Seasonality but Not Tree Age Determines Secondary Plant Metabolite Composition in Scots Pine Needles

doi:10.21203/rs.3.rs-3769558/v1

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Seasonality but Not Tree Age Determines Secondary Plant Metabolite Composition in Scots Pine Needles

https://doi.org/10.21203/rs.3.rs-3769558/v1

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Background and Aims:

Secondary plant metabolites, including organic acids and phenolic compounds, play key roles in plant physiology and response to environmental stressors. How the production of these compounds varies across tree age and seasonality are of considerable interest to understanding plant stress response.

Methods

Monthly, we collected fallen needles of Scots pine (Pinus sylvestris L.) across stands of five different age classes (20, 40, 60, 80, 100 years) for one year and measured organic acids and phenolic compounds.

Results

Seven low molecular weight organic acids and thirteen phenolic compounds were detected. No differences were observed across stand age. Significant seasonal differences were detected. Most compounds peaked during the growing season. Succinic acid was the most prevalent organic acid, comprising 78% of total organic acids (351.27 ± 34.27 ug g^− 1), and was 1.5 to 11.0 times greater in the summer than all other seasons. Sinapic acid was the most prevalent phenolic compound (42.15 µg g^− 1) representing 11% of total phenolic compounds and was 39.8 times greater in spring and summer compared to autumn and winter. Growing season peaks in needle concentrations were observed for all thirteen phenolic compounds and two organic acids (lactic, succinic). Citric acid exhibited a definitive peak in late winter into early spring.

Conclusion

Our results highlight the change in secondary metabolite production in response to seasonality, likely in response to UV-B radiation and the onset of the growing season. These compounds protect against environmental stressors through metabolic reactions within needles as trees acclimate to seasonal conditions.

Pinus sylvestris

litterfall

organic acids

phenolic compounds

Scots pine (Pinus sylvestris L.) is the most widespread species of the Pinus genus in the world (Eckenwalder 2009). It has been widely planted in the United States and occupies a range from Spain in the west to east Russia and from northern Scandinavia to southern Spain (Houston Durrant et al. 2016). In Poland, Scot's pine forests cover most of the country’s forest area (58.2%), constitute the largest share of forests managed by the State Forests (60.3%), and are slightly smaller in private forests (55.0%) (Bureau for Forest Management and Geodesy 2021). Scot's pine forests comprise over 20% of the productive forest area in Europe, exceeding 28 million hectares (Houston Durrant et al. 2016).

Organic acids and phenolic compounds are secondary metabolites that act as a carbon store while supporting physiological functions in plant metabolism, such as respiration, regulation of stomata, nutrient transport, regulation of plant and soil pH, and interaction with soil (Igamberdiev and Eprintsev 2016; Lehmann et al. 2016; Churakova et al. 2018). Phenolic compounds are the most diverse in function and structure of secondary metabolites. One or several aromatic rings containing one or several hydroxyl groups can be distinguished in their structure. Phenolic compounds play a role in physiological processes related to plant development and extension, including seed germination and cell division (Sharma et al. 2019; Kumar et al. 2019). Phenolic compounds play a significant role in plant resistance to various biotic and abiotic stress, defense mechanisms related to the antioxidative properties (e.g. scavenging and chelating ability) and play a structural function in the cell wall (Boudet 2007; Metsämuuronen and Sirén 2019). Moreover, for some classes of phenolic compound, the nutraceutical, clinical, antibacterial, and antifungal effects were observed (Venäläinen et al. 2004; Välimaa et al. 2007; Maimoona et al. 2011; Saxena 2015).

High-performance liquid chromatograph has been used to detect phenolic compounds in pine needles. Concentrations of organic acids have been observed to increase in needles with increasing temperature (Churakova et al. 2019) and in polluted soil environments (Magdziak et al. 2021), suggesting their role in needle maintenance and repair from heat stress.. However, our understanding of the variability in secondary metabolites in Scots pine needles in natural forest ecosystems is not well documented, nor do we have a clear understanding of the influence of tree age or season on this variation.

To address this knowledge gap we evaluated seven low molecular weight organic acids (citric acid, fumaric acid, lactic acid, malic acid, malonic acid, and succinic acid) and thirteen phenolic compounds (2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, caffeic acid, catechin, chlorogenic acid, ferulic acid, gallic acid, p-coumaric acid, protocatechuic acid, sinapic acid, syringic acid, t-cinnamic acid, and vanillic acid) in fallen pine needles (Pinus sylvestris L.) taken from five pine stands differing in age. The overall objective of the study was to determine differences in compounds with respect to tree age and seasonality of sampling. Results can be used to infer potential physiological and metabolic responses to age and season and to better understand which specific compound in needles could be dominating and play a specific role in “downstream metabolic processes.” The results of this study will contribute to a better understanding of the influence of the seasonal changes on the chemical composition of Scots pine needles, resulting in an in-depth knowledge of the species and possible future applications of needle-derived chemicals that would add economic importance in this multi-purpose crop.

2.1. Study site and litterfall sampling

Litterfall samples were collected in Scots pine stands in the Czarnobór Forest District in northwestern Poland during a field campaign reported in Ilek et al. (2021). Samples were collected across five forests differing in age (~ 20, 40, 60, 80, and 100 years old). Within each forest stand, one 100-meter-long transect was delineated and five litterfall traps were placed equidistant along each transect (25 litterfall traps in total). Pine needles were collected from the litterfall traps monthly for one year (12 sampling periods) in each stand, from April 2019 to March 2020. Pine needles were stored on ice and transported back to the lab where they were stored at 4℃ until chemical analyses. Characteristics of Scots pine stands, their location and information on the sampling date of needles have been described in detail by Ilek et al. (2021).

2.2. Extraction of organic acids and phenolic compounds from Scots pine needles

Litterfall samples were prepared for analysis of organic acids and phenolic compounds. For each monthly sample collection, approximately 2.5 g of needles were ground to powder in a mortar that was chilled using liquid nitrogen. Ground samples were collected in 50 mL centrifuge tubes and stored at -80°C until analysis according to the modified method of Sanità di Toppi et al. (2007) and Adeniji et al. (2010). For organic acids analysis, 5 mL of H₂O was added to the ground litterfall sample, then the mixture was heated in a water bath (60 min, 80°C) to denature the degradative enzymes. For phenolic compound analysis, ground litterfall samples were mixed with 80% methanol and HCl (99:1). Samples were sonicated and shaken for 5 hours at 40°C in a water bath (Bandelin Sonorex DL 102 H, Germany) and then shaken in orbital shakers for 8 hr (Ika KS 260 shaker, IKA-Werke GmbH & Co. Kg, Staufen, Germany). Aqueous and methanolic solutions of needle samples were centrifuged at 3600 rpm/min for 15 min at 25°C (Universal 320R Hettich Zentrifugen, Tuttlingen, Germany) then filtered through 0.2 µm nylon filters.

2.3. Chromatographic analysis

Organic acids and phenolic compounds were analyzed using a Waters Acquity H-class Ultra Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA). Separation was achieved on thermostated 35°C column Acquity UPLC BEH C₁₈ (150 mm × 2.1 mm, 1.7 µm, Waters, Milford, USA). Elution was performed with the mobile phase composed of A (water, containing 0.10% formic acid) and B (acetonitrile, containing 0.10% formic acid) at a flow rate of 0.4 mL/min. Detection was carried out at λ = 280 nm as the preferred wavelength for the following organic acids: acetic, citric, fumaric, lactic, malic, maleic, malonic, oxalic, quinic and succinic and phenolic compounds: gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, catechin, syringic acid, t-cinnamic acid) and at λ = 320 nm for the following phenolic compounds: caffeic acid, chlorogenic acid, sinapic acid, 2,5-dihydroxybenzoic acid, p-coumaric acid, ferulic acid) in a Waters Photodiode Array Detector (Waters Corporation, Milford, MA, USA). Five µL of liquor was injected into a UPLC BEH C18 column to determine organic compounds. Compounds were identified by comparing the retention time of the peaks with the retention time of standards or by adding a specific amount of the standard to the analyzed samples and repeated analysis. The results were expressed in micrograms per gram of fresh weight (f.w.) of needle samples [µg g^− 1 f.w.].

Statistical analysis

Statistical analysis and associated graphics were performed in Statistica 13.3 PL software (StatSoft Inc.). Data were checked for normality using the Shapiro-Wilk test and were found to be non-parametric. Therefore, we used Kruskal-Wallis and Dunn’s post hoc tests to identify significant differences in compound contents between stand age, sampling month, and season. Seasons were delineated following Ilek et al. (2021) where January-March are winter, April-June are spring, July-September are summer, and October-December are autumn. All tests were performed at a significance level of α = 0.05. Moreover, Spearman’s (non-parametric) correlation coefficient were computed for all measured compounds using the function “cor” in the “stats” package in R (R Core Team 2022) and plotted using the function “corrplot” in the “corrplot” package (Wei and Simko). All R analysis was done in version 4.2.2.

3.1. Organic acids content in Scots pine needles

Seven low molecular weight organic acids were detected in Scots pine needles. Among them, succinic acid was the most prevalent, comprising more than 78% of the total acid content at 351.27 ± 34.27 ug g^-1 (Table 1). Lactic, citric, and malic acids comprised 7%, 5%, and 5% of the total acid content found in needles, respectively, while malonic, fumaric, and acetic acids were found in very low concentrations. Stand age was not a significant predictor of organic acid content in needles (p > 0.05, Supplemental Fig. 1).

Table 1

Mean and standard deviation of organic acids and phenolic compounds in Scots pine needle samples. Values were computed across all ages of forest stands and across all months.
Organic Acid	Mean Content (ug g^-1)	Phenolic Compound	Mean Content (ug g^-1)
Succinic	351.27 ± 34.27	Sinapic Acid	49.18 ± 6.58
Lactic	33.28 ± 9.12	Gallic Acid	36.98 ± 2.42
Citric	23.51 ± 4.62	Syringic Acid	28.06 ± 4.50
Malic	23.06 ± 2.63	Vanillic Acid	18.69 ± 2.60
Malonic	9.30 ± 3.33	Catechin	15.49 ± 1.89
Fumaric	4.40 ± 0.25	Chlorogenic Acid	10.51 ± 0.99
Acetic	2.24 ± 0.50	4-hydroxybenzoic acid	8.64 ± 1.05
Total	447.07 ± 33.82	t-Cinnamic Acid	6.66 ± 0.79
		2,5-dihydroxybenzoic acid	5.94 ± 0.51
		Ferulic Acid	5.88 ± 0.61
		Caffeic Acid	5.24 ± 0.56
		Protocatechuic Acid	4.68 ± 0.54
		p-Coumaric Acid	3.89 ± 0.43
		Total	447.07 ± 33.82

In spring, the most prevalent organic acids present in needles were succinic acid (516.0 ± 81.7 µg g^− 1) followed by lactic acid (121.5 ± 33.5 µg g^− 1). Fumaric acid (5.2 ± 0.5 µg g^− 1) and acetic acid (2.0 ± 0.8 µg g^− 1) were observed in the smallest concentrations. Succinic acid was also the most prevalent organic acid in needles sampled during summer (752.5 ± 63.7 µg g^− 1) and autumn (88.8 ± 13.5µg g^− 1), while acetic acid, lactic acid, and malonic acid were all below detection levels. Citric acid was the most prevalent organic acid in needles sampled in winter (60.6 ± 15.5 µg g^− 1) followed by succinic acid (43.0 ± 10.5 µg g^− 1) and malic acid (31.9 ± 7.2 µg g^− 1). Malonic acid (2.8 ± 1.6 µg g^− 1) and fumaric acid (2.6 ± 0.4 µg g^− 1) were the least common organic acids found in winter needles.

Broadly speaking, needles collected in spring and summer had higher total organic acid content than needles collected in autumn or winter (Fig. 1a), reaching the highest monthly value in July (1287.29 µg g^− 1) (Supplemental Fig. 2a). Succinic acid content in needles was highest in summer (752.5 ± 63.7 µg g^− 1), which was 1.5 times greater than spring, and 11.5 times greater than autumn and winter (Fig. 3b). Succinic acid content in needles increased from January (41.84 ± 22.38 µg g^− 1), reaching its maximum in July (1266.30 ± 87.51 µg g^− 1), and then gradually decreased in the following months, reaching December, the lowest content (38.00 ± 9.35 µg g^− 1) (Supplemental Fig. 2b). Lactic acid needle content was highest in spring (121.47 ± 33.47 µg g^− 1), which was 10.5 times greater than needle concentrations in winter (Fig. 1c). Lactic acid was below detection limits in summer and autumn. The highest monthly concentrations of lactic acid were measured in March, April, May, and June (34.50 ± 9.75, 278.97 ± 79.76, 69.11 ± 36.94, and 16.34 ± 8.7 µg g^− 1, respectively) (Supplemental Fig. 2c). Citric acid content in needles was similarly high in both spring and winter seasons (30.28 ± 7.17 µg g^− 1), which was 39.5 times greater than values observed in summer and autumn (Fig. 1d). The largest monthly citric acid content was observed in needles collected in February (113.62 µg g^− 1) (Supplemental Fig. 2d). Fumaric acid content of needles collected in winter (2.6 ± 0.4 µg g^− 1) was 1.9 times lower than all other seasons (Fig. 1g). Malic acid content exhibited no seasonal trend but was intermittently higher in needles collected during transitional season months of February and March and September and October (Supplemental Fig. 2e). Malonic acid and acetic acid needle content exhibited no seasonal variation (Fig. 1e-h).

3.2. Phenolic compounds profile in Scots pine needles

Thirteen phenolic compounds were detected in Scots pine needles. The most dominant phenolic compounds were sinapic acid (42.15 µg g^− 1), gallic acid (36.93 µg g^− 1), and syringic acid (28.01 µg g^− 1) representing 11%, 8%, and 6% of the total phenolic compounds, respectively (Table 1). Mean needle content of vanillic acid (18.69 ± 2.60), catechin (15.49 ± 1.89), and chlorogenic acid (10.51 ± 0.99) all exceeded 10 µg g^− 1 while all other phenolic acids were very low, less than 2% of total phenolic compounds (Table 1). Stand age was not a significant predictor of phenolic compound content in needles (p > 0.05, Supplemental Fig. 2).

All phenolic compounds exhibited some degree of seasonal variation in needle content. In spring, sinapic acid was the most prevalent phenolic compound in needles at 129.4 ± 20.4 µg g^− 1, followed by gallic acid (49.6 ± 4.3 µg g^− 1) and vanillic acid (43.0 ± 9.0 µg g^− 1) while coumaric acid was the rarest phenolic compound (2.8 ± 0.5 µg g^− 1). Sinapic acid (62.1 ± 6.1 µg g^− 1) and gallic acid (48.2 ± 2.7 µg g^− 1) were also the most prevent compounds in needles sampled in summer, along with syringic acid (57.3 ± 6.9 µg g^− 1). Coumaric acid (8.6 ± 1.1 µg g^− 1) along with 4-hydroxybenzoic acid (7.6 ± 0.9 µg g^− 1), protocatechuic acid (7.5 ± 1.1 µg g^− 1), and ferulic acid (7.4 ± 0.9 µg g^− 1) occurred in the smallest concentrations in summer. Although found in smaller concentrations compared to other seasons, gallic acid (23.4 ± 3.5 µg g^− 1) and vanillic acid (10.8 ± 1.3 µg g^− 1) were most prevalent in autumn sampled needles while gallic acid (26.6 ± 6.6 µg g^− 1) and syringic acid (11.5 ± 1.9 µg g^− 1) were most prevalent in winter sampled needles. Protocatechuic acid was below detection thresholds across all aged stands in autumn. 2,5-DHBA (0.6 ± 0.2 µg g^− 1), t-cinnamic acid (1.4 ± 0.4 µg g^− 1), and ferulic acid (1.7 ± 0.3 µg g^− 1) exhibited the lowest concentrations in winter needles.

Needles sampled in spring and summer seasons had greater concentrations while needles sampled in autumn and winter seasons had lower concentrations. Caffeic and p-coumaric acid content were all significantly higher in needles sampled in summer compared to all other seasons (Fig. 2). Caffeic acid content of needles in summer (2.6 ± 0.5 µg g^− 1) was 1.8 times greater than caffeic acid in needles sampled in spring and 5.7 times greater than autumn and winter (Fig. 2b). p-coumaric acid content of needles was 4.2 times greater in the summer (8.6 ± 1.1 µg g^− 1) compared to the other seasons (Fig. 2c). 2,5-DHBA, protocatechuic, ferulic, t-cinnamic, chlorogenic, 4-hydroxybenzoic acid, catechin, gallic, syringic, and sinapic acid content in needles were all similarly higher in both the spring and summer seasons compared to lower average values in autumn and winter seasons. (Fig. 2). For needles sampled in summer and spring, their acid content of 2,5-DHBA (10.41 ± 1.0 µg g^− 1) was 10.0 times greater, protocatechuic (6.8 ± 1.1 µg g^− 1) was 3.4 times greater, ferulic (9.5 ± 1.4 µg g^− 1) was 4.2 times greater, t-cinnamic (10.3 ± 1.8 µg g^− 1) was 3.6 times greater, chlorogenic (14.7 ± 2.2 µg g^− 1) was 2.3 times greater, 4-hydroxybenzoic acid (13.1 ± 2.2 µg g^− 1) was 3.4 times greater, catechin (24.3 ± 5.1 µg g^− 1) was 3.2 times greater, gallic (48.9 ± 3.5 µg g^− 1) was 2.0 times greater, syringic (46.1 ± 5.3 µg g^− 1) was 4.5 times greater, and sinapic (95.8 ± 13.3 µg g^− 1) was 39.8 times greater compared to needles sampled in autumn and winter. Vanillic acid content of needles was the only phenolic compound that had similarly high levels throughout spring, summer, and autumn (21.7 ± 3.8 µg g^− 1) that was 2.3 times greater than vanillic acid content in needles sampled during the winter (Fig. 2l).

Spearman’s (non-parametric) correlation coefficients were calculated and are shown in the (Fig. 3). Correlations were stronger between phenolic compounds compared to organic acids. Within the phenolic compounds, the strongest positive correlations were between sinapic acid and syringic acid (r = 0.70, p < 0.001), gallic acid and syringic acid (r = 0.68, p < 0.001), and ferulic acid and sinapic acid (r = 0.66, p < 0.001). 4-hydroxybenzoic acid was positively correlated with every other phenolic compound while coumaric acid was only correlated with seven of the twelve other phenolic compounds. Correlations among organic acids and between organic acids and phenolic compounds were less common. Among organic acids, citric acid and malonic acid had the strongest positive correlation (r = 0.38, p < 0.001), followed by acetic acid and malic acid (r = 0.35, p < 0.001), citric acid and malic acid (r = 0.30, p < 0.001), and acetic acid and citric acid (r = 0.23, p < 0.001). There were also negative correlations among organic acids. Succinic acid was negatively correlated with both lactic acid (r = -0.48, p = 0.013) and citric acid (r = -0.45, p = 0.003). Citric acid was also negatively correlated with fumaric acid (r = -0.25, p = 0.004) and fumaric acid was negatively correlated with malic acid (r = -0.37, p < 0.001). Lactic acid was correlated, both positively and negatively, with nine phenolic compounds, exhibiting the strongest correlations with protocatechuic acid (r = 0.30, p < 0.001). Fumaric acid exhibited the strongest positive correlation with the phenolic acid gallic acid (r = 0.33, p < 0.001). Citric acid exhibited the strongest negative correlation with the phenolic acid t-cinnamic (r = -0.36, p = 0.022). Acetic acid was the only organic acid not correlated with any phenolic compounds.

In this study, we evaluated differences in organic acid and phenolic compounds in Scots pine needle leaves collected monthly across stands of different ages. We found that both organic acid and phenolic compounds in pine needles varied monthly and seasonally but did not vary across stand ages. Summed together, organic acid needle content was highest in spring and summer and declined in autumn and winter, which was largely driven by succinic acid. In contrast, citric acid formation was highest in winter and spring. Temporal trends of phenolic compounds were similar, with peak values during growing season months. However, phenolic compounds occurred more evenly, with several compounds (sinapic acid, gallic acid, syringic acid, and vanillic acid) occurring in near equal proportions.

Physiological functions of plants including respiration, osmotic regulation, transport of nutrients, and pH regulation in plant cells and the rhizosphere require the presence of carbon compounds in the form of sugars, organic acids, phenolic compounds, and amino acids (Desalme et al. 2017; Churakova et al. 2018). These organic acids are essential molecules secreted by plant roots, taking part in processes such as the detoxification of heavy metals, adaptation to the environment, and facilitation of nutrient uptake. Essential physiological functions of organic acids ensure redox balance, support ionic gradients on membranes, and acidify the extracellular environment (Igamberdiev and Eprintsev 2016). Furthermore, the accumulation, biosynthesis, transport, and secretion of organic acids in the rhizosphere increase in response to environmental stress (López-Bucio et al. 2000). The most frequently occurring acids in plants (roots, bark, leaves/needles) are citric, malic, succinic, and fumaric acids. Citric acid, depending on the intensity of reactions related to the conversion of this compound (Igamberdiev and Eprintsev 2016), is necessary for the acquisition and transport of iron in the plant (Meyer et al. 2010).

Organic acids play an important role in other plant organs where they are produced in mitochondria and stored in vacuoles (Winter and Smith 1996; López-Bucio et al. 2000; Black and Osmond 2003). Here these molecules are involved in the processes of anabolic and catabolic metabolic pathways, which are necessary for primary metabolism, stomatal regulation, and storage of bound carbon compounds (Hettmann et al. 2005; Lehmann et al. 2016; Churakova et al. 2018). Succinic acid has a powerful antioxidant effect (Chen et al. 2015) and is essential to the citric acid cycle and helps produce respiration energy (Steuer et al. 2007; Khan et al. 2020). Interestingly, we found a strong negative relationship between succinic and citric acids, which may imply that they are produced in greatest concentrations in opposite seasons, alternating when each is near its peak. Malic acid is involved in the transfer of redox equivalents between cellular compartments, playing the role of osmolyte and anion in compensating the positive charge of potassium, which is particularly important in stomatal responses (Meyer et al. 2010; Geigenberger and Fernie 2014; Maurino and Engqvist 2015). Citric, succinic, and fumaric acids are the main components of the tricarboxylic acid pathway. Lactic acid is a signaling molecule, a precursor of glucose production, and a source of energy for mitochondria (Gilliland 1990). It has also been shown that lactic acid fermentation under the influence of bacteria significantly affects plants' total content of phenols. However, the biochemical production of these compounds depends on plant species, environmental conditions (e.g. drought, presence of metals), stage of plant development, and age (Hettmann et al. 2005). As such, understanding these relationships, especially in field settings, is lacking and requires further investigation.

Our research identified differences in the seasonal variability of individual organic acids in needles of Scots pine at a compound-specific level. Luethy-Krause et al. (1990) showed that malic acid has a clear seasonal tendency, with concentrations being low in summer and high in winter, as was observed in our study. In turn, Churakova et al. (2018) observed citric and malic acids decreasing from June to October, at which point the lowest concentration of these acids was observed. We also observed a decline in citric acid throughout the growing season. The general increase in citric and malic acid during winter provides equivalent reductions (Finkemeier and Sweetlove 2009) and is associated with increased metabolic activity at the onset of the growing season (Luethy-Krause et al. 1990). Citrate can also be used as a carbon-transporting metabolite transporting carbon to sinks such as roots, facilitating the transport of nutrients such as iron towards the needles (Igamberdiev and Eprintsev 2016). Confirmed relationships, where seasonal variations in the content of organic acids (especially malic and citric acids) are observed, suggest increased investment in maintenance and repair mechanisms. Therefore, the seasonal cycle of organic acids can be considered an indicator of modified carbon metabolism in leaves and possibly in other tree tissues (Churakova et al. 2018, 2019). Succinic acid and fumaric acid were the only two organic acids that peaked in summer months in our study. Succinic acid primarily normalizes cell metabolism and supports the formation of new ones, so the observed increase aligns temporally when the elongated needle process was most intense.

Needle phenolic compounds have also been documented in other studies evaluating Scots pine (Oleszek et al. 2002; Häggman et al. 2009; Antonova et al. 2011). The composition of phenolic compounds in trees is related to genetic and environmental parameters, including species, age, tissue type, and environmental conditions (Willför et al. 2003; Inderjit and Duke 2003; Venäläinen et al. 2004; Külheim et al. 2011; Šežiene et al. 2017; Smeds et al. 2018). Karapandzova et al. (2015) identified different classes of phenolic compounds in four species of Pinus (P. peuce, P. nigra, P. mugo and P. sylvestris), including flavonoid glycosides, phenolic acids, and procyanidins. In P. halepensis needles, the dominant phenolic compound was protocatechuic acid, which was only observed in very low concentrations in our study needles. Häggman et al. (2009) noted that catechin, phenolic glycosides, and their derivatives were the main compounds in different tissues of Scots pine.

Exposure to UV-B radiation generates reactive oxygen species, which affect DNA and damage protein. In response, plants synthesize phenolic compounds to protect tissue and regulate the antioxidant mechanisms at cellular and whole-organism levels (Daayf and Lattanzio 2009; Sembi et al. 2019; Neugart et al. 2021). In Poland where our study was conducted, UV radiation is the lowest from November to March (autumn, winter). The sharp increase in most of the phenolic compounds in needles coincided with the increase in UV-B radiation and the onset of the growing season. During summer, the synthesis of phenols stabilized and decreased in the autumn and winter. Considering a more detailed analysis of seasonal changes, the spring surge of most of the compounds analyzed was followed by a decrease in their content and another spike. Such changes may result from changes in the amount of radiation and periods of drought in the summer. Production of phenolic acids to repair cell damage from air pollutants (ozone, sulfur dioxide, nitrogen dioxide) during summer months may also occur (Pasqualini et al. 2003). Additionally, catechin has been shown to increase under elevated CO₂ treatments, where extra carbon from enhanced photosynthesis is available for the production of secondary metabolites (Booker and Maier 2001). Phenolic acids also have strong antibacterial and antimicrobial activity (Puupponen-Pimiä et al. 2001; Naz et al. 2007; Silva et al. 2010; Masek et al. 2016). Ferulic, protocatechuic, and coumaric acids have antifungal activity that inhibits pathogen growth during the growing season (Ganthaler et al. 2017) (Perradin et al. 1983). In total, we did not observe direct relationships between phenolic compounds and tree age, instead our results suggest that Scots pine produce compounds more broadly in response to seasonal physiology and environmental stressors.

We observed strong correlations between many of the phenolic compounds that were not present among organic acids. This is likely due to shared biosynthesis pathways in which the compounds are produced. For example, the strong correlation found between sinapic and syringic acids may be attributed to the fact they are derivatives of the same phenylpropanoid pathway that involves the conversion of phenylalanine into cinnamic acid, which in turn is converted into sinapic and syringic acids (Dixon et al. 2002). Furthermore, correlations between phenolic compounds and organic acids are likely related to their shared response mechanisms and their roles in stress-related defenses.

Results of this study inform our understanding of secondary metabolite production in a common tree European tree species. Most notably, we observed high concentrations of compounds in spring and summer, regardless of age. Future research could leverage findings from our study to contribute to the valorization of Scots pine as a source of natural antioxidants and find applications for its components in nutraceuticals and cosmetics to prevent damage caused by oxidative stress in the human body. Our results may also inform watershed management. Ilek et al. (2021) showed that needle hydrologic and nutrient properties followed a similar trend, whereby needle wettability, carbon, and nitrogen content increased in the growing season across all ages of Scots pine trees. However, further investigation is necessary across a larger geographic distribution to determine the full extent of the trends in organic acid and phenolic compound production in Scots pine needles.

Our study offers valuable insights into the seasonal dynamics of organic acids and phenolic compounds within Scots pine needles across stands of varying ages. We found distinctive variations in organic acid and phenolic compound concentrations over time but did not observe differences across stand ages. The seasonal patterns revealed higher levels of organic acids, notably succinic and fumaric acids during the summer months, coinciding with increased metabolic activity and needle elongation. Conversely, citric and malic acids showed a peak in winter months and declined during the growing season, potentially indicating altered carbon metabolism and increased investment in maintenance and repair mechanisms. Furthermore, our investigation highlighted the change in phenolic compounds throughout the seasons, likely in response to UV-B radiation and the onset of the growing season. These compounds, known for their antioxidant properties, serve as protective agents against environmental stressors and potentially harmful air pollutants.

While our study contributes to comprehending the secondary metabolite dynamics in this common European tree species, further research across diverse geographical locations is necessary to develop a comprehensive understanding of the trends observed in organic acid and phenolic compound production. Such future investigations can enhance our understanding of these compounds' potential applications and their role in environmental adaptation and stress response in trees.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Gąsecka – conceptualization, methodology, formal analysis, investigation, writing – original draft, writing – review & editing. Ilek – conceptualization, methodology, formal analysis, investigation, writing – review & editing. Magdziak – conceptualization, methodology, formal analysis, investigation, writing – original draft, writing – review & editing. Saitanis – conceptualization, methodology, formal analysis, investigation, writing – review & editing. Siegert – writing – review & editing.

Data availability

The datasets generated during and/or analyzed during the current study are available as electronic supplementary materials.

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Seasonality but Not Tree Age Determines Secondary Plant Metabolite Composition in Scots Pine Needles

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Abstract

Background and Aims:

Methods

Results

Conclusion

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1. Introduction

2. Materials and methods

2.1. Study site and litterfall sampling

2.2. Extraction of organic acids and phenolic compounds from Scots pine needles

2.3. Chromatographic analysis

3. Results

3.1. Organic acids content in Scots pine needles

3.2. Phenolic compounds profile in Scots pine needles

4. Discussion

5. Conclusions

Declarations

References

Supplementary Files

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