Syringyl unit in the lignin of Ginkgo biloba leaves

To elucidate site-specific lignin in Ginkgo biloba leaves, the chemical architecture of lignin in leaf veins (LV) and petioles (LP) was analyzed by combining thioacidolysis and 2D Heteronuclear Single Quantum Correlation (HSQC) NMR. LV and LP were separated from the leaves, and polysaccharides were enzymatically digested to give residual lignin (EL). Acetylated EL (ELAc) and milled-wood lignin (MWL) were also prepared. As a result of thioacidolysis, guaiacyl (G) units were the predominant monomers in all samples. In LV and LP samples, the p-hydroxyphenyl (H) unit (approx. 1–2%) and a syringyl (S) unit were detected. The S-unit was observed higher in LP (approx. 2%) than in LV (trace). In thioacidolysis dimeric product analyses, major G–G products and a few G–H products were yielded; additionally, LP samples contained a β-1′ dimer incorporating S-units. 2D HSQC NMR spectra agreed with these trends. LV-ELAc exhibited β-O-4′, β-5′, and β-β′ linkages with H and G aromatics, whereas LP-MWL additionally showed an S-aromatic signal. Some unidentified resonances in LV-ELAc and LP-MWL were observed but not assigned to specific molecules. The S-derived monomers and dimers in petioles suggest a site-specific lignin potentially tailored to petiole mechanics or physiology.

Lignin is based on a phenylpropane skeleton, and its chemical structure is regulated according to tissue and cell type, conferring various functions and properties such as mechanical strength, hydrophobic barrier formation, and UV protection. Recently, C-lignin polymerized solely from caffeic alcohol [1, 2] and tricin lignin incorporating the flavonoid tricin [3, 4], which are further extensions of the conventional guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units, have been reported, demonstrating the diversity of lignin structures.

The xylem lignin of Ginkgo biloba exhibits a typical G-type lignin, with H-unit detected in the compression wood, and is treated similarly to coniferous lignin. On the other hand, the ability to form S-units has been confirmed in ginkgo cultured cells [5], indicating that the biosynthetic pathway potentially allows for S-units. Examples of ginkgo lignin analysis include wood normal tissue [6, 7], and compression and roots [8], where G-units were predominant, with a small amount of H-units detected in the compression wood.

Several studies have reported the detection of S-units in the lignin of ginkgo xylem [9, 10]. These studies report the detection of S-units in the tips of young branches and sapwood of gymnosperms, including ginkgo, using thioacidolysis, nitrobenzene oxidation, pyrolysis gas chromatography–mass spectrometry (GC–MS), and ¹³C nuclear magnetic resonance (NMR). Based on the evolutionary process of the related enzymes mentioned above, it is reasonable to conclude that many gymnosperms can possess S-units. However, the specific location of the S-unit or its functional role is unknown.

Ginkgo leaves contain various pharmacologically active components, including flavonoids, and are utilized as materials for health foods. However, there are only a few reports on the lignin structure in leaves. Leaf tissues contain large amounts of non-lignin compounds such as waxes and proteins, leading to overestimation of lignin content using the conventional Klason’s method. Therefore, more selective and quantitative methods for structural analysis are required. As a result of lignin analysis of ginkgo leaves using nitrobenzene oxidation and ozone decomposition, reports have detected trace amounts of S-units in ginkgo leaves [11, 12]. Previous studies have cautiously discussed the detection of S-unit in ginkgo leaves, noting the possibility that they may originate from compounds other than lignin, and further verification was necessary.

Based on the above background, this study focused on the possibility that ginkgo leaves contain special lignin different from that in the stem xylem, and compared and analyzed lignin in wood and leaves. In particular, leaf veins (LV) and leaf petioles (LP) removed from leaves were used in the experiments. After freezing and grinding, the solvent-extracted samples were quantified for lignin content using the gravimetric rapid thioglycolic acid (grTGA) method. Structural units were analyzed using the thioacidolysis and GC–MS method, and side–chain patterns such as β-O-4′, β-5′, and β-β′ bonds, as well as aromatic ring composition, were evaluated using 2D Heteronuclear Single Quantum Correlation (HSQC)–NMR.

Reagent

Chemical reagents were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan), Kishida Chemical Co., Ltd. (Osaka, Japan), Merck KGaA (Darmstadt, Germany), and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Dimethyl sulfoxide (DMSO) and N-methylimidazole were used after drying with activated molecular sieves 3A.

Plant sample preparations

In June 2023, a 7-year-old ginkgo tree was felled from a field within the Nagoya University campus, and the stem was cut every 20 cm. The stem diameter was from 30 to 25 mm. The stem parts were quickly immersed in methanol preheated to 65 °C and subjected to a 7 h hot methanol treatment. After manually peeling off the bark, the current year xylem region was peeled using a peeler and preserved by immersion in acetone. This sample was designated as stem xylem. Green leaves were collected in June 2023, and yellow leaves were collected in November 2022, immediately after leaf fall, from ginkgo trees on the Nagoya University campus. The leaf surfaces were washed with distilled water, frozen, and stored. Leaves were used as mixtures from multiple individuals in the experiments. The samples obtained from green and yellow leaves will be described separately in the following sections. The separation of the leaf blade and petiole is shown in Fig. 1.

To observe the cross section of the leaf and the petiole, sections were prepared using a sliding microtome (REM-710; Yamato Koki Co., Ltd.) by Kawamoto’s film method [13, 14] and stained with toluidine blue. Microscopic observations were performed using a BX50 microscope (Olympus). The resultant cross-sectional images are shown in Fig. 1.

The stem xylem was dried and cut into powder using a rotor speed mill (p-14, FRITSCH). After Soxhlet extraction (acetone for 8 h, hot water for 8 h) and freeze-drying, the wood meal was ground using a planetary ball mill (P-6, FRITSCH). In a 45-mL zirconia pot, 100 g of Φ5 mm zirconia beads and 1.0 g of extracted wood meal were added. The mixture was milled at a rotation speed of 600 rpm, with a milling time of 2 min and a pause of 2 min per set, for a total of 180 sets. Thus, the actual milling time was 6 h. After milling, the sample was sieved through a 100-mesh sieve, and the resulting wood powder was designated as ball milled wood (BMW).

For the leaves, the freeze-dried leaves were separated into the leaf blade and the LP parts. At this time, to make it easy to collect the resultant LV samples, approximately 3 mm of the LP was left on the leaf blade. Therefore, the cut LP sample did not contain LV. The leaf blade portion was treated with a 0.25 M NaOH solution at 80 °C for 20 min to soften tissues other than LV, then mesophylls were carefully removed using a toothbrush. The remaining petiole-containing portion was removed from the washed part, yielding a sample consisting solely of LV. The obtained LV and LP were washed with distilled water and freeze-dried. After drying, the samples were freeze-ground for 2 min to produce a powder. The powder was subjected to Soxhlet extraction (hexane for 6 h, acetone for 8 h). Further extraction was performed in a flask using 80% ethanol at 60 °C, followed by washing with distilled water and freeze-drying. BMW was prepared using the same method as for the stem xylem. The separation yield of LV from the leaf blades based on dry weight was ca. 11% for green leaves and ca. 8% for yellow leaves.

Preparation of MWL, EL, and ELAc

The preparation of Milled-wood lignin (MWL) was performed according to previous reports [15,16,17]. The extracted powder sample was added to an 80 mL zirconia pot along with 32 Φ10 mm zirconia beads and 44 mL of toluene. Planetary ball milling (P-6, FRITSCH) was performed at a rotation speed of 600 rpm for 40 h. A 10-min cooling period was introduced every 30 min of milling to prevent overheating of the pot. Subsequent purification procedures were carried out according to the literature. Briefly, the milled powder was extracted with a 90% dioxane aqueous solution, and the supernatant was recovered by centrifugation. The crude MWL obtained by concentration was purified by dissolving it in a 90% aqueous acetic acid solution, followed by reprecipitation into ice-cold water. After freeze-drying, the insoluble portion was removed by adding a 2:1 mixture of 1,2-dichloroethane:ethanol, followed by reprecipitation in cold diethyl ether and washing with petroleum ether, yielding the purified MWL. The yield of MWL from the extracted powder was approximately 7% in the stem xylem, 2% in Green LP, and 0.4% in Yellow LP.

To enhance the detection sensitivity of lignin, enzyme treatment was performed based on the method of Kim et al. [18]. BMW 1.0 g, 25 mL of acetic acid buffer (pH = 5.0), and 50 mg of Cellulysin (cellulase from Trichoderma viride) were added to a 100-mL conical flask with baffles, and the mixture was shaken at 35 °C and 120 rpm for 6 days using a shaking incubator. The mixture was transferred to a 50-mL centrifuge tube every 2 days, centrifuged at 800 g for 20 min (800 g, 20 min), and the supernatant was removed. The mixture was then returned to the flask, and an equal volume of acetic acid buffer and Cellulysin was added. After shaking, the sample was collected in a 50 mL centrifuge tube, centrifuged to remove the supernatant, and washed three times with distilled water. The recovered sample was freeze-dried to obtain enzyme lignin (EL).

EL from LV and LP had poor solvent solubility, so acetylation was performed according to the method of Lu et al. [19]. In a 30 mL flask, 2 mL of dimethyl sulfoxide and 1 mL of N-methylimidazole were added, then the atmosphere was replaced with nitrogen. Next, 100 mg of EL was added, the flask was sealed, and the mixture was stirred at room temperature for 3 h. Then, 0.6 mL of anhydrous acetic acid was slowly added using a syringe, and the mixture was stirred in the dark for 24 h. After stirring, the mixture was added dropwise to 350 mL of ice water. After stirring for about 1 h, the sample was recovered by filtration and washed three times with distilled water. The recovered sample was freeze-dried to obtain acetylated EL (ELAc).

grTGA method

The grTGA method is developed to quantify the lignin content in the herbal or leaf samples [20]. It is reported that the grTGA method is superior to Klason’s method for quantifying lignin in leaves and is also applicable to xylem samples. The grTGA method was performed using 40–80 mg of extracted powder samples, based on the method of Shimada et al. [20]. Three measurements were conducted for every sample, and average and standard deviations (S.D.) were calculated.

Thioacidolysis and GC–MS

Thioacidolysis selectively cleaves the β-aryl ether bonds of lignin. Therefore, the analysis of the lignin-derived monomeric products can evaluate the type and amount of the lignin structural units involved only in β-aryl ether bonds [21]. Furthermore, the lignin-derived dimers are obtained after desulfurizing the thioacidolysis monomeric products over Raney nickel [22,23,24]. The monomeric and dimeric products are analyzed by GC–MS (QP2010, Shimadzu, Japan) as previously reported [23, 25,26,27,28]. Referring to these literatures, we used a response factor of 1.5 for monomeric products and 1.0 for dimeric products. The mass spectra of the signals appearing in the chromatogram were compared with those previously reported. The characteristic ions are designated in Figures S1, S2, and S3, and the dimeric product chemical structures are summarized in Figure S4. Three measurements were conducted for every sample, and average and S.D. were calculated. The dimer involving the S-unit had a weak signal intensity and overlapped with the adjacent signal, making it difficult to calculate the area (Figures S2 and S3). Therefore, we used the ratio of the total ion count over the selected ion count calculated from the reference standard [29] for the β-1′-linked G–S dimer and multiplied it by the area in the selected ion chromatogram at m/z = 448 to convert it to an area value equivalent to total ion count.

NMR

¹H-¹³C HSQC measurements were performed using a Bruker 400 MHz NMR spectrometer (AVANCE 400). The sample concentration was prepared at 70–110 mg/mL, and the number of scans was set to 20. The stem xylem ELAc was measured using CDCl3, the LV ELAc was measured using a CDCl3/DMSO-d₆ (1/1) mixed solvent, and the LP-MWL was measured using DMSO-d₆. The spectra were analyzed using TopSpin (Bruker, Ver. 4.5), and chemical shift values were corrected using the methyl signal of tetramethyl silane (TMS) or the signal of the solvent DMSO. Signal assignment was performed by referring to literature and the online database [18, 30,31,32,33,34].

Evaluation of lignin content by EL yield and grTGA methods

Table 1 shows the EL yield and lignin quantification results using the grTGA method for each sample. The lignin content obtained from the stem xylem was approximately 30%, a typical value for gymnosperm wood. The yield from leaf veins and petioles was 10–20%, lower than that from stem xylem, and the EL yield tended to be higher than that from the grTGA method, other than that of Green LV. This result suggests that the lignin content determined by the grTGA method may have been overestimated due to the influence of other components in the green LV, or that some high-molecular-weight substances that cannot be removed by enzymatic treatment may have accumulated in the yellow LV due to internal changes during the leaf-falling period. No significant difference in the yield was observed between green and yellow LP samples.

Analysis of lignin monomer units by thioacidolysis

Table 2 shows the results of thioacidolysis monomeric product analysis for each sample. To confirm the effects of alkaline treatment during LV preparation and enzymatic saccharification, thioacidolysis was also performed on the mesophyll sample as a residue of the LV removal, and stem xylem powder subjected to the same alkaline treatment (0.25 M NaOH solution at 80 °C for 20 min).

No lignin monomer units were detected in the mesophyll sample. In the stem xylem, G-unit was dominant, and H-unit was detected in small amounts. The monomer yield in the alkaline-treated stem xylem was higher than without treatment. This may be due to the swelling of cell walls or the solubilization of hemicellulose by the alkaline treatment, making it easier for the attacking reagent to penetrate the cell wall. There are two types of interpretation. If H-units are originally abundant at the terminal ends of lignin [35, 36], H-unit should be sufficiently cleaved by thioacidolysis even without the alkaline treatment. When the alkaline treatment improves the reactivity of the cell wall, it may result in increased yield from G-units and a corresponding decrease in the H-unit ratio. The alternative interpretation could be the partial lignin elution by the alkaline treatment and the high H/G ratio in the removed lignin portion.

EL analysis of green LV and yellow LV revealed abundant G-unit, small amounts of H-unit, and trace amounts of S-unit monomers. To confirm signals originating from H and S-units, GC–MS results for green LV EL are shown in Figure S1a. Multiplying the total monomer yield by the yield during EL preparation (green 0.128, yellow 0.174) estimated the original yields from the leaf samples of 28.2 μmol/g for green LV and 99.2 μmol/g for yellow LV, as shown in parenthesis in Table 2. Thus, the green LV value is approximately one-tenth of the stem xylem (254 μmol/g). Based on the grTGA results, where the green LV value was approximately half that of the stem xylem, if there is no significant difference in the proportion of β-O-4′ bond ratio in lignin, it is considered that the decomposition products of green LV obtained by the grTGA method contain a significant amount of impurities other than lignin. On the other hand, the yellow LV value was nearly three times that of green LV, suggesting that the ratio of lignin may have increased due to the withdrawal of leaf contents associated with leaf fall. However, it was still less than half of the stem xylem.

H, G, and S-units were detected from the EL and MWL of green and yellow LP samples. S-units were detected at the same level as H-units. The GC–MS results for green LP EL are shown in Figure S1b to confirm the signals originating from H and S-units. Multiplying the total monomer yield of green LP EL by the EL yield (0.211) results in 98.8 μmol/g, significantly higher than green LV (28.2 μmol/g for green LV). Compared to LV, LP is mainly responsible for water and photosynthetic product transport; it is likely to contain more lignin. This is consistent with the cross-sectional observation results of LV and LP (Fig. 1). The results for green LP MWL were slightly higher than those for green LP EL.

When the total monomer yield of yellow LP EL is multiplied by the EL yield (0.233), the result is 78.4 μmol/g, which is slightly lower than that for green LP (98.8 μmol/g). The monomer yield of yellow LP-MWL (184 μmol/g) was only 27% of that for green LP-MWL (678 μmol/g). This suggests that in the preparation of EL and MWL, samples derived from yellow LP contained more non-lignin residues, with this effect being particularly pronounced in MWL. On the other hand, the MWL yield was 2% for green LP and 0.4% for yellow LP, with yellow LP being significantly lower. This difference suggests that the lignin fraction in yellow LP may be more difficult to extract. To discuss the lignin extraction efficiency and optimize the procedures for green and yellow LP samples, it is necessary to consider possible seasonal changes in the chemical composition of the leaves [37, 38].

Analysis of lignin dimer units by thioacidolysis and desulfurization

Table 3 shows the thioacidolysis dimeric product analysis results obtained after Raney-nickel reduction. The results are shown as the ratio of the area corresponding to each signal, with the total set to 100. The μmol/g values calculated using a response factor of 1.0 are shown in Table S1. Results for alkaline-treated stem xylem and LP MWL samples are described in Tables S1 and S2.

The following structures were detected in each sample: 5–5′ type, 4-O-5′ type, β-1′ type, β-5′ type, and β–β′ type. When examining the relative area of the detected dimer structures, the β-1′ type G–G had the most significant area ratio, which was common to all samples. Compared to stem xylem, leaf samples showed a tendency toward higher proportions of 5–5′ type (stem 16%, leaf 24–33%) and β-5′ type (stem 26%, leaf 27–34%), and lower proportions of β-1′ type (stem 52%, leaf 32–39%). The 4-O-5′ type ratio was small but increased slightly in leaf samples, while the β–β′ type was difficult to detect.

Dimer involving H-units (5–5′, G–H; β-1′, G–H) was detected from green and yellow LV EL samples. In addition to dimers involving H-units, dimers involving S-units (β-1′, G–S) were detected in green and yellow LP-EL and MWL samples. To confirm the signals originating from these dimers containing H-units and S-units, the GC–MS results for green LP-EL are shown in Figures S2 and S3. The results show that structures present at the lignin end via β-O-4′ bonds or structures containing S-units placed between β-O-4′ bonds were detected in the LP samples.

2D HSQC NMR

2D HSQC NMR analysis was performed using ELAc and MWL samples to obtain further chemical structural information. Figure 2 shows enlarged views of the aliphatic and aromatic regions of the HSQC NMR spectra obtained from the MWL of stem xylem, green LP, and yellow LP, overlaid on each other with different colors. The overall data from the ELAc (Figures S5, S6) and MWL (Figures S7, S8) measurements of stem xylem, as well as the LV and LP measurements of green (Figures S9–12) and yellow (Figures S13–16) samples, and the overlaid figure of the ELAc data as shown in Fig. 2 (Figure S17) are presented as Supplementary Figures.

Signals derived from the side chains of major bonds such as β-O-4′, β-5′, and β–β′ were detected in all three samples. Signals derived from dibenzodioxocin (DBDO) were weak; some were detected while others were not. Signals presumed to be H-units were detected in all three samples, but they were unclear, especially in leaf samples, due to overlapping unknown signals. Signals originating from the S-unit were detected in the LP sample but not in the LV sample. These trends are consistent with the results of the thioacidolysis.

Many unknown signals were detected in LP-MWL. In the side chain region, a signal adjacent to α(β-O-4′) (¹H/¹³C, 4.75/73.5 ppm) was characteristic because it was not detected in the xylem sample. Numerous unknown signals were observed in the aromatic region. These unknown signals were significantly different in the ELAc spectra, suggesting that they may have been influenced by the acetylation, and may have structures containing hydroxyl groups in near positions. Some signals also showed differences between green and yellow leaves, for example, signals at 3.51/70.2, 4.26/62.3, 4.3/52.0, 4.75/73.5, 5.17/69.2, and 6.78/128.9 ppm, possibly reflecting seasonal changes within the leaves. These signals were investigated using ginkgo extract references [39,40,41,42,43]. However, we were unable to assign these signals to specific molecules. In the sample preparation for this study, LP samples could only be measured as MWL so that these unknown peaks might be generated during MWL adjustment. However, since these signals were not detected in the stem xylem MWL, they are likely specific to leaves.

Although HSQC measurements are not quantitative, they can be used to compare the relative amounts of similar structures, such as signal integral volumes, and to compare samples. The results of comparing the signal volume ratios of the α and β-position of β-O-4′, β-5′, β-β′, and DBDO are summarized in Table 4, and ratios using G2-H as a reference are in Table S3. When comparing stem xylem and leaf samples, the overall intensity of side-chain bonding patterns relative to G2 units showed a tendency for β-O-4′ to be lower and β-β′ to be higher, but the overall trend was consistent. Additionally, there was little difference between green and yellow leaves.

S-unit in the ginkgo leaf lignin

As indicated by the lack of detection of lignin-derived monomeric products in mesophyll by thioacidolysis, most of the lignin in leaf tissue is present in LV. LV lignin contained a small amount of H-units, and trace amounts of S-units were detected. On the other hand, thioacidolysis dimeric product analysis revealed that LP lignin contained S-units in addition to H-units.

In angiosperm trees, it is known that xylem vessels primarily transport water and nutrients efficiently and tend to have a low S/G ratio [44]. This trend is interpreted because lignin dominated by G-units should be more robust and superior in mechanical strength, pressure resistance, and hydrophobicity than lignin containing S-units. It is reported that broad leaf lignin has a lower S/G ratio than that in xylem [45].

The amount of S-units detected in this study is small, and it is unlikely to have a significant effect on the cell wall properties of the entire petiole. Therefore, S-units may be distributed uniformly at very low concentrations throughout the petiole, but it is also possible that S-units are concentrated locally in specific cells or tissues. Examples of finely controlled lignin structures in cells or tissues are reported [46, 47]. Such localized structural differences may be involved in their mechanical or chemical properties. More detailed histological analysis and high spatial-resolution techniques for local molecular mapping are required to clarify these possibilities. Furthermore, lignins in ginkgo leaves and xylem have different lifetimes of 1 year and much longer, respectively. This difference may influence their structural design.

In this study, the lignin structure in the cell walls of ginkgo leaves was evaluated in detail by dividing the leaf into the leaf vein (LV) and petiole (LP) regions. The results revealed that S-units were explicitly identified in the petiole region. Additionally, unidentified components specific to leaf tissue were found, and it is speculated that these may be associated with leaf-specific functions. Further analysis is necessary to elucidate the roles and dynamic changes of these tissue-specific lignin structures and associated components.

The data sets analyzed during the current study are available from the corresponding author on reasonable request.

BMW:

Ball milled wood

DBDO:

dibenzodioxocinEL

EL:

Enzyme lignin

ELAc:

Acetylated EL

GC-MS:

Gas chromatography-mass spectrometrygr

TGA:

Gravimetric rapid thioglycolic acidLP

LP:

Leaf petiole

LV:

Leaf vein

MWL:

Milled-wood lignin

NMR:

Nuclear magnetic resonance

SD:

Standard deviations

TMS:

Tetramethyl silane

JSPS KAKENHI Grant Numbers JP18H03959, JP23K26967, JP23K26968, and JP24H00056 supported this work.

Authors and Affiliations

1. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464‐8601, Japan

   Shori Imamura, Dan Aoki, Masato Yoshida & Kazuhiko Fukushima

2. Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, 183‐8509, Japan

   Yasuyuki Matsushita

Contributions

SI, DA, and KF conceived the research. SI conducted sample preparations. SI and MY performed microscopic observations. SI, DA, and YM conducted chemical analyses. All authors read and contributed to the manuscript.

Corresponding author

Correspondence to Dan Aoki.

Competing interests

The authors declare that they have no competing interests.

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