Phytochemistry 101 (2014) 40–51

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Exogenously applied 24-epi brassinolide reduces lignification and alters cell wall carbohydrate biosynthesis in the secondary xylem of Liriodendron tulipifera Hyunjung Jin a, Jihye Do b, Soo-Jeong Shin c, Joon Weon Choi d, Young Im Choi e, Wook Kim b,⇑, Mi Kwon b,⇑ a

Department of Biosystems Engineering, Korea University, Seoul 136-701, Republic of Korea Department of Biotechnology, Korea University, Seoul 136-701, Republic of Korea Department of Wood and Paper Science, Chungbuk National University, Cheongju 361-763, Republic of Korea d Department of Forest Sciences, Seoul National University, Seoul 151-742, Republic of Korea e Division of Forest Biotechnology, Korea Forest Research Institute, Suwon 441-350, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 15 October 2013 Received in revised form 30 December 2013 Available online 25 February 2014 Keywords: Yellow poplar Liriodendron tulipifera Magnoliaceae Cell wall Secondary xylem Brassinolide (BL) Lignin Cellulose Crystallinity Hemicellulose Arabinan Galactan Pectin

a b s t r a c t The roles of brassinosteroids (BRs) in vasculature development have been implicated based on an analysis of Arabidopsis BR mutants and suspension cells of Zinnia elegans. However, the effects of BRs in vascular development of a woody species have not been demonstrated. In this study, 24-epi brassinolide (BL) was applied to the vascular cambium of a vertical stem of a 2-year-old Liriodendron, and the resulting chemical and anatomical phenotypes were characterized to uncover the roles of BRs in secondary xylem formation of a woody species. The growth in xylary cells was clearly promoted when treated with BL. Statistical analysis indicated that the length of both types of xylary cells (fiber and vessel elements) increased significantly after BL application. Histochemical analysis demonstrated that BL-induced growth promotion involved the acceleration of cell division and cell elongation. Histochemical and expression analysis of several lignin biosynthetic genes indicated that most genes in the phenylpropanoid pathway were significantly down-regulated in BL-treated stems compared to that in control stems. Chemical analysis of secondary xylem demonstrated that BL treatment induced significant modification in the cell wall carbohydrates, including biosynthesis of hemicellulose and cellulose. Lignocellulose crystallinity decreased significantly, and the hemicellulose composition changed with significant increases in galactan and arabinan. Thus, BL has regulatory roles in the biosynthesis and modification of secondary cell wall components and cell wall assembly during secondary xylem development in woody plants. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Brassinosteroids (BRs) refer to a group of polyhydroxylated plant steroidal lactones involved in various developmental events, including embryogenesis, cell division, skotomorphogenesis, photomorphogenesis, vascular system differentiation, reproductive development, and stress tolerance processes (Clouse and Sasse, 1998; Grove et al., 1979). BR-deficient mutants display characteristic phenotypes such as dwarfism, reduced fertility, abnormal vascular apparatus, and altered stress responses (Li et al., 1996; Szekeres et al., 1996). 24-epi Brassinolide (BL(1)) (Fig. 1), the most bioactive compound among BRs, and its precursor castasterone, ⇑ Corresponding authors. Address: Department of Biotechnology, College of Life Science and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136701, Republic of Korea. Tel.: +82 2 3290 3482. E-mail address: [email protected] (M. Kwon). http://dx.doi.org/10.1016/j.phytochem.2014.02.003 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

initiate a signaling cascade on direct or indirect binding to the brassinosteroid insensitive 1 (BRI) receptor, which is a leucine-rich repeat receptor kinase located in the plasma membrane (Friedrichsen et al., 2000; Li and Chory, 1997). A similar membrane-bound kinase BRI-ASSOCIATED KINASE1 (BAK1) binds to BRI to form a heterodimer in the BR receptor complex (Nam and Li, 2002). BRI1 functions as positive regulator because overexpression of BRI1::GFP causes BR-induced cell elongation of Arabidopsis and increases the number of BL(1) binding sites in the membrane fraction (Wang et al., 2001). BAK1 is implicated as a positive regulator in the BR signaling pathway, because a T-DNA insertional mutant of BAK1 causes weak dwarfism in Arabidopsis and the dominant mutation bak1-1D partially suppresses the dwarf phenotype of bri1–5 (Nam and Li, 2002). Genetic screens using brassinazole (brz), which is a BR biosynthetic inhibitor, led to isolation of two dominant mutations, brassinazole resistant 1 (bzr1, Wang et al., 2002) and bri1-EMS-Suppressor1 (bes1/bzr2, Yin et al., 2002). The

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Fig. 1. Application of BL(1) 24-epibrassinolide(1) to the stems of 2-year-old Liriodendron. (A) The structure of BL(1). (B) A mixture of BL(1)in lanolin was applied to debarked stem tissues with application sites protected with aluminum foil. (C) Growth promotion of BR(1)-treated stems (1 ng BL(1) per unit area 5 cm2) for 3 weeks. Growth promotion of BL(1)-treated Liriodendron was obvious after 1 week of treatment, with the best effects observed after 3 weeks under the conditions tested.

nuclear-localized transcription factor BZR1 is regarded as a positive regulator because the dominant mutation bzr1-D suppresses BR-deficient det2 (De-etiolated2), BR-insensitive bri1 (Nam and Li, 2002), and bin2 (BR-insensitive 2) mutants (He et al., 2002). BES1, a BZR1 homolog, is a positive regulator because the dominant mutant bes1-D displays constitutive BR responses and exhibits an elongated phenotype (Yin et al., 2002). Several negative regulators, such as BIN2 and BRS1, also are reported. The semidominant mutant bin2 results in a BR-insensitive dwarf phenotype (Li et al., 2001). Genetic and biochemical analyses of Arabidopsis BIN2 demonstrate its negative effects on BR signaling by controlling phosphorylation status of the BIN2 substrates BZR1 and BES1 (He et al., 2002; Li and Nam, 2002; Wang et al., 2002). Recent studies suggest that BR might be an important regulator for vascular development. For example, brz suppresses xylem differentiation but promotes phloem differentiation in cress (Nagata et al., 2001). The transcripts of several tracheary element (TE) differentiation-related genes (i.e., TED2 and TED3) accumulate in cultured Zinnia cells in media containing uniconazole, which is an inhibitor of BR synthesis (Yamamoto et al., 1997). In addition to evidence from studies of cultured cells, the phenotypic analyses of Arabidopsis BR mutants strongly support a possible role of BR in xylem differentiation in plants. For example, the BR-deficient mutant dwf7-1 has fewer and irregularly located vascular bundles, whereas wild-type Arabidopsis contains eight evenly spaced vascular bundles (Choe et al., 1999). The BR signaling mutants bri1 and bin2 contain a modified number of vascular bundles (Ibañes et al., 2009). Three BRI1 homologs (BRL1, BRL2/VH1, and BRL3) show preferential expression in vascular cells (Caño-Delgado et al., 2004; Clay and Nelson, 2002). Many studies suggest that BR might be an essential regulator of the biosynthesis of cell wall components, such as cellulose and lignin. Several BR-dependent CesA genes have been isolated in Arabidopsis by chromatin immune precipitation experiments (Xie et al., 2011). In the case of lignin, the sterol-deficient Arabidopsis mutant seedlings fackel (fk), hydra 1 (hyd1), and sterol methyltransferase1/cephalopod (smt1/cpd) accumulate ectopic lignin (Schrick et al., 2004). The T-DNA insertional loss-of-function mutant of DIM1/DWF1/CBB1 is involved in the conversion of 24-methylen-

echolesterol to campesterol (Klahre et al., 1998), and displays a dwarf phenotype with up to 38% and 23% reduction in total lignin and cellulose, respectively (Hossain et al., 2012). Arabidopsis overexpressors harboring 35S::BRI1-GFP and 35S::BES1-GFP contain an increased amount of cellulose, which is up to 7% and 3% higher, respectively, than that of the wild-type counterpart (Xie et al., 2011). The CesA promoter-driven GUS expression is increased on BR-containing media (Xie et al., 2011). Although xylem differentiation and associated cell wall biosynthesis is regarded as a typical example of BR-dependent development, experimental data to support the role of BR in secondary cell wall formation during xylem development have been generated primarily from cell cultures or Arabidopsis thaliana mutants. Plant growth is severely deficient in the Arabidopsis BR mutants, which presents difficulties for the evaluation of a direct role of BR in cell wall biosynthesis during xylem differentiation. Thus, this study was designed to determine the role of BR in secondary xylem development in a tree species, which contain the most well developed plant vascular system in the secondary xylem. 24-epi Brassinolide BL(1) has pleiotropic effects on the biosynthesis and gene expression of secondary cell wall components in Liriodendron. This suggests an essential role of BRs in secondary xylem development in woody plants. Results and discussion Exogenously applied BL(1) induces growth in Liriodendron stem BL(1) is the most active BR in plants and promotes growth (Choe, 2004). In some plant species, castasterone, a direct precursor of brassinolide, is an active BR and a final product in the BR biosynthetic pathway (Kim et al., 2008, 2004). To determine whether BL(1) (Fig. 1A) is active and causes typical BR-induced growth promotion in Liriodendron, it was applied directly to the secondary xylem of 2-year-old Liriodendron stems after debarking (Fig. 1B). A preliminary study was performed to determine the optimum amount of BL(1) that induced cell elongation. The typical BL(1) response was induced by 0.1 to 50 ng BL(1) in 5 ml lanolin paste (which corresponded to 1 ng per 5 cm2). Therefore, this amount

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was evenly applied to the exposed vascular cambium (Supplementary Fig. S1). The optimum amount of BL(1) that induced BR-effect phenotypes in Liriodendron stem was observed with 1 ng BL(1) in lanolin paste per 5 cm2 of exposed xylem application area (Supplementary Fig. S1). Therefore, 1 ng BL(1) in lanolin paste per 5 cm2 was applied in a time-course study for up to 3 weeks (Fig. 1C) in a greenhouse with the average temperature maintained at 20– 22 °C and natural light conditions. Growth was clearly promoted in the BL(1)-treated Liriodendron after 1 week of treatment (Fig. 1C). However, the best effects were observed at 3 weeks under the same cultivation conditions (Fig. 1C). The length of xylary cells (fiber and vessel elements) in the newly formed xylem was measured to determine whether the increased growth was achieved via cell elongation. To avoid latewood of secondary xylem that formed during the previous year of BL(1) treatment, secondary xylem was carefully harvested from the outmost layers under a microscope with a digital camera (Zeiss Axio Imager M1). As shown in Fig. 2, the lengths of fiber and vessel elements increased slightly in BL(1)-treated samples compared to those of the corresponding mock controls (Fig. 2). Statistical analysis indicates that both types of xylary cells were significantly elongated in BL(1)-treated secondary xylem compared to that in controls. After 1 week, fiber and vessel elements were 13% and 7% longer in BL(1)-treated samples, respectively, and these differences were significant (Table 1). After 3 weeks, fiber and vessel elements were 10.9% and 10.1% longer in BL(1)-treated samples than in controls (Table 1). Although there were some variations of individual plants between the 1-week and 3-week samples, the increase in cell length in response to BL(1) treatment seems to be its effect, because differences are statistically significant in both cell types (Table 1). These results show that the exogenously applied BL(1) promotes growth via cell elongation in the secondary xylem of 2-year-old Liriodendron. The effects of exogenous BL(1) treatment are cell type-specific Next, cross-sections of 2-year-old Liriodendron stem from four individual lines for each treatment were prepared and observed under light microscopy to determine gross morphological changes

Table 1 Statistical analysis of cell length. Lengths of fibers and vessel elements increased significantly by BL(1) application. The biological replicate was three plants for each treatment. Light photomicrographs were taken of more than 100 views in each individual plant. Approximately 100 individual fiber cells and 50 individual vessel elements were measured for each plant after maceration. (Comparison between BL(1)-treated and corresponding mock control.) Weeks

Fiber lengtha (lm)

Length of vessel elementa (lm)

Mock

BL(1)

Mock

BL(1)

1 3

817.77 ± 4.13** 786.48 ± 4.29 

923.95 ± 4.97** 871.93 ± 4.45 

499.88 ± 4.72** 496.36 ± 4.45*

534.70 ± 4.37** 546.31 ± 4.08*

a Data are means ± standard deviations for three individual plants. Significance was analyzed using a nested ANOVA procedure. ** P < 0.01. * P < 0.05.   P < 0.1.

in response to BL(1) treatment. As shown in Fig. 3A, secondary xylem developed normally under the debarking treatment and the vascular cambium recovered in 3 weeks after debarking (Fig. 3A, left panel). The amount of secondary xylem increased in the BL(1)-treated sample compared to that of the mock control (Fig. 3A). The diameter of the fibers increased significantly, whereas the diameter of the vessel elements decreased slightly in the developing xylem of BL(1)-treated stems compared to the control (Fig. 3A). The reduction in vessel element diameter was more visible after 3 weeks, and made it difficult to distinguish between fiber and vessel elements in cross-section images (Fig. 3A). Because BR controls cell division (Clouse and Zurek, 1991; Hu et al., 2000), the number of cells in the newly developing xylem was counted in cross-sections (Fig. 3B). Twenty spots were analyzed in four individuals for each treatment. The number of fibers increased significantly, whereas the number of vessel elements decreased in BL(1)-treated samples (Fig. 3B). However, the overall number of xylary cells per unit area increased in BL(1)-treated samples compared to that of the mock controls (Fig. 3B, bottom). This was achieved primarily by increases in the number of fiber cells in the developing secondary xylem (Fig. 3B). These results indicate that growth promotion in BL(1)-treated stems was

Fig. 2. Representative images of macerated xylary cells in the newly developing xylem of Liriodendron stem. Newly developing xylem was separated into individual cells using Schulze’s reagent. BR(1)-treated sample displayed increase in length of fiber compared to that of the mock controls. The biological replicates were three plants for each treatment. Light photomicrographs were taken for more than 100 views in each individual plant. Abbreviations: f, fiber cell; v, vessel elements. Bars = 200 lm.

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Fig. 3. BL(1)-induced secondary xylem development with increased cell division. (A) Toluidine blue stained cross-sectional views of a stem after exogenous application of BL(1). (B) Number of cells per unit area (100 lm2). Significance levels indicated by one asterisk (⁄P < 0.05). Cell number was investigated using cross-sectional views. A total of 20 spots of 100 lm2 was selected on a picture from 5–8 sections for each application. Abbreviations: dsx, newly developing secondary xylem; lw, latewood; f, fiber cell; v, vessel elements. Bars = 200 lm.

achieved by cell elongation and by accelerating cell division with preferential increase in fiber cell types. Exogenous BL(1) causes reduced lignification of secondary xylem Several previous studies using cell cultures (Yamamoto et al., 1997) and Arabidopsis (Goda et al., 2004; Schrick et al., 2004) suggest that BR controls lignin biosynthesis. For example, the steroldeficient Arabidopsis mutant fackel displayed ectopic lignin deposition (Schrick et al., 2004). Hossain et al. (2012) demonstrated that loss-of-function mutation of DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis and similar to the cell elongation factor, caused a dwarf phenotype with up to an 38% reduction in lignin compared to that in a wild-type control. Therefore, histochemical staining was performed with phloroglucinol–HCl and Mäules reagent. No differences were observed between BL(1)-treated samples and mock controls at 1 week when stained with phloroglucinol–HCl, which mainly stains coniferaldehyde (G) units in hardwood species (Fig. 4). However, after 3 weeks, developing secondary xylem was more weakly stained in the BL(1)-treated sample compared to that of the mock control (Fig. 4, bottom). Weaker staining was more prominent in fiber cell types (Fig. 4, bottom), which suggests a reduction in G units of lignin primarily in the fibers, rather than in vessel elements. This result indicates that the effect of BL(1) in the reduction of G units is cell type specific. A reduced number of vessels with decreased diameter was observed in the cross-sections (Fig. 4), consistent with Fig. 3B. Next, when the tissues were stained with Mäules reagent, which stains sinapyl aldehyde (S) units in hardwood lignin, significantly weak stains were observed in vessel elements, especially at 3 weeks after BL(1) treatment compared to that of the mock control (Fig. 5). Similarly to the preferential decrease of G units in the fiber cell type, the decrease in S units occurred mainly in vessel elements in BL(1)-treated samples (Fig. 5). Therefore, exogenously applied BL(1) caused a reduction in overall lignin deposition in the developing secondary xylem in a cell type-specific manner. The histochemical analysis suggested a reduction in lignin content and altered lignin monomeric composition in different cell types. Chemical analysis was performed to determine the amount

and composition of lignin. First, Klason lignin was analyzed from the tissues at the application site together with those of the upper region (up to 10 cm from the application site), because tissues at the application site were insufficient for Klason lignin analysis. Prior to including the tissues from the upper region for Klason lignin analysis, the effects in the upper region of exogenously applied BL(1) were tested by measuring the cell lengths of both types of xylary cells. Exogenously applied BL(1) was assumed to be transported acropetally along the xylem based on an autoradiographic examination of uptake and transport of 14C-labeled BL(1) in intact seedlings of cucumber and wheat (Nishikawa et al., 1994). The same pattern of cell elongation of fibers and vessel elements was consistently observed in the upper region and at the application site (data not shown). Total Klason lignin content decreased slightly by up to 6.7% and 19.4% in the BL(1)-treated stems at 1 and 3 weeks, respectively, compared to those of mock controls (Fig. 6). Because the previous histochemical analysis indicated a change in monolignol composition after BL(1) treatment (Figs. 4 and 5), the lignin monomeric composition was analyzed using derivatization followed by reductive cleavage (DFRC). The DFRC method was selected owing to efficient cleavage of arylglycerol-b-aryl (b-O-4) ether linkages in lignins (Lu and Ralph, 1997). The total lignin content was significantly lower in BL(1)-treated samples than in control samples at 1 and 3 weeks (Table 2), consistent with the histochemical analysis (Figs. 4 and 5). The S/G ratio between BL(1)-treated samples was similar to that of the controls at 1 week (Table 2). After 3 weeks of application, the reduction of the S/G ratio was up to 30% greater than that of mock control, which suggests a greater reduction of S unit than G unit. Taken together, the results of total lignin content and lignin monomeric composition are in agreement with the histochemical analysis of lignin (Figs. 4 and 5). Effects of exogenously applied BL(1) on the expression of lignin biosynthetic genes BL(1) application significantly reduced the total lignin content and altered the monomeric composition at the cellular level. Therefore, expression of lignin biosynthetic genes was analyzed

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Fig. 4. Lignin histochemical analysis using phloroglucinol–HCl reagent for p-hydroxyphenyl (H) and coniferaldehyde (G) units in the newly developing xylem of the BL(1)treated stem. Newly developing secondary xylem was more weakly stained in the BL(1)-treated samples compared to that of the mock control, particularly at 3 weeks. Abbreviations: dsx, newly developing secondary xylem; lw, latewood; f, fiber cell; v, vessel elements. Bars = 200 lm.

Fig. 5. Lignin histochemical analysis using sinapyl aldehyde (S) unit specific Mäule’s stain in newly developing xylem from BL(1)-treated stem. Mäule’s stain showed that lignin deposition decreased slightly in both types of xylary cells. Abbreviations: dsx, newly developing secondary xylem; lw, latewood; f, fiber cell; v, vessel elements. Bars = 200 lm.

by quantitative real-time RT-PCR. The Liriodendron EST was previously constructed from the specialized secondary xylem of 2-yearold Liriodendron, and the sequence information of the lignin biosynthetic genes is available (Jin et al., 2011). Using the primers listed in Jin et al. (2011), quantitative real-time RT-PCR was conducted in tissues harvested only from the application site.

The expression of most of the lignin biosynthetic genes in the BL(1)-treated stem was significantly down-regulated at the transcriptional level compared to that in the control at 1 week (Fig. 7A). However, transcriptional levels gradually increased up to the level of mock control over time (Fig. 7A). The expression of putative COMT homologs was significantly affected by the BR

H. Jin et al. / Phytochemistry 101 (2014) 40–51

Fig. 6. Changes in total cell wall polysaccharides and lignin by BL1 24-epi brassinolide(1) application. Bars represent percentage (%) of the major component without minor components including non-polar solvent and hot-water soluble extracts (polar extracts). Total cell wall polysaccharides increased slightly, whereas lignin content decreased in the BL(1)-treated stem compared to that in controls. ⁄ Numerical values of total polysaccharides and lignin were calculated using a correction factor according to Shin and Cho (2008).

Table 2 Lignin monomeric compositions analyzed by derivatization followed by reductive cleavage. BL(1) treatment decreased total lignin content and altered the S/G ratio. In 3 week BL(1)-treated samples, lignin deposition and amount of major angiosperm monolignol (S+G unit) were significantly decreased, and these changes were mainly attributed to S unit change. Two replicates with four individual plants were used. Week

G unit (lmol/g)

S unit (lmol/ g)

S+G (lmol/ g)

S/G ratio

1

Mock BL

120.7 ± 2.8 101.6 ± 6.0

165.3 ± 4.1 132.2 ± 2.4

284.7 ± 1.3 232.8 ± 3.6

1.4 ± 0.1 1.3 ± 0.1

3

Mock BL

128.8 ± 1.1 108.1 ± 10.4

140.5 ± 4.3 84.2 ± 0.6

268.2 ± 5.3 191.6 ± 9.9

1.1 ± 0.0 0.8 ± 0.1

treatment (Fig. 7). For example, the transcript level of CL83Contig2 was lower in the BL(1)-treated sample at 1 week, but higher at 3 weeks (Fig. 7). By contrast, the expression of CL145Contig1 was lower in BL(1)-treated samples than in controls, and the differences became larger at 3 weeks (Fig. 7). For CL570Contig1, the expression was lower in BL(1)-treated samples than in controls at 1 week, but became similar between BL(1)-treated samples and controls at 3 weeks (Fig. 7). Expression of TW3-1a-T3_G01 was slightly lower in the BL(1)-treated sample at 1 week, but higher in the BL(1)-treated sample at 3 weeks (Fig. 7). Expression of TW12-4a-T3_H18, which is phylogenetically similar with PtrFOMT1 (flavonoid O-methyltransferase, XP_002312933.1), was significantly higher at 1 week but lower at 3 weeks in BL(1)-treated samples (Fig. 7A). Therefore, BL(1) treatment induces differential effects for COMT homolog expression in the stem of Liriodendron. Differential effects of COMT expression by BL(1) treatment suggest a possible role of BR in the control of the degree of monolignol methoxylation. Among five COMTs that were originally isolated from the EST constructed from 2-year-old Liriodendron stem (Jin et al., 2011), CL145Contig1 and CL83Contig2 were the major COMTs in the stem of 2-year-old Liriodendron because their expression levels were relatively higher than others as determined by copy numbers under normal growth conditions (Fig. 7B and Supplementary Table S2). Interestingly, two major wood-abundant COMTs, CL145Contig1 and CL83Contig2 are highly homologous to

45

At5g54160 (AtCOMT1), which has been proposed as 5-hydroxyconiferaldehyde O-methyltransferase that is involved in syringyl lignin synthesis for the methylation of both 5hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compounds (Nakatsubo et al., 2008). Therefore, reduction of S/G ratio correlated to the down-regulation of two major COMTs at transcriptional level in the BL(1)-treated sample. It is also interesting that decrease of F3H expression is much significant that that of C3H (Fig. 7). Since C-3 and C-5 positions are needed to be hydroxylated prior to methylation in the biosynthesis of coniferyl (G) and sinapyl (S) units, the remarkable reduction of F3H expression than that of C3H are also possible to attribute the reduction of S/G ratio in BL(1)-treated sample. Therefore, alteration of C3H and F3H expression upon BL(1) treatment needs to be analyzed further for BLmediated alteration of monolignol composition. In addition to the monolignol biosynthetic genes, genes for the monolignol coupling process were significantly affected at the transcriptional level by exogenously applied BL(1) (Fig. 7A). For example, LtLac2-1 (U73103), LtLac2-3 (U73105), and LtLac2-4 (U73106), which have been functionally characterized in cell wall lignification in Liriodendron (LaFayette et al., 1999), were significantly down-regulated at the transcriptional level during BL(1) application. A putative laccase (CL474contig1) isolated from the EST constructed from Liriodendron secondary xylem (Jin et al., 2011) was significantly down-regulated in response to BL(1) treatment. Therefore, BR affects monolignol biosynthesis and the dehydrogenative polymerization process during cell wall lignification at the transcriptional level. Taken together, BR down-regulated most of the lignin biosynthetic genes except COMTs. This may have contributed to the reduction of total lignin content and changes in monolignol composition in the developing secondary xylem. BR induces modification of the cell wall carbohydrate composition in secondary xylem The analysis of BR- and sterol-deficient mutant seedlings suggests that BR might up-regulate cellulose biosynthesis in Arabidopsis. The BR-deficient mutant dim1 has a dwarf phenotype with up to a 23% reduction in cellulose (Hossain et al., 2012). The reduction in cellulose was caused by BL(1), based on the result of CesA promoter-driven GUS expression in Arabidopsis and the increased amount of BRI1-GFP in BL-containing medium (Xie et al., 2011). Therefore, the amount of cell wall carbohydrates was measured to determine whether BR regulates cellulose biosynthesis in the secondary xylem of tree species. The same tissues (from the application site including the upper region) that were used for lignin analysis were used for the cell wall carbohydrate analysis. The total polysaccharide content increased slightly by 5% and 9.3% in BL(1)treated stems at 1 and 3 weeks, respectively (Fig. 6). The amount of cellulose, which was calculated based on the amount of glucan (Shin and Cho, 2008), did not increase at 1 or 3 weeks, but was reduced by BL(1) application compared to that in mock controls (Table 3). Because secondary xylem contains other cell wall polysaccharides, (hemicellulose and pectin), the amount of various hexoses (galactose and mannose), pentoses (xylose and arabinose), and galacturonic acid were analyzed. As shown in Table 3, the amount of xylan, the main hemicellulose backbone and a major non-cellulosic polysaccharide in angiosperms (Ebringerová and Heinze, 2000), increased in BL(1)-treated secondary xylem by up to 16.8% and 5.6% compared to that of the corresponding controls at 1 and 3 weeks, respectively. The amounts of galactan and arabinan, which are major components for pectin and the side chain of hemicellulose, increased significantly in BL(1)-treated secondary xylem of Liriodendron. Galactan increased up to 3.2% and 34.6% in response to BL(1) treatment compared to that in the corresponding controls at 1 and 3 weeks, respectively. Arabinan increased up to

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H. Jin et al. / Phytochemistry 101 (2014) 40–51

Fig. 7. Expression analysis of selected genes for monolignol biosynthesis and dehydrogenative polymerization processes in response to BL(1) application. (A) Fold-changes of lignin biosynthetic gene expression by BL(1) treatment compared to that in controls for 1 week. Most genes, except TW12-4a-T3_H18, were significantly down-regulated at 1 week after BL(1) application. (B) Fold-changes of lignin biosynthetic gene expression by BL(1) treatment compared to that in controls for 3 weeks. CL83Contig2 expression was significantly higher in BL(1)-treated samples than in controls at 3 weeks. The real-time PCR experiments were repeated three times with five replicates for each set of experiment. (C) Quantitative RT-PCR analysis of transcript copy numbers of four COMT genes isolated from the stem of 2-year-old Liriodendron. Among four COMTs, CL83Contig2 and CL145Contig1 were abundantly expressed in stems of 2-year-old Liriodendron stem under normal growth conditions. Transcript abundances of CL83Contig2 showed the strongest differences between mock and BL(1)-treated tissues. The real-time PCR experiments were repeated three times with five replicates.

Table 3 Analysis of cell wall carbohydrates. The amounts of galactan and arabinan significantly increased in BL(1)-treated stems of Liriodendron. Three replicates for more than four individual plants were used. Unit: %. Week

Glucan

Xylan

Mannan

Galactan

Arabinan

Uronic acid

1

Mock BL

59.9 ± 0.3 54.8 ± 0.1

30.9 ± 0.2 36.1 ± 0.5

1.9 ± 0.1 1.0 ± 0.8

3.1 ± 0.3 3.2 ± 0.0

3.0 ± 0.2 3.3 ± 0.2

1.6 ± 0.1 1.7 ± 0.1

3

Mock BL

59.3 ± 0.3 54.2 ± 0.8

32.1 ± 0.3 33.9 ± 0.2

1.6 ± 0.3 2.2 ± 0.1

2.6 ± 0.2 3.5 ± 0.2

2.8 ± 0.2 4.6 ± 0.4

1.6 ± 0.1 1.6 ± 0.1

All values in this table indicate the percentage of each component, with total polysaccharide regarded as 100%.

10% and 64.3% in response to BL(1) treatment compared to that in the corresponding controls at 1 and 3 weeks, respectively. Therefore, the analysis of cell wall carbohydrates strongly suggests that BL(1) application results in an increasing amount of non-cellulosic cell wall carbohydrates such as hemicellulose and pectin, rather than cellulose.

The increase amount of non-cellulosic carbohydrates, i.e., xylan, galactan, and arabinan, may reflect that BL(1) affect secondary cell wall integrity. For example, xylan influences the helicoidal orientation of the cellulose microfibril (Reis and Vian, 2004) and is an important component for cell wall strength (Mortimer et al., 2010). By contrast, galactan and arabinan are important side chains

H. Jin et al. / Phytochemistry 101 (2014) 40–51 Table 4 Lignocellulose crystallinity index measured by X-ray diffraction spectroscopy. The crystalline index (CI) was calculated by the Segal method as follows: CI ¼ 100  ½ðI200  IAM Þ=I200 , using the height of the 200 peak (I200, 2h = 22.16°) and the minimum between the 200 and 110 peaks (IAM, 2h = 18°). Lignocellulose crystallinity was significantly lower in BR-treated secondary xylem than that in the control. Comparison between control and BL(1)-treated sample for each treatment. 1 week

Mock BL Reduction (%)

3 weeks

I200

IAM

CI

I200

IAM

CI

220 260

135 191

38.6 26.5 31.3

254 254

146 152

42.5 40.6 4.5

of pectin and hemicellulose. In addition, lignocellulose crystallinity has been proposed to be closely associated with increases in arabinan (Xu et al., 2012). An increase of arabinan in BL(1)-treated stems of Liriodendron is an important result because pectic arabinans, together with pectic galactan, have been proposed to contribute to cell wall properties in terms of matrix porosity, maintenance of flexibility, mechanical properties, and cell adhesion events (Devaux et al., 2005; Fenwick et al., 1999; Ha et al., 2005; Jones et al., 2003; Verhertbruggen et al., 2009). In addition, recent studies propose that some of the components of hemicellulose are essential for secondary cell wall integrity, as Arabidopsis irregular xylem8 mutants deficient in these components have a collapsed xylem and a dwarf phenotype (Peña et al., 2007; Persson et al., 2007). Therefore, increased amounts of xylan, galactan, and arabinan in BL(1)-treated samples provide an extensive pectin and hemicellulose network to contribute, at least partially, to maintenance of cell wall integrity.

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BR caused reduction of lignocellulose crystallinity in secondary xylem Since analysis of cell wall components imply that BL(1) affects cell wall integrity, lignocellulose crystallinity was measured by X-ray diffraction analysis, a well-established method for determining the crystallinity of partially crystalline materials such as wood (Andersson et al., 2003). The cellulose crystallinity was calculated as crystallinity index (CI), the ratio of the amount of crystalline cellulose to the total amount of sample including crystalline and amorphous cellulose, lignin, hemicellulose, pectin, etc. With the intensities of the diffraction bands (Supplementary Fig. S2), CI was calculated based on the equation, CI ¼ 100  ½ðI200  IAM Þ=I200 , using the height of the 200 peak (I200, 2h = 22.16°) and the minimum between the 200 and 110 peaks (IAM, 2h = 18°) according to the Segal method (Segal et al., 1959). Lignocellulose crystallinity was significantly lower in BL(1)-treated secondary xylem than that of the control, with 31.3% and 4.5% reductions at 1 and 3 weeks, respectively (Table 4). Because the wood sample has different crystallinity mainly by the arrangement of cellulose microfibrils, changes in crystallinity believe to be caused by the reduction of cellulose crystallinity in wood samples. Therefore, BR affect the amount and composition of cell wall carbohydrates, and the overall cell wall network including lignocellulose crystallinity, especially at an early stage of secondary cell wall development. BL(1)-induced reduction of lignocellulose crystallinity is desirable trait for lignocellulose conversion to biofuel. Therefore, quantitative real-time RT-PCR was performed to isolate genes that might be important for these modifications. Even with limited information of genes involved in cell wall modification in Liriodendron, it is noteworthy that several pectin methylesterase homologs are significantly down-regulated at the transcriptional

Fig. 8. Expression analysis of selected genes for cell wall carbohydrate biosynthesis and BR signaling cascade. (A) Fold-changes of genes involved in cell wall biosynthesis and modification. (B) Fold-changes of genes involved in brassinosteroid signaling pathway. Expression of positive regulators including BRI1, BAK1, BES1/BZR1 homolog protein, and BIM2 increased slightly at 1 week after BL(1) application. Negative regulators, such as BIN2 and BRS1, did not increase expression significantly at 1 week after BL(1) treatment; their expression level was regulated in a time-dependent manner during the long-term application. The real-time PCR experiments were repeated three times with five replicates for each set of experiment. Asterisks indicate significant differences compared with the mock sample (⁄⁄⁄P < 0.001; ⁄⁄P < 0.01; ⁄P < 0.05; t-test and wilcoxon test).

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level by BR treatment i.e., BL(1) (Fig. 8A), since pectin methylesterase and pectin esterase inhibitor were proposed to be involved in BR-mediated cell wall extensibility (Wolf et al., 2012). Wolf et al. (2012) proposed that plant cell wall relaxation and stiffening are controlled by BR-mediated feedback regulation, in which pectin methylesterases and pectin methylesterase inhibitor protein play important roles by controlling the formation of Ca2+-pectate crosslinks in the cell wall. One homolog for a-expansin was analyzed because it is involved in the dissociation and cell wall loosening of some types of polysaccharide complexes (Xu et al., 2012). The transcript level of a-expansin was down-regulated by BL(1) treatment (Fig. 8A). Previously, a-expansin genes were induced by BL(1) in chickpea epicotyl section (Sánchez et al., 2004). Transcription analysis of a-expansin with only one homolog is not enough to provide any insight for the possible involvement of BR in cell wall extensibility. However, a-expansin was reported to have a synergistic effect with pectin lyase for cell wall loosening and thus extensibility in vitro (Wei et al., 2010). Because the number of ESTs was limited in the Liriodendron stem, further analysis of the transcript network and functional genomics might be necessary to gain insight into the regulation of BR-induced cell wall modification and growth in the secondary xylem of tree species. Exogenously applied BL(1) induced BR signaling in the stem of Liriodendron The method of applying BL(1) herein makes it difficult to determine the actual amount of absorbed BR. Overdoses of applied BR can cause a negative effect; therefore, the expression of positive and negative components in the BR signaling cascade was analyzed at the transcriptional level. Because the EST for only a few BR signaling components such as BES1/BZR1, BIM2, and BIN2 were available (Jin et al., 2011, Supplementary Table S1), quantitative real-time RT-PCR was performed using tissues harvested from the BL(1) application site. The expression of positive BR signaling regulators (i.e., BRI1, BAK1, BZR1/2, and BIM2) increased at 1 week after BR treatment (Fig. 8B), suggesting that exogenously applied BL(1) induced BR signaling for 1 week. However, expression of the negative regulators BIN2 and BRS1 did not increase their transcriptional level for 1 week (Fig. 8B). Therefore, exogenously-applied BL(1) induced BR signaling in the debarked stem of 2-year-old Liriodendron as expected. However, as exposure time to BR was extended to 3 weeks, expression levels of BAK1, BES1, and BIM2 were significantly reduced compared to those of controls, whereas BRI1 still maintained a higher level in the BR-treated sample than in the control sample (Fig. 8B). On the other hand, transcript levels of two BIN2 homologs and one BRS1 homolog were slightly higher in the BL(1)-treated sample than in control after 3 weeks of treatment. BRS encodes serine carboxypeptidase, which mediates BR inactivation at the cell surface (Zhou and Li, 2005). The higher expression of BRS1 in the BR-treated sample than in the control suggests an inactivation of exogenous BL(1) during long-term exposure. These data suggest that the BR signaling cascade is induced by exogenously applied BL(1). Since debarked stem is not be able to synthesize BR endogenously and BR is rarely detectable even in normal stems with bark tissue, as determined by DWARF4 (DWF4) transcript level (Kim et al., 2006) and expression of the b-glucuronidase (GUS) reporter gene driven by the CPD (CONSITUTIVE PHOTOMORPHOGENESIS AND DWARFISM) promoter (Mathur et al., 1998). Therefore, anatomical and chemical modification associated with BL(1)-induced growth promotion is a result of exogenously applied BL(1) rather than the action of endogenous BL(1).

Concluding remarks Increasing evidence indicates that BRs are involved in the regulation of lignin and cellulose biosynthesis during xylem formation in plants. However, most studies are based on results obtained from herbaceous plants and cultured cells (Hossain et al., 2012; Kubo et al., 2005; Peng et al., 2002; Schrick et al., 2004; Xie et al., 2011). In this study, the effects of exogenously applied BL(1) were first investigated in the stem (secondary xylem) of a tree species that contains more complicated, heterogeneous, and well-developed secondary xylem that greatly differs from that of herbaceous plants. When BL(1) was exogenously applied directly to the debarked stem, pleiotropic changes in cell wall components as well as gross anatomical changes were observed at the cellular level. Exogenously-applied BL(1) induced growth in the stems of 2-year-old Liriodendron that primarily involved increased cell elongation in fiber and vessel elements (Table 1 and Fig. 2) and at least partially accelerated cell division (Fig. 3), as in other herbaceous plants. BL(1) induced alteration of major cell wall components during secondary xylem formation, i.e., reduction of lignin content, decrease of lignocellulose crystallinity, and alteration of hemicellulose composition. In case of lignin content, it can be explained by down-regulation of most of the lignin biosynthetic genes at the transcriptional level (Fig. 7A). A previous microarray analysis of 7-day-old Arabidopsis seedling demonstrated that cinnamyl alcohol dehydrogenase (CAD), which converts p-coumaraldehyde, coniferaldehyde, and sinapyl aldehyde to corresponding alcohol moieties in the lignin biosynthetic pathway, is down-regulated by BR (Goda et al., 2004). Therefore, exogenously applied BL(1) acts as a negative regulator of lignin biosynthesis at transcription level. This result is not consistent with previous research with zinnia cultured cells, which suggest that BR increases lignin biosynthesis, and is involved in the initiation of TE differentiation during xylem formation (Yamamoto et al., 1997). The discordance with previous data obtained from zinnia cultured cells and Arabidopsis dwarf mutants was probably caused by the in vivo nature of secondary xylem of tree species. Alternatively, it can be interpreted that the reduction in lignin and increase in wall polysaccharides is a re-distribution of carbons due to the BL(1) application. Most interestingly, when BL(1) was applied directly to the debarked stem of Liriodendron, the cellulose content in the BL(1)treated stem did not significantly increase compared to that of the control (Table 3). Instead, lignocellulose crystallinity decreased dramatically by BL(1) treatment (Table 4 and Supplementary Fig. S2). On the other hand, the content of xylan, arabinan and galactan were significantly increased upon BL(1) treatment. Since these components are proposed to be associated with cell wall integrity, reduction in lignin content and lignocellulose crystallinity seems to be compromised by the modification of non-cellulosic carbohydrate composition such as xylan, galactan and arabinan upon BL(1) treatment. In conclusion, exogenously-applied BL(1) induced BR signaling successfully and results in cell elongation and cell division in the stems of 2-year-old Liriodendron. Exogenously-applied BL(1) induced significant changes in the chemical composition of secondary xylem, including reduced lignin with alteration of monolignol composition in a cell type-specific manner, modification of hemicellulose composition, and lignocellulose crystallinity. The observed changes upon exogenous BL(1) application may not mirror BR action in intact plants. However, it is clear that exogenously applied BL(1) caused pleiotropic effects on cell wall biosynthesis at least locally. Thus, BL(1) have important regulatory roles in the biosynthesis and modification of secondary cell wall components during secondary xylem development in woody plants.

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Experimental

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under a Zeiss Axio Imager M1 microscope with a digital camera (Carl Zeiss, Oberkochen, Germany).

Reagents 24-epiBrassinolide BL(1) (Fig. 1A) was purchased from Sigma– Aldrich (St. Louis, MO, USA), with stock solutions prepared in aqueous ethanol and stored at 20 °C until application. Just prior to application, BL(1) dissolved in EtOH was mixed with melted lanolin paste according to a previously published protocol (Funada et al., 2008) and applied with a brush. The same amount of EtOH without BL(1) was mixed with melted lanolin and applied in the same way for the mock treatment. Plant materials and BL(1) application Sixty 2-year-old Liriodendron tulipifera plants derived from the same clone were provided by the Korea Forest Research Institute. Each tree was planted in March 2011 in an individual pot (26 cm height  28 cm diameter) containing mixed soil (bed soil and peat moss), and maintained in a glasshouse facility under natural light conditions. Forty plants with similar heights and stem diameters were selected for BL(1) treatment in May 2011. Based on a pilot experiment to determine the optimum amount of BL(1) (Supplementary Fig. S1), lanolin paste containing BL(1) (4 ng) was applied once to a 20 cm2 debarked stem area in the middle of the vertical stem. Lanolin alone was applied as the mock control (Fig. 1B). The same experiment was performed again in March 2012 in the same greenhouse facility. Measuring the lengths of xylary cells The outmost xylem of the stem that formed after BL(1) treatment was carefully harvested with a razor blade and cut into small pieces under a microscope (2 mm  1 mm  3 mm). The specimens were immediately soaked in Schulze’s reagent [6% (w/v) KClO3 in 50% (v/v) nitric acid], and were stored in Schulze’s reagent at room temperature for 1 week. After heating at 60 °C for 30 min, the specimens were washed three times with distilled H2O and shaken vigorously. The dissociated cells in distilled H2O were analyzed and photographed under a Zeiss Axio Imager M1 microscope with a digital camera (Carl Zeiss, Oberkochen, Germany). The length of more than 100 individual cells was measured for fiber and vessel elements, respectively. Length was recorded as means ± standard deviation of at least two independent experiments with more than three replicates. The SAS program was used for calculating the one-way or nested analysis of variance (ANOVA) of cell length data (SAS Institute, Cary, NC, USA). A nested ANOVA procedure was used for the experiment, with the main factor of ‘‘treatment’’ (two levels, mock and BL(1) application), and the nested factor of ‘‘individual plant’’ (three individual plants within each main level), and 100 cell length replicates. The multiple comparison tests were performed with Tukey’s honestly significant difference (HSD) amongst length means of each treatment group.

Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was purified from the stems at the application site as described in Chang et al. (1993). First-strand cDNAs were synthesized from total RNA using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time RT-PCR was performed using a CFX-96™ Real-Time PCR Detection System (Bio-Rad, Munich, Germany). Amplification conditions for PCR consisted of 95 °C pre-incubation for 10 min and 45 cycles of denaturation at 95 °C for 10 s, annealing at 56–60 °C for 5 s, extension at 72 °C for 9 s, with a final extension step at 72 °C for 5 min followed by a temperature ramp from 72 to 95 °C, with a 5 s hold at each 1 °C step for melting curve analysis. For each reaction, diluted cDNA (2 ll) was used in a ready-to-use PCR reaction mixture (20 ll) containing iQ™ SYBRÒ Green Supermix (Bio-Rad, Munich, Germany). Gene expression fold-changes in response to BL(1)-lanolin-treated tissues relative to that of lanolin-treated tissues were analyzed using the delta-delta CT method (Livak and Schmittgen, 2001). Expression of the actin gene (JG567591) served as an internal control to determine RT-PCR amplification efficiency. For lignin biosynthetic, BR(1) signaling, and cell wall carbohydrate-related genes, the same primers reported in Jin et al. (2011) were used. Primers for cellulose synthase-like protein (JZ164902), b-D-galactosidase (JZ164903), and three BIN2 homologs (JZ164904, JZ164905, and JZ164906) were designed based on the Liriodendron expressed sequence tag (EST); these primers are listed in Supplementary Table S1. Real-time data with biological replicates for each experiment analyzed for statistical analysis according to the method described by Yuan et al. (2006). T-Test and wilcoxon test was used to assess significant difference in gene expression. These tests with DCt were performed using SAS program (SAS Institute, Cary, NC, USA). Transcript copy number analysis of COMT The molecules containing the target PCR product of each caffeic acid 3-O-methyltransferase (COMT) were constructed using pGEM-T Easy Vector system (Promega, Madison, WI, USA). The concentration of the PCR product was estimated by OD260, and the number of copies/ll of standard were calculated according to the method described by Yin et al. (2001). Plasmid DNAs containing target PCR product of each COMT gene were used as the standards for establishing a quantitative correlation between the copy numbers and the Ct values. Transcript copy numbers were calculated from the plasmid concentrations after a serial dilution (10, 102, 103, 104, 105, 106, 107, and 108 copies per ll). To estimate the copy number of COMTs, quantitative real-time RT-PCR analysis was performed using the previously described method. The ANOVA procedure and Tukey HSD test using SAS program was used for assessing significant difference of COMT copy number data (SAS Institute, Cary, NC, USA).

Anatomical and histochemical analysis

Analysis of total lignin content and cell wall monosaccharides

Wood blocks from the application site were harvested and processed for histochemical analysis according to the method described by Jin and Kwon (2009). After infiltration and embedding with paraffin, thick sections (10 lm) were obtained and stained either with toluidine blue or for lignin histochemical analysis. The serial sections for histochemical analysis of lignin were stained with either phloroglucinol–HCl (Krishnamurthy, 1999) or Mäule’s reagent (Meshitsuka and Nakano, 1979). Photographs were taken

Minor components in wood cells were first removed with acetone and hot-water extraction according to Moon et al. (2011). Briefly, tissues (2 g) from the application site and upper sites (up to 10 cm from the application site) were mixed to prepare sufficient wood tissue for chemical analysis, ground through a 40–60 mesh, and then extracted with acetone ((20 ml) 99.9%, reagent grade) for 8 h at room temperature. After filtration through Whatman no. 2 filter paper, the acetone-extracted samples were

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extracted again in boiling H2O for 2 h and filtered. Prepared extractive-free samples (200 mg) were air dried and allowed to react with 72% H2SO4 (1.5 ml) for 2 h at 20 °C. The reaction solution was diluted to 3% H2SO4 with boiling distilled H2O, the sample solution was boiled for 3 h, and then cooled to room temperature overnight to precipitate Klason lignin. After filtering through a 1G4 porcelain crucible filter, the residue in the filter was assessed for Klason lignin content (TAPPI Standard 222 om-88). Wood powder (0.2 g) from the application site and the upper stem were hydrolyzed with 24.0N H2SO4 and diluted with deuterium oxide (D2O) as a nuclear magnetic resonance (NMR) solvent for 1HNMR spectroscopic analysis to analyze cell wall monosaccharide composition (Shin and Cho, 2008). The specific NMR (500 MHz) conditions were as follows: Broadband Observe Probe type, 30 °C, 90°, 11 lm pulses, 10 s delay between pulses, 2.73 s acquisition time, and 10 ppm sweep width. All data were mean values of three determinations. Monosaccharide composition in cell walls was calculated based on the interpretation of the 1H-NMR spectra at the anomeric hydrogen peak integrals as described previously (Shin and Cho, 2008).

Acknowledgments We thank Dr. Insik Kim and Youngwook Kim at the Korea Forest Research Institute for providing Liriodendron seedlings for these experiments, and Dr. Ryo Funada at Tokyo University of Agriculture and Technology for providing a detailed hormone treatment protocol for tree stems. This study was supported by a Korea Research Foundation Grant (2012R1A1A3001418) funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) and with financial support of ‘Forest Science & Technology Projects (S111212L18010) Grant provided by the Korea Forest Service.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 02.003.

References Analysis of lignin monomeric composition For analysis of lignin monomeric composition, the DFRC analysis was performed as described by Lu and Ralph (1997). Briefly, a sample (20 mg) was added to a 10 ml round-bottom flask containing AcBr stock solution [CH3COOBr/CH3COOH, 8:92, (v/v)]. The mixture was gently stirred for 2 h at 50 °C. Finally, the solvent was removed completely via rotary evaporation below 50 °C under a stream of air, which appeared to yield satisfactory results. The residue was then dissolved in an acidic reduction solvent of mixed dioxane/AcOH/H2O [5:4:1, (v/v/v)]. Zinc dust (100 mg) was added to the well-stirred solution, and stirring continued for 30 m. The mixtures were quantitatively transferred into a separatory funnel with CH2Cl2 (10 ml) and saturated NH4Cl (10 ml); 0.3 mg of internal standard (tetracosane in methylene chloride) was added. The pH of the aqueous phase was adjusted to 3 with addition of 2 M HCl. The mixture was then vigorously mixed to separate the organic layer. The aqueous phase was extracted CH2Cl2 (10 ml  2). The combined CH2Cl2 fractions were dried (Na2SO4), and the filtrate was acetylated for 2 h in Ac2O (0.5 ml) and pyridine (0.5 ml). All volatile components were completely removed via co-evaporation with EtOH under reduced pressure. The degraded products were then dissolved in CH2Cl2, and 1–2 ll of this solution was employed for GC analysis. Degraded monomers were quantitatively determined by GLC (Hewlett Packard 5980): column, 0.20–30 mm DB5MS (Agilent); He carrier gas, 1 ml min1; 30:1 split ratio; injector 220 °C; flame ionization detector (FID), 300 °C. The amounts of individual monomers, including p-acetoxycinnamyl acetate, coniferyl diacetate, and sinapyl diacetate (H-, G-, and S unit), were determined by response factors (RF) derived from pure monomer standards using tetracosane as an internal standard.

Analysis of lignocellulose crystallinity Lignocellulose crystallinity was measured by X-ray diffraction spectroscopy using a simple ground powder (Bruker D5005, MA, USA) according to a method described previously (Segal et al., 1959). The diffraction pattern was scanned from 3–40° 2h in 0.02° steps for 1 min per 1°. The crystalline index (CI) was calculated by the Segal method (Segal et al., 1959) as follows: CI ¼ 100  ½ðI200  IAM Þ=I200  using the height of the 200 peak (I200, 2h = 22.16°) and the minimum between the 200 and 110 peaks (IAM, 2h = 18°).

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Exogenously applied 24-epi brassinolide reduces lignification and alters cell wall carbohydrate biosynthesis in the secondary xylem of Liriodendron tulipifera.

The roles of brassinosteroids (BRs) in vasculature development have been implicated based on an analysis of Arabidopsis BR mutants and suspension cell...
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