Source to Sink of Lignin Phenols in a Subtropical Forest of Southwest China

Abstract

In biodiverse forest ecosystems, plant diversity has been reported to increase plant-derived lignin accumulation and soil organic carbon (SOC) storage. However, little is known about the fate of lignin and its degradation dynamics from plant to soil. This process is critical for the formation of SOC, especially in natural forest ecosystems with diverse plant species. This study presents the lignin biomarker characteristics of several common plant species and in mixed litter. The study was conducted in 45 plots along a plant species diversity gradient in a subtropical forest located in southwest China. Our results demonstrate that lignin content and its biochemical characteristics in plant leaves vary among species, while different plant species also alter the content of lignin and its monomeric compounds in the litter. Lignin compounds are gradually disintegrated from plant leaf to litter and then to soil, further indicating that plant-derived lignin from plant sources contributes to the formation and accumulation of forest SOC. These findings provide novel information on the linkage between tree species diversity and lignin accumulation while indicating the role of plant-derived lignin on SOC storage. These results may be useful in predicting forest soil C dynamics in Earth system models.

1. Introduction

Terrestrial ecosystems, especially forest soil C pools, impact global climate change by driving global C sequestration [1,2,3]. Previous studies have found that karst forests of Southwest China have a strong carbon sink effect [4,5]. Soil organic C (SOC) plays a key role in the dynamics of the global C cycle as an important component of the C pool of terrestrial ecosystems. The focus of soil C sequestration research is SOC formation pathways and stabilization mechanisms [6]. SOC is a complex system composed of identifiable biomolecules, which are formed mainly from plant and microbial sources, including plant residues, microorganisms (e.g., necromass, metabolites, and secretions), and animal residues [7,8]. It is generally believed that the forest vegetation type and the amount of plant litter affects forest SOC composition and storage. Meanwhile, plant diversity includes the species richness and functional group richness of plants. Plant diversity plays an important role in maintaining a normal ecosystem function and strongly influences the diversity of forest litter and soil microorganisms, which in turn affects SOC stocks. Forest SOC formation originates through a plant-regulated pathway, which in part demonstrates that high plant production may promote the transformation of plant-derived organic matter (OM) inputs and further enhance SOC accumulation [9,10,11,12]. Therefore, exploring the linkage of plants and SOC in forest ecosystems can help to better understand the dynamics of the soil C cycle under global biodiversity change.

SOC is a general term for all types of organic compounds in soil in various states of degradation. The sources of SOC mainly include plants (above-ground litter and underground root secretions) and microorganisms (residues, metabolites, and extracellular secretions) [6]. The sources and composition of SOC are very complex and, from different biological (i.e., plant and microbial) sources, vary widely in terms of chemical stability, biodegradability, and response to environmental change. Lignin represents typical plant-derived biomarkers in the soil environment, and its distribution and degradation characteristics can indicate the mechanisms of plant-derived C sequestration and environmental regulation, which can help distinguish the relative contributions of microorganisms and plants to SOC and their different responses to environmental changes and provide a theoretical basis for understanding the dynamic processes of soil C in the context of global changes. Lignin phenols have traditionally been considered critical SOC components and the major source of a stable SOC pool due to their chemical recalcitrance and structural complexity [13,14]. As a complex biopolymer of terrestrial vascular plants, the basic monomers of lignin can be used to characterize angiosperms and gymnosperms. Their fate in soils commonly represents the transformation of plant-derived OM, especially in forest ecosystems [13].

Due to the complex structure and phenol-based network of the lignin macromolecules, a chemical analysis of lignin is challenging [13]. We can distinguish three main types of single-ring lignin monomers (i.e., V-, vanillyls; S-, syringyls; and C-, cinnamyls) based on CuO oxidation method. Although the method has some shortcomings for the quantification of lignin, the analysis of the products has proved to be a valuable approach to the different chemical structures of lignin and the plant types from which lignin originates [15]. Recently, emerging evidence has confirmed that lignin compounds are also important in the context of plant–soil–microorganism interactions. Although lignin is a complex biopolymer of terrestrial vascular plants, it is an easily used C source for soil microbes as its biodegradation is faster than previously thought [15,16,17]. The degree of lignin degradation in the soil is determined by the vegetation type and the molecular chemical structure of lignin monomers in different litter materials. These different types of lignin present differences in their stability at the initial stage of entry into the soil [15], and their fate in soils commonly presents transformational signatures of plant-derived OM [9,14,18,19]. The aldehyde, ketone, and acid substitutions of these three types of phenols are generally used to indicate the degree of lignin decomposition and oxidation status. For instance, the acid/aldehyde ratio (Ad/Al) of the vanillyl and syringyl increases as lignin gradually degrades in plant litter [20]. (Ad/Al)_V&S in fresh vegetation generally ranges from 0.1 to 0.3, while in soils, the measured lignin (Ad/Al)_V ratio is greater than 0.6, indicating a high degree of biodegradation or transformation of lignin [17,21]. In addition, the more V-type phenols that are present, the more stable the lignin is, while C-type phenols are preferentially degraded. The ratios C/V and S/V decrease to a certain extent as degradation progresses [13]. Studies have also reported that the characteristics of lignin degradation vary significantly between regions and types of plants, as well as plant tissues [14,22,23]. The stability and biodegradability of lignin from plant leaf to litter during decomposition and transfer into the soil is a topic of much debate in the field of soil biogeochemistry [21,24]. It remains unclear how lignin phenol content and its oxidation status change during the decomposition from plant to soil. This information gap constrains our ability to precisely predict soil C dynamics in biogeochemical cycles.

Karst ecosystems are an important component of surface ecosystems, accounting for about 15% of the global land area. Karst ecosystems are even more prevalent in China, accounting for about 36% of the Chinese land area [25]. The southwest region hosts one of the three major concentrated and contiguous distribution areas of karst ecosystems worldwide. Karst forest ecosystems have a series of special characteristics. Their special topography with complex and diverse microhabitats contributes to the diversity and complexity of plant community structure and species composition [26,27]. The diversity of forest plant species in these subtropical forest ecosystems provides a rich source of quality and quantity of litter and contributes a large amount of complex plant-derived C to the soil C pool [28,29,30]. However, since lignin is a part of the stable soil C pool, there is increasing evidence that the properties of lignin are more susceptible to change with plant species. Therefore, elucidating the distribution pattern of the fate of plant-derived lignin in soils and its relationship with various plant species can help to provide insight into the stability of lignin in soils and its sequestration mechanisms specific to this study area, especially along natural diversity gradients.

For this purpose, we selected a typical subtropical forest as our study site in Southwest China, which hosts several hundred plant species. The objectives of this study were to (1) Expand the lignin biomarkers of pure plant leaves by analyzing dominant tree species in a subtropical forest; (2) Compare data on lignin biomarkers from plant litter to topsoil in 45 plots of various plant diversities; (3) Reveal how lignin monomer composition and ratios change and reflect their degradation status. Overall, this study provides crucial information to understand the ultimate fate of plant-derived lignin and provides new insights into SOM transformation processes under various plant diversity in forest ecosystems.

2. Materials and Methods

2.1. Study Site and Sampling

This study site is located at the Mulun National Natural Reserve (25°06′09″–25°12′25″ N, 107°53′29″–108°05′45″ E) in Huanjiang County of Guangxi Zhuang Autonomous Region, Southwest China. This area covers a typical subtropical monsoon climate, with mean annual temperatures of 19 °C and mean annual precipitation of 1500 mm, respectively [26]. The landscape of this subtropical forest corresponds to a typical karst landscape with gentle valleys flanked by steep hills, and the bedrock is dominated by karst ecosystems underlain by limestone, dolomite, or mixtures thereof, and the soils are calcareous with types being Cambisols, Luvisols, or Leptosols [31]. This subtropical forest type has a complex tree community structure and rich diversity, mainly comprising evergreen and deciduous broad-leaved mixed trees, and the plant species are distributed in aggregated or random patterns [32]. The dominant tree species are Cryptocarya concinna, Itoa orientalis, Platycarya strobilacea, and Lindera communis [33]. This reserve was established in 1991 and developed after clear cut at the end of the 1950s. Due to human disturbance during the early stage of forest development and soil heterogeneity, there is a clear spatial difference in plant diversity.

A 25 ha (25°8′ N, 108°0′ E, 625 quadrats, designed in 500 m × 500 m) site was established in the Mulun Reserve in April 2014, with the first census completed in December 2014 following the standard field protocol of the CTFS (Center for Tropic Forest Sciences). Field surveys and sampling were carried out between 28 July and 1 August 2020. We selected a total of 45 plots (each measuring 20 m × 20 m cells) along a plant diversity gradient (tree species richness ranging from 4 to 61, and a Shannon–Wiener index ranging from 0.15 to 3.57) for this study (Figure S1). The distance between every two adjacent plots was 40 m with an elevation range of 446 m to 521 m to minimize the potential effects of elevation on biogeochemical cycling. More details on soil sample collection and processing can be found in our previous study [34]. In total, 62 plant species were selected, representing approximately more than 50% of the species composition, with the highest occurrence in the selected plots with different plant diversity. About 25 fresh leaves were randomly collected standing green from the branches of each plant species, then placed in paper envelopes. Litter samples were randomly collected from a 20 × 20 cm wire square in each plot (16 random locations), then thoroughly mixed into one composite sample and pooled into a litter trap. After litter collection and removing any possible organic layer, soil samples at depths of 0–10 cm were collected from 16 random locations in each plot, using a stainless-steel auger with an inner diameter of 5 cm. All the samples were immediately transported to the laboratory, the plant leaves and litter samples were subsequently evenly chopped into lengths of 1 cm and oven-dried at 70 °C, and then they were ground into fine powders using a mortar and pestle prior to determining the chemical properties and lignin content.

2.2. Lignin Biomarkers

The classical method for determining lignin phenols was used in conjunction with the cupric oxide (CuO) oxidation method [35]. The principle is that under alkaline conditions, copper oxide catalyzes the oxidation of lignin macromolecules to break the intermolecular ether bonds, thus releasing a series of simple phenolic compounds [20]. Briefly, the samples (accurately 0.50 g) were oxidized under the alkaline condition with 10 mL of NaOH solution (2 M) at 170 °C for 2 h in sealed Teflon-lined bombs. The ethylvanillin was added to the solution as an internal standard (IS) after the bombs were cooled to control the recoveries during the procedure. The aqueous solutions were transferred to a centrifuge tube and centrifuged for 5 min at 6000 rpm, then the supernatant was acidified to pH = 1 with 6 M HCl to remove the humic acid. The extracts of lignin phenols were purified using a C₁₈-cartridge (500 mg/6 mL) in a solid phase extraction system and eluted with 5 mL of ethyl acetate. The residues were redissolved in 0.05 mL of pyridine and derivatized with 0.1 mL bis-(trimethylsilyl)-trifluoroacetamide prior to analysis after being dried by a stream of nitrogen gas. The concentration of major lignin phenols was detected by GC–FID (Agilent 7890A, Wilmington, DE, USA) with an HP-5 column (30 m × 0.25 mm × 0.32 μm) [34]. Lignin monomers included V-type phenols (vanillin, acetovanillone, and vanillic acid); S-type phenols (syringaldehyde, acetosyringone, and syringic acid); C-type phenols (ferulic and p-coumaric acids); P-type phenols (p-hydroxybenzaldehyde, p-hydroxyacetophenone, and p-hydroxybenzoic acid); and 3,5-dihydroxybenzoic acid (3,5-Bd).

The absolute concentration of lignin was defined as the sum of the eight lignin phenols (VSC) per unit of sample (mg g⁻¹). The contribution of lignin-derived phenols to organic C was assessed as organic C-normalized lignin phenols content (mg 100 mg⁻¹ OC). The acid to aldehyde (Ad/Al) ratios of V- and S-type phenols and C/V and S/V ratios, P/(V + S), as well as 3,5-Bd/V, were calculated to reflect the lignin or soil organic matter (SOM) degradation status.

2.3. Analyses of Auxiliary Variables

Leaf or litter organic C and N were analyzed using an elemental analyzer (EA 3000; EuroVector, Milano Italy). To represent the ability to depolymerize lignin, activities of plant litter extracellular oxidase, including peroxidases (PER) and polyphenol oxidases (PPO), were measured using a colorimetric microplate assay using the substrate of L-3,4-dihydroxy phenylalanine (DOPA). The activities of PER and PPO were expressed as nmol h⁻¹ g⁻¹ soil.

2.4. Statistical Analyses

One-way ANOVA with Tukey’s HSD post-hoc test was used to assess the differences in lignin phenols content and degradation characteristics between different samples at a significance level of p < 0.05. The effects of soil enzyme activities (e.g., peroxidase) on lignin content were examined using Pearson correlation. In addition, a principal component analysis (PCA) was further performed to capture the variations of eight detected lignin phenols across three types of samples. All statistical tests were conducted using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and figures were performed using Origin 2023 (OriginLab, Northampton, MA, USA).

3. Results

3.1. Lignin Phenol Contents

The absolute content of lignin phenols in the investigated leaf and litter samples ranged from 1.60 to 33.4 mg g⁻¹ and 4.51 to 26.4 mg g⁻¹, with average values of 15.8 ± 7.25 mg g⁻¹ (mean ± SD) and 10.7 ± 4.52 mg g⁻¹, respectively (Figure 1). The average lignin content of plant leaves was significantly higher than that of litter, while the lowest lignin content was found in soil. The average content of V-, S-, and C-type phenols was significantly higher in leaf than in litter and soil, but the content of C-type phenols was comparable between litter and soil (Figure 1). The average organic C-normalized lignin content in leaf, litter, and soil was 3.47 ± 1.65 mg 100 mg⁻¹, 2.67 ± 1.16 mg 100 mg⁻¹, and 3.58 ± 1.05 mg 100 mg⁻¹, respectively (Figure 1). More details about plant species and chemical properties are reported in Table S1 in Supplementary Information.

For clarity, only the first and second principal components (PC1 and PC2) were used to construct the ordination graph depicting the relationships between the concentration of lignin phenols in different samples. The first two components contain 43% and 16.4% of the variations in the data, respectively (Figure 2). Overall, PCA analysis showed that lignin phenols in plant litter and soil samples were clearly separated along the first axis, but there was no clear separation of the leaf and litter samples in these studied plots.

3.2. Lignin Degradation Characteristics

The S/V ratio is commonly used to assess the contribution of angiosperm species to the SOM, while the C/V ratio can indicate the relative importance of woody versus non-woody parts of plants in the SOM. The mean values of the S/V ratio in leaf, litter, and soil were 1.12 ± 0.85, 0.40 ± 0.16, and 1.06 ± 0.34, while C/V ratios were 0.05 ± 0.02, 0.93 ± 0.09, and 0.12 ± 0.02, respectively. The C/V ratio depended on the sample types, with the ratio in the leaf being significantly higher than that in litter and soil, while the S/V ratios were not significantly different among the three sample types (Figure 3a).

The 3,5- Bd/V ratio, used as a characteristic indicator of the degree of SOM oxidation, did not differ significantly in leaf, litter, and soil (Figure 3b). The P/(V + S) ratio has been used as an indication of the degradation state of SOM since the degradation process leads to a selective loss of methoxy groups in S- and V-type lignin phenols without affecting the p-hydroxy phenols. Moreover, since the functional groups of aldehyde can be oxidized to carboxylic acid during degradation, the (Ad/Al) ratio of S- and V-type lignin phenols is often used as an indicator of the degradation status of SOM, which increases with microbial and photochemical degradation processes. In the present study, P/(V + S) and (Ad/Al)_v&s ratios showed a trend of soil > litter > leaf, with significant differences (Figure 3b,c). In addition, oxidase activity in litter and soil showed they significantly and positively correlated with their respective (Ad/Al)_v ratios (Figure 4a,b).

4. Discussion

As previously mentioned, plant species affect not only the lignin content in soil but also the characteristics [14,19]. Previous studies have demonstrated that lignin-derived phenolic components in soils are closely related to their respective source plants, reflecting the preservation of characteristic lignin patterns in soils [9]. Therefore, the different chemical properties of lignin attributed to different plant species in each plot may influence the dynamics of lignin in soils [13]. The turnover rate of lignin in soil is faster than that of total soil organic carbon [6,12], and its degradation process is complex due to its aromatic ring structure [13,16], which is also considered to be an important component in the soil C pool. Lignin often accumulates during the degradation of plant litter, and its content is negatively correlated with the degradation rate of plant litter [9]. In this study, the lignin content in the plant leaf was significantly higher than that in litter samples. This indicates that lignin phenols may undergo a certain degree of degradation, such as photo–oxidative degradation [36] and microbial degradation, before the plant-derived lignin transfers into the soil layer [13,14,15]. Therefore, lignin in the soil is determined by a balance of plant-derived OM inputs and degradation, which involves both biotic and abiotic metabolic processes [37]. In particular, this involves fungi and bacteria that degrade lignin participate in the SOM degradation process by producing extracellular enzymes such as lignin peroxidase, manganese peroxidase, multifunctional peroxidase, and laccase [38]. Since these enzymes are key to lignin degradation, as our previous study found, there is a significant positive relationship between oxidase activity and the degree of lignin degradation (Figure 4), further illustrating the effect of microorganisms on lignin content in litter and/or soil across the sampling plots.

Despite the chemically recalcitrant structure of lignin macromolecules, the content and ratios of lignin CuO by-products (monomeric phenolic compounds) are strongly influenced by degradation processes, and lignin biomarkers in soil represent the combined lignin profile of multiple plant fragments after the humification process [20]. V- and S-type monomers are involved in the formation of the plant cell wall and are located in the interior of the cell wall as the main structural component, whereas C-type monomers are located outside the cell wall and are more susceptible to microbial attack. Therefore, they are preferentially degraded. Consistent with our findings, we found lower C/V ratios in litter and soil compared to purely plant-derived leaves (Figure 3). Our study also found that the S/V ratios decreased or remained constant with increasing degradation (Figure 3), and the results were consistent with the previous study [14]. Accordingly, the ratios of S and V monomers and C and V monomers can be used to determine the plant origin of lignin phenols. However, differences in the priority of lignin monomers during degradation can lead to bias in determining regional vegetation types. In addition, both (Ad/Al)_v and (Ad/Al)_s ratios in soil samples were significantly higher than those measured in plant leaf and litter samples (Figure 3), due to the increased proportion of acid derived from the oxidation of lignin. This was also related to the high degree of SOM degradation in soil [13,15,20].

Furthermore, we observed P/(V + S) ratios significantly higher in soil than in leaf and litter samples, due to the fact that p-hydroxyphenols are produced not only by the oxidation of lignin but also by compounds such as aromatic amino acids [39]. The proportional contributions of woody and non-woody plant parts, as well as the respective contributions of angiosperms and gymnosperms to forest succession and ecosystem change, may have an impact on the P/(V + S) ratio measured in forest soils, and the ratio of P/(V + S) can be used to indicate the process of demethoxylation [14]. Plant-derived OM may persist in soils for thousands of years [15], so high P/(V + S) ratios in soil samples may also be due to the fact that the main source of SOM in this forest ecosystem was not fresh plant-derived lignin. In this study, 3,5-Bd/V ratios showed no differences in leaf, litter, and soil samples compared to P/(V + S) ratios, mainly because 3,5-Bd is not a compound derived from lignin but from tannins and flavonoids in undecomposed plant tissues [40]. It is worth noting that both P/(V + S) and 3,5-Bd/V ratios were influenced by vegetation type and source. Forest SOM degradation by lignin monomers needs to be considered and compared in an integrated manner [15].

Although the role in the formation of SOM with long persistence was exerted from the soil microbial necromass [8], plant-derived OM can also be stabilized with long residence times in soil according to Sokol et al. (2019) [41]. Hence, soil lignin-derived phenols, as biomarkers, have been used to study the dynamic processes of SOM in forest ecosystems [13]. The lignin alkaline CuO oxidation used in this study is a reliable method for obtaining information on the source and content of lignin in soil samples. However, it only represents a small fraction of the total lignin present in the soil, thus underestimating the contribution of actual lignin, especially in the mineral fraction [41]. It is crucial to accurately quantify lignin, and its contribution to the soil C pool in addition to characterizing specific components since the interpretation of lignin biomarker data is complicated. Microbial degradation is crucial in the carbon cycle of forest ecosystems through the process of lignin transfer from plant to apoplast to soil during SOM formation and is a key factor influencing the compositional changes in SOM [15]. Numerous studies over the past years have shown that the plant-derived lignin and its characteristics in soil are related to plant origin, land use, soil properties, and environmental conditions [15]. These studies also pointed out some limitations of CuO biomarkers obtained by the lignin CuO oxidation method and commonly used to identify gymnosperm sources and angiosperm sources from SOM. These markers do not allow the distinguishing of plants from the same taxonomic unit, especially with the input of mixed vegetation apoplasts in forest soils with complex plant diversity. In addition, using the CuO oxidation method to obtain lignin phenol monomers does not consider monomers derived from other sources than lignin [14,40]. Common lignin degradation indicators may also be influenced by the contribution of non-lignin phenols to soil lignin properties. Our study highlights the fact that lignin monomers and their characteristics are mainly useful to provide general characteristics of vegetation input. There are still significant differences in different types of samples (e.g., leaf, litter, and soil), which provide important indications of the source and the degree of degradation of SOM.

5. Conclusions

In summary, lignin is one of the important plant-derived organic C in the soil C pool, and its degradation and accumulation directly affect the stability and storage capacity of the SOC pool. Our study provides a characterization of the monomer content of plant-derived lignin from different species of leaves to litter and to the soil, as well as the extent of degradation. The results of this study illustrate that the fate of lignin in forest ecosystems is very complex, and plant species greatly affect the lignin characteristics of their leaves. Meanwhile, the degradation properties of lignin can provide information on the accumulation and regulatory mechanisms of SOC, and our findings showed the extent of lignin degradation in mixed-species litter under different plant diversity plots depended on oxidase activity and further influenced SOC storage. Future research should focus on the effects of dominant plant species in specific forest ecosystems on SOM dynamics, thus providing more precise information for forest management as well as soil C sequestration. The results of the above research could be incorporated into Earth system models to better predict the dynamics of soil C pools in response to differences in forest plant species and diversity.