0.05), leading to decreased contents of chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoids (Car), and the Chl a/b ratio. The exogenous application of organic acids has been demonstrated to effectively alleviate this damage. The application of different concentrations of organic acid treatments resulted in a mitigated decline in chlorophyll content in seedlings. Comprehensive analysis showed that 5.0 mmol·L⁻¹ was the optimal concentration for most treatments, which was determined based on the comprehensive cold resistance indices of seedlings (cell membrane stability, antioxidant capacity, osmotic adjustment capacity) rather than a single photosynthetic pigment index. Although chlorophyll content was relatively high under the treatment with 25 mmol·L⁻¹ oxalic acid, the MDA content and relative electrical conductivity significantly increase at this concentration (p<0.05), posing a risk of cell membrane damage. Therefore, 5.0 mmol·L⁻¹ is more suitable for actual seedling cultivation. The protective effects of the three organic acids on photosynthetic pigments showed differences,and the recovery amplitude of total chlorophyll content and Chl a content under most concentration treatments was as follows: CA > MA > OA (Table 1).

4. DISCUSSION

4.1 Protective Effects of Organic Acids on the Cell Membrane System of P. koraiensis Seedling Needles Under Low Temperature

Cell membrane stability is a core indicator of plant cold resistance, which is usually evaluated by relative electrolyte leakage and MDA content[9]. Low temperature causes the membrane lipids of cell membranes to transform from a liquid crystalline state to a gel state, increasing membrane permeability; meanwhile, reactive oxygen species (ROS) accumulate in large quantities, attacking membrane lipid molecules and triggering peroxidation reactions, leading to an increase in MDA content. In a low-temperature environment, organic acids may help maintain membrane stability by regulating membrane lipid composition and fluidity. Under cold stress, the accumulation of organic acids (e.g., CA and MA) can alleviate membrane lipid peroxidation, thereby protecting the cell membrane[10]. Although LMWOAs are mainly distributed in the rhizosphere, foliar-applied LMWOAs can be rapidly absorbed by needle stomata and cuticle of P. koraiensis, and transported to the whole plant through the phloem; they act as signal molecules to activate the cold resistance pathway and chelate intracellular mineral elements to enhance antioxidant enzyme activity, which is a direct physiological regulation independent of rhizosphere nutrient activation.This study compared three non-salicylic acid (non-SA) low-molecular-weight organic acids, revealing a protective efficacy ranking of CA > MA > OA. Citric acid contains 3 carboxyl groups, and its ability to chelate metal ions and scavenge ROS is stronger than that of oxalic acid and malic acid, which contain 2 carboxyl groups. Additionally, citric acid is an intermediate product of the tricarboxylic acid cycle and can directly participate in cellular energy metabolism, providing ATP for the synthesis of cold-resistant substances[7]. This finding aligns with the mechanism proposed by Song et al. (2012) for Larix olgensis seedlings, where CA exhibited the strongest protection against low-temperature-induced membrane damage. While the optimal concentration for SA in previous studies is typically 0.1-8.0 mmol·L⁻¹ (e.g., Lin et al. 2004[11]; Lü et al. 2004[12]),the optimal concentration range for the three organic acids tested here was 1.0-5.0 mmol·L⁻¹, with higher concentrations (25-50 mmol·L⁻¹) diminishing the protective effect. When the concentration of organic acids is ≥25 mmol·L⁻¹, it will disrupt the intracellular pH homeostasis and inhibit the activity of membrane-bound enzymes; at the same time, excessive organic acids accumulate in the cytoplasm, causing osmotic stress and reducing the cold resistance of seedlings. This difference may stem from variations in chemical structure: CA, a tribasic acid with more carboxyl groups than dibasic acids OA and MA, may more effectively acidify the rhizosphere and activate nutrients like Fe and Mg[13],indirectly supporting membrane stability. In this study, foliar spraying was used, where organic acids can be directly absorbed by the epidermal cells of needles, maintain membrane lipid fluidity by regulating intracellular ion balance, and do not need to rely on the rhizosphere nutrient activation process. This conclusion has been verified by the research of Lin et al. [10] on citrus needles. Conversely, high concentrations might disrupt cellular pH balance and inhibit membrane protein activity[14]. When the concentration of organic acids is ≥25 mmol·L⁻¹, the cytoplasmic pH value deviates from the normal physiological range (5.5-7.0), the activity of membrane-bound ATPase decreases, transmembrane ion transport is blocked, and ultimately, cell membrane damage is aggravated. This finding provides a crucial concentration reference for the precise application of organic acids in forestry seedling cultivation under cold stress.

4.2 Regulation of Organic Acids on Antioxidant Enzyme Activity of P. koraiensis Seedling Needles Under Low Temperature

The response of antioxidant enzyme activities to low temperature shows species specificity in plants. Under low-temperature stress, the metabolism of reactive oxygen species (ROS) in plants becomes imbalanced, leading to the massive accumulation of ROS such as superoxide anions (O₂⁻) and hydrogen peroxide (H₂O₂), which directly attack biological macromolecules. SOD catalyzes the dismutation of O₂⁻ to form H₂O₂, while POD, CAT, and APX are responsible for scavenging H₂O₂, forming a synergistic defense network for ROS scavenging[15,16]. In this study, 4°C cold stress induced slight increases in SOD, POD, and APX activities but caused a significant decrease in CAT activity in P. koraiensis seedlings. This finding suggests that, while the seedlings intrinsic enzymatic system exhibits a partial response to cold stress, its capacity to scavenge H₂O₂ is inadequate. Exogenous organic acid treatment significantly enhanced the activities of all four antioxidant enzymes (SOD, POD, CAT, APX), with efficacy positively correlated with their nutrient activation potential (CA > MA > OA). Compared with SA, non-SA LMWOAs (e.g., citric acid) have a stronger regulatory effect on the antioxidant enzyme system of P. koraiensis seedlings, and the optimal concentration range (1.0–5.0 mmol·L⁻¹) is wider, which is more suitable for field application in cold regions of Northeast China. Although this study did not directly determine the content of mineral elements in needles, the activities of CAT and POD depend on Fe²⁺ as a cofactor, and the activity of SOD depends on Cu²⁺/Zn²⁺. Organic acids can chelate metal ions stored in cells, providing a guarantee for the conformational stability and functional performance of enzyme proteins, which is consistent with the "organic acid - metal ion - enzyme activity" regulatory model proposed by Kumari et al. [18].This consistency lends support to the findings of Song et al. (2008)[17] on enhanced P and Fe availability by organic acids in dark brown soil, as well as the mechanism proposed by Venugopalan, Visha, Kumari et al. (2022)[18,19] which implicates Fe as a key cofactor for CAT and POD, Furthermore, organic acids can act as signaling molecules to induce the transcriptional expression of SOD and APX genes, increasing the synthesis of enzyme proteins; at the same time, they provide ascorbic acid, a substrate for APX, to further enhance its efficiency in scavenging H₂O₂. This direct regulatory mechanism has been confirmed by Wang et al [20]. in their study on rapeseed. This finding suggests that organic acids may enhance cold resistance not only through direct signalling (e.g., Wang et al., 2024[20]) but also via an indirect "nutrient activation → enhanced enzyme synthesis" pathway. Based on the statistics of the average activities of the four antioxidant enzymes at their optimal concentrations, the average increase in enzyme activity in the citric acid treatment group was 42.3%, which was significantly higher than that in the malic acid group (38.5%) and the oxalic acid group (31.2%) (p<0.05). Although the increase in POD activity by malic acid was slightly higher than that by citric acid, its regulatory effect on the three key enzymes SOD, CAT, and APX was weaker than that of citric acid. Therefore, the comprehensive ranking is CA > MA > OA. The results of the present study stand in contrast to those obtained by Wu et al. (2002)[21], who found that high SA concentrations inhibited SOD and CAT in Frassinus mandshurica. In contrast, the three organic acids tested here showed no inhibition within 1.0–5.0 mmol·L⁻¹. The observed discrepancy may be attributable to enhanced tolerance of P. koraiensis to non-SA organic acids or variations in signalling pathways (e.g., promoting enzyme synthesis directly rather than inhibiting CAT to accumulate H₂O₂ signals). This provides a novel insight for the analysis of cold resistance signalling pathways unique to forest trees.

4.3 Effects of Organic Acids on Osmotic Adjustment Substances in P. koraiensis Seedling Needles Under Low Temperature

Proline (Pro) and soluble proteins (SP) have been identified as critical osmotic adjustment substances in response to low-temperature stress. Under low-temperature stress, cell dehydration can disrupt the stability of proteins and membrane structures. Proline (Pro) and soluble proteins (SP) can enhance the cold resistance of plants by reducing cell osmotic potential, maintaining cell turgor pressure, and protecting the conformation of biological macromolecules[22,23]. In this experiment, cold stress was found to increase Pro content in P. koraiensis seedlings. The application of organic acid treatment at 5.0 mmol·L⁻¹ has been shown to result in a substantial increase in Pro levels. In the present study, the 5.0 mmol·L⁻¹ citric acid treatment was found to have the most significant promoting effect on Pro accumulation, increasing it by 50.5% in comparison with the low-temperature control group. This was followed by malic acid (34.5%) and oxalic acid (21.5%).This finding is consistent with those of studies on alfalfa (Zhang Caixia, 2010[24]), blueberry (Zhang et al., 2020[25]), and Larix olgensis (Song et al., 2012[7]), which underscores the vital role of organic acids in improving osmotic adjustment capacity under cold stress in P. koraiensis. The molecular mechanism by which organic acids promote Pro accumulation is as follows: they upregulate the gene expression of the key enzyme for Pro synthesis (Δ¹-pyrroline-5-carboxylate synthetase, P5CS) and simultaneously inhibit the activity of the Pro-degrading enzyme (proline dehydrogenase, ProDH), thereby achieving the net accumulation of Pro[24]. The results demonstrate that low temperature led to decreased SP content, which is inconsistent with the findings reported in some studies (for example, watermelon (Citrullus lanatus) Lü et al., 2004[12]; alfalfa (Medicago sativa) Zhang, 2010[24]), but they are consistent with those of Song et al. (2012)[7]on conifers. This discrepancy may be attributable to differences in plant species and organic acids. Notably, the application of organic acid treatment resulted in a substantial augmentation of SP content, Within the concentration range of 0.2-5.0 mmol·L⁻¹, the SP content increased linearly with the increase in organic acid concentration, reaching a peak at 5.0 mmol·L⁻¹. The SP content in the citric acid treatment group was 46.4% higher than that in the low-temperature control group. This suggests that these treatments enhance the pool of protein-like osmotic adjustment substances and improve seedling cold adaptability[26,27]. Organic acids may promote the de novo synthesis of stress-responsive proteins by activating the transcription of ribosome-related genes, and the specific molecular mechanism of this process needs to be further verified by transcriptome sequencing. The involvement of de novo protein synthesis in this process requires further investigation.

4.4 Effects of Organic Acids on Chlorophyll Content of P. koraiensis Seedling Needles Under Low Temperature

Chlorophyll content directly influences photosynthetic capacity. Low-temperature stress reduces chlorophyll content through two pathways: first, it inhibits the activity of key enzymes in chlorophyll synthesis (such as δ-aminolevulinic acid dehydratase, ALAD), hindering the synthesis of chlorophyll precursor substances; second, it accelerates reactive oxygen species-mediated chlorophyll degradation, damaging the structure of photosynthetic pigments[28]. In this study, Low temperatures caused a decrease in chlorophyll content in P. koraiensis seedlings, and organic acid treatment significantly mitigated this decline. The mechanisms involved may include the following: (1) Organic acids participate in the metabolism of chlorophyll precursors (glutamic acid, α-ketoglutaric acid)[29]; (2) Organic acids significantly enhance the activity of multiple enzymes in plants [30], including nitrate reductase (NR) and glutamate synthase (GOGAT). These two enzymes are key rate-limiting enzymes for the synthesis of glutamate, a chlorophyll precursor, and their increased activity can directly promote chlorophyll biosynthesis[31]. Additionally, organic acids may promote chlorophyll biosynthesis by enhancing the activity of key enzymes involved in the chlorophyll synthesis pathway[31]. (3) Mineral elements are crucial for chlorophyll synthesis. Trace elements such as Fe, Mn, Cu, Zn, and Mg influence chlorophyll biosynthesis indirectly or directly, either by serving as enzyme cofactors or by directly contributing to chlorophyll molecule composition[31-34]. In this study, foliar spraying was adopted. Organic acids can promote the release of Mg²⁺ and Fe²⁺ stored in leaf vacuoles into the cytoplasm, and form soluble complexes through chelation, which are then transported to chloroplasts to participate in chlorophyll synthesis. This process does not rely on soil nutrient activation and has no necessary connection with fertilization measures. Organic acids enhance the activity and plant availability of soil Fe, Mn, Cu, Zn and Mg through mechanisms including acidification, solubilisation, chelation, redox reactions and reduced adsorption[35,36]. The specific modes of action are as follows: ① Acidification: reducing the pH value of the leaf apoplast and increasing the solubility of metal ions; ② Chelation: forming stable chelates with metal ions to prevent their precipitation and inactivation; ③ Reduction: reducing Fe³⁺ to Fe²⁺, which is more easily utilized by plants. In this study, AC demonstrated the most effective results in maintaining chlorophyll content, followed by malic acid and oxalic acid, consistent with their relative abilities to activate soil nutrients. Although the total chlorophyll content was the highest in the 25 mmol·L⁻¹ malic acid treatment group, the MDA content and relative electrical conductivity significantly increased at this concentration, resulting in damage to the cell membrane system. In contrast, although the chlorophyll content in the 5.0 mmol·L⁻¹ citric acid treatment group was slightly lower than that in the 25 mmol·L⁻¹ treatment group, it showed the best performance in terms of comprehensive indicators such as membrane stability and antioxidant capacity. Therefore, 5.0 mmol·L⁻¹ is recommended as the optimal concentration for field application. Notably, the protective effect of organic acids is exhibited to be concentration-dependent. Excessively high concentrations may negate their positive effects or even produce adverse impacts[12].

4.5 Practical Implications

Based on the research results, 5.0 mmol·L⁻¹ citric acid is recommended as a cold hardiness regulator for P. koraiensis seedlings in the cold-temperate forest region of Northeast China. Foliar spraying with 10 mL per seedling is suggested 3–5 d before the predicted spring cold snap, which can effectively improve the cold resistance of seedlings with the advantages of simple operation, low cost and easy popularization in field production. This method can be directly applied in the artificial afforestation and seedling raising of P. koraiensis in Heilongjiang and other cold regions. In addition, the mixed application of citric acid with other plant growth regulators (e.g., melatonin, uniconazole) can be further explored in future research to enhance the cold resistance effect of P. koraiensis seedlings, and the long-term effect of exogenous organic acid application on the growth and development of P. koraiensis seedlings also needs to be further verified by field trials.

4.6 Limitations and Future Research Directions

The current study has confirmed that exogenous non-SA LMWOAs can significantly enhance the cold hardiness of P. koraiensis seedlings by regulating cell membrane stability, antioxidant enzyme system, osmotic adjustment and photosynthetic pigment maintenance. However, the proposed "nutrient activation-physiological regulation" synergistic mechanism has not been directly verified due to the lack of quantitative data on foliar mineral nutrient elements, and remains a testable scientific hypothesis to be confirmed. To fully verify this synergistic mechanism and clarify its causal relationship, four levels of supplementary experiments are required in future research:

(1) Core quantitative verification of foliar mineral elements: Quantitative analysis of the total content, chemical speciation (available/unavailable forms) and subcellular distribution of key mineral elements (Mg, Fe, Cu, Zn, Mn) in pine needles under different LMWOA treatments, using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), sequential chemical extraction and differential centrifugation. This experiment will directly verify the "nutrient activation" effect of exogenous LMWOAs in leaf cells, which is the core premise of the synergistic mechanism.

(2) Causal relationship verification of the synergistic mechanism: Through metal ion chelation inhibition experiments (LMWOAs + Na₂-EDTA co-treatment) and key mineral element deficiency-rescue hydroponic experiments, to confirm that the cold resistance regulation effect of LMWOAs depends on their nutrient activation effect, and clarify the causal synergistic relationship between "nutrient activation" and "physiological regulation".

(3) Molecular mechanism verification of the synergistic mechanism: Using quantitative real-time PCR (qRT-PCR) to determine the expression levels of genes related to mineral element transport and assimilation, and Western Blot to quantify the protein expression levels of key antioxidant enzymes. This will reveal the molecular basis of the synergistic mechanism at transcriptional and translational levels.

(4) Field long-term effect verification: Setting up long-term positioning field experiments in the P. koraiensis nursery of Liangshui National Nature Reserve, to monitor the dynamic changes of foliar mineral nutrients, seedling growth and cold resistance-related physiological indicators throughout the growing season, and verify the stability of the synergistic mechanism under natural field conditions.

5. CONCLUSIONS

This study explored the regulatory effects and physiological mechanisms of exogenous non-salicylic acid (non-SA) low-molecular-weight organic acids (LMWOAs) on the cold hardiness of P. koraiensis seedlings under 4°C low-temperature stress. The results showed that 5.0 mmol·L⁻¹ was the optimal concentration of LMWOAs to improve the cold hardiness of P. koraiensis seedlings, with citric acid exhibiting the best regulatory effect, followed by malic acid and oxalic acid. Exogenous LMWOAs enhanced the cold hardiness of P. koraiensis seedlings mainly by mitigating membrane lipid peroxidation, activating the antioxidant enzyme system (SOD, POD, CAT, APX), promoting the accumulation of osmotic adjustment substances (proline and soluble protein) and maintaining the content of photosynthetic pigments. This study proposed and systematically discussed the "nutrient activation-physiological regulation" synergistic mechanism of non-SA LMWOAs regulating the cold hardiness of P. koraiensis seedlings as a testable scientific hypothesis, and provided a reliable theoretical basis and technical support for the cold hardiness cultivation of P. koraiensis seedlings in northern cold regions.

The phenotypic characteristics of P. koraiensis seedlings under different treatments are shown in Supplementary Materials Figure S1, and the temperature dynamic changes in the experimental process are shown in Supplementary Materials Figure S2.

6. ACKNOWLEDGEMENTS

The authors would like to thank Dr. Wang Yafei for his help in data processing and graphics production. We thank the Innovation Project of the State Key Laboratory of Tree Genetics and Breeding (TGBFRF202521) for its support. We would like to thank the Instrument Sharing Platform of State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University for providing instrument and equipment support, as well as Liangshui National Nature Reserve for their assistance in experimental sites and seedling management.

7. AUTHOR CONTRIBUTIONS

Chen Meixuan: Methodology, Investigation, Data curation, Formal analysis, Writing-original draft preparation. Sun Zhihu: Resources, Software, Validation. Sun Hailong: Visualization, Investigation, Data curation. Wang Yafei: Formal analysis, Validation, Writing-reviewing and editing. Chen Liming: Conceptualization, Supervision, Project administration, Funding acquisition, Writing-reviewing and editing.

8. CONFLICT OF INTEREST STATEMENT

The authors declare that they hold no competing interests.

9. DECLARATION OF GENERATIVE AI IN PREPARATION OF MANUSCRIPT

During the preparation of this work, the author(s) used ChatGPT-4 to improve the readability and language of the manuscript. After using this tool, the author(s) reviewed and edited the content as necessary and take(s) full responsibility for the publication's content.

10. FUNDING

This research was financially supported by the Innovation Project of the State Key Laboratory of Tree Genetics and Breeding (Grant Number TGBFRF202521).

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