Role of monolignol glucosides in supramolecular assembly of cell wall components in ginkgo xylem formation

3.5.1 Role of glycone moiety d-Glc in formation of HCs and H₂O₂ generation

From the MLGs [Figure 11, (1)] monolignol and d-Glc are liberated by β-glucosidase [Figure 11, (2)] that present in the lignifying cell wall (Marcinowski et al. 1979, Samuels et al. 2002). The major part of the d-Glc may be returned into cytosol [Figure 11, (3)] and converted to various HCs in Golgi apparatus [Figure 11, (4)] (Pauly et al. 2013; Rennie and Scheller 2014) that may be fused with plasma membrane under high turgor pressure (Fricke et al. 2000) at night. Hosoo et al. (2006) observed deposition of amorphous material containing glucomannans and xylans on the innermost surface of the tracheid of C. japonica during the dark period. The ginkgo shoot tracheids under present experimental conditions showed diurnal change of turgor pressure cleary as shown in Figure 10.

By an another hypothetical route, the UDP-D-Glc may be formed from the glycone d-Glc in the apoplast by UDP-Glc synthesizing enzyme that located in the apoplast or associated with plasma membrane (Kleczkowski et al. 2010) [Figure 11, (5)]. Then, conversion of UDP-D-Glc to UDP-D-Glc A by dehydrogenase coupled with NAD⁺ → NADH, and the NADH converts O₂ to H₂O₂ [Figure 11, (6)].

When dual radio-labeled CF, at -CH₂OH of position C6 of glycone Glc and Cβ of aglycone coniferyl alcohol with ³H and ¹⁴C, respectively, [Glc-6-³H,¹⁴Cβ]CF, ³H/¹⁴C:13.2, was administered to a growing shoot of Magnolia kobus for 7 days, the ratio ³H/¹⁴C of the newly formed xylem was 3.1 (Matsui et al. 1995). This significant drop of the ³H/¹⁴C ratio may support the hypothetical scenario that the UDP-D-Glc A is formed by the selective dehydrogenation of UDP-D-Glc at position 6 [Figure 11, (6)]. According to the radio-tracer study on the incorporation of [U-¹⁴C]Glc into tracheids of Japanese ceder (C. japonica D. Don), little HC is deposited in the early stage of SW thickening, and in the later stage of S₂ thickening it is intususseptively deposit interior of the preexisting cell wall (Takabe et al. 1981). Deposition of structurally different glucuronoxylan during the Eucalyptus xylem cell wall formation has been observed (Magina and Evtuguin 2019). This kind of modification of HCs may occur in the apoplast [Figure 11, (9), (10)], or Golgi apparatus may also participates in the modification.

The apoplastic H₂O₂ generation stimulated by NADH has been shown in cultured spruce cells (Kärkönen et al. 2009), and a part of the NADH may modify the side chain of coniferyl alcohol to give dihydroconiferyl alcohol units in lignin (Holmgren et al. 2006). The H₂O₂ can be generated in situ by the peroxidase-catalysed oxidation of NADH that is produced by a cell wall bound malate dehydrogenase (Elstner and Heupel 1976; Gross 1977; Halliwell 1978). However, Olson and Varner (1993) failed to detect any effect of malate on H₂O₂ production in lignification zones of fresh unwounded tissue of Zinnia elegans L., and Savidge et al. (1996) could not detect a pool of H₂O₂ in lignifying xylem of conifer. It was suggested recently that the apoplastic H₂O₂ may be produced from apoplastic O₂ via two-step enzymatic reactions by NADPH oxidase, or respiratory burst oxidase homolog (RBOH) protein on the plasma membrane (Leitinen et al. 2017; Tobimatsu and Schuetz 2019) [Figure 11, (7)]. The UDP-D-Glc A is decarboxylated to give UDP-D-Xyl [Figure 11, (8)], and HCs including glucuronoxylan, glucomannan, xyloglucan, etc. [Figure 11, (10)] may be formed [Figure 11, (9)]. Some modification of the HCs formed mainly in Golgi apparatus [Figure 11, (4)] (Ye and Zong 2022) may occur also in apoplast to give modified HCs [Figure 11, (4), (10)].

3.5.2 Role of the HCs originated from glycone moiety d-Glc

The -COOH on the HCs contributes to transfer inorganic elements such as Ca²⁺ from cell plate to lignin depositing site [Figure 11, (11)] (Terashima 1990; Terashima et al. 1993). Calcium is an essential component for hard gel formation in the cell plate composed of pectic substances in the early stage of cell wall formation, and the Ca²⁺ is known to promote effective dehydrogenation of monolignols by peroxidase. The Ca²⁺ attracts phenolic monolignol and–COOH of glucuronoxylan to mediate deposition of lignin preferably on the HCs [Figure 11, (20), (26), Figure 12B, D, Figure 13A]. The deposition of xylan in the early stage of cell wall formation is spatially consistent with the early stage of lignin deposition in the tracheid cell wall of Cryptomeria (Kim et al. 2010), and lignin deposition occurs at the same time as the deposition of xylan in differentiating xylem of beech (Awano et al. 2002).

Figure 12:

Supramolecular assembly of HG-lignin on the xyloglucan, pectin and CMF-bundles at cell corner (CC), middle lamella (ML) and primary wall (PW), before (A, B) and after (C, D) mild selective removal of lignin. Globular HG oligolignol micelles form 3D grape-like cluster connected by condensed unit linkages (B, +). They are held by the polysaccharides networks (C, D) consist of small CMF bundles, pectin and xyloglucan that clamps CMF-bundles each other (D). Reproduced from the article by Terashima (2013).

Figure 13:

A hypothetical concept of supramolecular assembly of G-lignin on HC-Ca-HC gel and large CMF-bundles at SW of ginkgo tracheid, modified from the article by Terashima et al. (2009). The Ca²⁺ attracts glucuronoxylan(–COOH) and lignol(–OH) to deposit lignin preferentially on the HC resulting in the frequent bonding between lignin and the HC (+) (A). The removal of Ca²⁺, water and enzymes from the swollen HC gel during the maturation of cell wall causes anisotropic shrinkage. The globular G-lignin micelles (A) with few condensed bonds (+) are deformed into flattened disks (B) by the anisotropic shrinkage.

Formation of phenoxy radical, then quinone methide [Figure 11, (12), (13)] just on the glucuronoxylan mediated by Ca²⁺ will promote bonding of HC to Cα of lignols [Figure 11, (13), (14), Figure 13A]. This bonding has been shown by chemical and NMR analyses (Balakshin et al. 2011; Imamura et al. 1994; Nishimura et al. 2018; Watanabe et al. 1989; Xie et al. 2000).

In the present study, about a half of the glycone moiety [U-¹³C]Glc liberated from [U-¹³C-Glc]CF is incorporated into HCs in ginkgo tracheid (Figures 7 and 11), and play an essential role in the synchronized assembly of HCs and lignin on the CMF bundles under the significant influence of turgor pressure (Figures 10, 12, and 13).

3.5.3 Role of aglycone H and G monolignols in supramolecular assembly of cell wall polymers at CCML

The H- and G-monolignols liberated at lignin depositing site at CCML form its radicals by laccase + O₂ [Figure 11, (12)], then oligolignols with H units on their growing ends due to the higher oxidation potential of H unit [Figure 11, (16)]. Then large globular micells of folded HG oligolinols are formed with phenolic OH of H-units on their outer region [Figure 11, (17)]. On the network of small bundle of CMFs [Figure 11, (18)] and HCs [Figure 11, (4) + (10), Figure 12C, D], grape-like cluster of HG lignin micells with bonds between the micells, at the positions shown by X, are formed by laccase and O₂ [Figure 11, (19), Figure 12A, B]. Due to the high content of 3–3′ and 3–5′ bonds, the HG lignin cluster is mechanically rigid [Figure 11, (20), Figure 12B]. Removal of water, Ca²⁺ and enzymes from the HG-lignin-HCs-CMF layer accelerated by the increase of turgor pressure during the maturation of cell wall results in a little or no isotropic shrinkage [Figure 11, (21)] to give pressure-resistant layer at CCML [Figure 11, (22)] as shown in Figure 12.

3.5.4 Role of aglycone G monolignol in supramolecular assembly of cell wall polymers at SW

A hypothetical concept of supramolecular complex of G-lignin on HC-Ca-HC gel and CMF-bundles at SW is shown in Figure 13. At SW, G-lignol is dehydrogenated by peroxidase and H₂O₂ to give folded oligolignols (Figure 11, (23)). Then micells are formed under hydrophilic environment (Figure 11, (24)). On the large bundles of CMFs (Figure 11, (25), Figure 13A), that laid down during daytime, 4-O-methyl-glucuronoxylan biosynthesized in Golgi apparatus (Figure 11,(4)) is laid down during night when the exocytosis from the Golgi apparatus is promoted under high turgor pressure (Figure 10). The direction of glucuronoxylan molecule, crossing over the CMF-bundles (Figure 13), has been observed under high resolution FE-SEM (Terashima et al. 2009), and this direction may be controlled by the exocytosis point from Golgi apparatus moving with the cytoplasmic streaming at night. G-lignin micells are formed only on the glucuronoxylan molecule mediated by Ca²⁺ as shown in Figure 13A. Removal of Ca²⁺, H2O and enzymes from the swollen G-lignin-HCs-CMF layer causes anisotropic shrinkage (Figure 11, (27)) to give supramolecular complex composed of flattened G-lignin disks between HC-CMFs (Figure 11, (28), Figure 13B). The estimated thickness of the G-lignin-HCs layer surrounding CMF-bundle is 3–4 nm, and the lignin moiety occupies space of about 2–2.5 nm (Terashima et al. 2009). The average shape and size of ligninosulfonate macromolecules were estimated to be irregular flexible disk of about 10 nm in diameter with an average thickness of 2 nm (Goring et al. 1979).

3.5.5 Role of MLGs in posture control of erectly standing tree

As shown by the microautoradiogram in Figure 14, ginkgo cell wall is formed via quite different assembly of lignin-HC-CMF in an early stage at CCML,and in a later stage at SW (Fukushima and Terashima 1991a; Terashima et al. 2004, 2009, 2012; Terashima and Yoshida 2006). H-MLG (p-glucocoumaryl alcohol) and G-MLG (CF) play different roles in the assembly as shown in Figures 12 and 13, respectively.

Figure 14:

Different roles of H-MLG (p-glucocoumaryl alcohol) and G-MLG (CF) in ginkgo cell wall formation. (Microautoradiogram was reproduced from Fukushima and Terashima 1991a). Formation of rigid, pressure resistant HG lignin-HC-CMF layer at CCML allows expansion of new cell by the elevation of turgor pressure [(1)–(3)]. Deposition of deformable G lignin-HC-CMF layer, at SW, and shrinkage of this layer causes shortening of the cell length [(4)–(6)], resulting in generation of contraction force on the xylem in the lateral and longitudinal directions. Shrinkage of the upperside tracheids of inclined stem (7) is greater than that of underside tracheids (8).

In the cambial zone of the growing tracheids, new cells are formed by cell division [Figure 14, (1)], and 3D clusters of globular HG condensed oligolignol are formed on the 3D network of small bundles of CMFs, pectin and xyloglucan which clamps CMFs each other [Figure 12, Figure 14, (2)]. This lignin-rich (>60 %), waterproof and pressure resistant CCML layer allows expansion of the new cells by elevation of turgor pressure at night [Figure 14, (3)]. With the increase of cell size, the CCML area increases quickly, so that a large amount of monolignols must be quickly supplied, and the xyloglucan molecules that clamps CMFs must be frequently cleaved and reconnected by xyloglucan transferase with new additional supply of Glc and Xyl that may be from the glycone Glc [Figure 11, (4), (10)]. Presence of this xyloglucan transferase and its role has been found in the epicotyls of Vigna bean (Nishitani and Tomonaga 1992). On-demand quick supply of H, G monolignols and HCs including xyloglucan is achieved by a large stock of their glucosides, p-glucocoumaryl alcohol and CF in the cambial zone and in an early stage of differentiating xylem where the main lignification in SW has not occurred yet (Aoki et al. 2016; Fukushima et al. 1997; Tsuyama and Takabe 2014; Yoshinaga et al. 2016).

At SW, large CMF bundles are formed during daytime [Figure 11, (25), Figure 14, (4)]. At night, HCs biosynthesized in Golgi apparatus, which may be fused with the plasma membrane (Fricke et al. 2000), are deposited on the CMF-bundles (Hosoo et al. 2003, 2006; Terashima et al. 2004, 2009; Yoshida et al. 2000) [Figure 11, (26), Figure 13A, Figure 14, (4)]. The high turgor pressure at night (Figure 10) and new deposition of hydrophobic G-lignin on the hydrophilic swollen gel of CMF-HC-Ca-HC-CMF causes removal of water, Ca²⁺ and enzymes toward lumen side across the apoplast that result in anisotropic shrinkage of globular G-lignin micelles in the direction perpendicular to CMF layer to give flattened disks [Figure 11, (27), Figure 13B, Figure 14, (5), (6)] (Terashima 1990). This anisotropic shrinkage has been detected by the parallel orientation of lignin aromatic rings to CMFs by Atalla and Agarwal (1986); Agarwal and Atalla (1986) employing Raman microspectrometry, and by Salmén et al. (2012) employing FTIR microspectrometry.

The HG lignin in CCML region does not show any anisotropic shrinkage (Salmén et al. 2012). This mechanically rigid CCML lignin is formed by laccase [Figure 11, (16), (17), (18), (19)]. Laccase dehydrogenates not only monolignols (Freudenberg 1960) but also oligolignols including condensed structures such as dibenzodioxocin (Hilgers et al. 2018; Kudanga et al. 2009) to form high molecular lignin molecules. One of the reasons for the participation of peroxidase, not laccase, in the major G-lignin deposition at SW may be to scavenge the toxic H₂O₂ produced in the course of effective utilization of glycone Glc [Figure 11, (6)] in the apoplast. Each of peroxidase and laccase plays essential roles in the cell wall formation in a cooperative manner.

The shrinkage of the G-lignin-HC gel between CMF bundles at SW causes contraction of the tracheid not only in the lateral but also longitudinal directions [Figure 14, (6)]. This is because, at a rough estimate, one tracheid (3–6 mm long) contains 4000–12,000 terminal ends of CMF bundles (average DP: 1000–1500, 0.5 μm–1.5 μm long) in longitudinal direction of the tracheid, and shrinkage occurs at those many spaces between the terminal ends of CMF bundles. This shrinkage occurs also between the axial ends of tracheids that result in the total shrinkage of the newly formed xylem in both lateral and longitudinal directions of the upright tree stem. Maturation of the newly formed gymnosperm xylem mainly composed of those shrinking tracheids increases contraction force in lateral and longitudinal directions. This contraction force contributes posture control of the erectly standing tree stem that is always under bending force by wind. The main driving force of SW shrinkage is the increase of cohesion force of water followed by the increase of Van der Waals force and hydrogen bonding between cell wall components caused by the removal of water and Ca²⁺ (Terashima 1990, 2013).

Those observations support the hypothetical scenario that lignin and a part of HCs derived from aglycone and glycone moieties of MLGs deposit at the same time and same location under the influence of diurnally changing turgor pressure. Synchronized deposition of lignin on the HCs on CMFs-bundles of lignifying ginkgo tracheid has been observed under high resolution FE-SEM (Terashima et al. 2009).

3.5.6 Role of MLGs in the posture control of inclined tree stem

In the underside of an inclined stem of most gymnosperms, thick HG-lignin-rich layer is formed in outer SW of the tracheids (Fukushima and Terashima 1991b) by laccase (Hiraide et al. 2021). This layer may allow larger expansion of the underside tracheids than that of upperside tracheids by the uneven increase of turgor pressure at night that is controlled by the ion and water channels on the plasma membrane in response to the gravity and light, and the shrinkage of the underside tracheid is smaller than that of upperside tracheid as shown in Figure 14, (7), (8). Though the posture control of inclined stem may be understood by combination of a variety of biochemical and histological factors such as number and size of tracheid, CMF angle etc. (Yamamoto et al. 1998), the contribution of glycone and aglycone moieties of a large amount of H- and G-MLG is essential by the strategy similar to formation of CCML and SW layers in normal wood. Large amount storage of CF in the cambial sap (Terazawa et al. 1984), differentiating zone (Aoki et al. 2016; Fukushima et al. 1997; Tsuyama and Takabe 2014; Yoshinaga et al. 2016) is necessary for on demand quick supply of this MLG.

On the other hand, in the cambial sap of growing angiosperm trees except for Magnoliaceae and Oleaceae, MLGs were not detected (Terazawa et al. 1984). This is because angiosperms control the posture of stem and branches by means of the strategy different from that of gymnosperms. In angiosperms, stem and branches are mechanically supported mainly by fiber cells and subsidiarily by vessels. The length of the fiber cell of beech wood is 0.5–1.8 mm and the axial length of the CMF estimated from the degree of polymerization of hardwood cellulose (DP ≑ 800–1200) is about 0.4–0.6 μm. A fiber cell contains about 800–4500 ends of CMF bundles in the axial direction. Shrinkage of HCs surrounding those CMF bundles at their side and 800–4500 end parts by removal of water and Ca²⁺ from the HC gel caused by increase of turgor pressure and deposition of syringyl (S)-rich G lignin and acidic HCs at night will result in the shrinkage of the fiber cell in its axial direction. Shrinkage of the vessel may be small, because vessel contains pressure-resistant, waterproof HG lignin for transport of water. Because the structure of SG lignin is almost linear (Ralph et al. 2019), the plasticity of globular micelle of SG oligolignols will be higher than that of HG or G lignin macromolecules. Then the deposition of S-rich SG lignin on the HCs-CMFs of fiber cell wall helps the shrinkage of CMF-HCs gel by its high plastic and hydrophobic properties. Posture control of the inclined angiosperm stem is achieved by the difference in the degree and number of shrinking fibers (and vessels) between upper and lower side of inclined stem in response to the gravity. The fact that gelatinous layer of the fiber cells of tension wood does not contain lignin or contain only a small amount of lignin indicates that lignin deposition is not always essential for the posture control of angiosperms via shrinkage of fiber cells generated by water cohesion force and Van der Waals attractive force between CMF bundles. This scenario of shrinkage has been experimentally supported by Okuyama et al. (1994) and Yoshida et al. (2002). Some amount of SG-lignin is essential for hardwood xylem to form plastic and viscoelastic layer between CMF bundles and between fibers and vessels. Because low content of SG-lignin and HCs is enough for posture control of stem by generation of contraction force, most angiosperm trees do not need to store a large amount of MLGs in the cambium region (Terazawa et al. 1984). However, monolignols must be delivered as their glucosides (Terashima et al. 2016; Tsuyama and Takabe 2014), and both of the glycone and aglycone moieties of MLGs play different essential roles in cooperative manner for posture control of growing angiosperms.