Specific Lignin Accumulation in Granulated Juice Sacs of Citrus maxima

Article pubs.acs.org/JAFC

Specific Lignin Accumulation in Granulated Juice Sacs of Citrus maxima Jia-Ling Wu, Teng-Fei Pan,* Zhi-Xiong Guo, and Dong-Ming Pan* College of Horticulture and Institute of Storage Science and Technology of Horticultural Products, Fujian Agriculture and Forestry University, Fuzhou 350002, China ABSTRACT: Juice sac granulation occurring in pummelo fruits [Citrus maxima (Burm.) Merr.] is an undesirable trait, and the underlying mechanism remains unresolved. Previous studies have shown that lignin metabolism is closely associated with the process of juice sac granulation. Here, a method suitable for lignin isolation from pummelo tissues is established. Acetylated lignins from different pummelo tissues and cultivars were analyzed by HSQC NMR. The results showed that lignins in granulated juice sacs were characterized by an extremely high abundance of guaiacyl units (91.13−96.82%), in contrast to lignins from other tissues, including leaves, stems, and segment membranes. The abnormally accumulated lignins in granulated juice sacs were specific and mainly polymerized from coniferyl alcohol. No significant difference was found in lignin types among various cultivars. These findings indicated that the mechanism of juice sac granulation might be similar among various cultivars, although very different degrees of juice sac granulation can be observed. KEYWORDS: pummelo fruits, juice sac granulation, nuclear magnetic resonance, ultraviolet−visible spectroscopy, heteronuclear single-quantum coherence

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with another monolignol radical, β-5-, β-β-, and β-O-4- form the three main linkages in lignin polymers. In addition, other phenylpropanoids can be incorporated into lignin at various levels. The composition and amount of lignin may vary among species, tissues, cell types, and developmental stages.5,11 It is unknown which lignin metabolic pathway is dominant during juice sac granulation and whether it differs among cultivars. In previous studies, a series of analytical methods for lignin detection have been reported, including thioacidolysis (TA), pyrolysis−gas chromatography−mass spectrometry (Py-GCMS), derivatization followed by reductive cleavage (DFRC), ultraviolet (UV) and Fourier transform infrared spectroscopy (FT-IR spectra), and nuclear magnetic resonance (NMR). These analytical methods have revealed that lignin structures have various degrees of complexity.11−14 Recent advances in two-dimensional solution-state NMR techniques have made them the most useful methods for structural analysis of lignins: they provide unambiguous information for specific structures as well as quantitative structural information without lignin destruction. Among them, two-dimensional heteronuclear single-quantum coherence (2D-HSQC) NMR has attracted significant attention due to its versatility in illustrating structural features and transformations of isolated lignin fractions.15 Before lignin analysis, the main challenge is to obtain high yields of lignins from pummelo tissues without structural changes, because lignin intimately interpenetrates the other major components (cellulose and hemicelluloses)11 and the lignin content is low in pummelo fruits compared with woody plants. Although several lignin isolation procedures have been

INTRODUCTION Pummelo [Citrus maxima (Burm.) Merr.] is a distinctive citrus fruit characterized by its thick peel, large size, and high vitamin C content.1 The occurrence of juice sac granulation during the harvest season and storage period is widespread among citrus, especially in pummelo fruits. Granulated juice sacs are abnormally enlarged, stiffened, and dried, with thinned out flavor.2 ‘Guanximiyou’ [Citrus maxima (Burm.) Merr. ‘Guanximiyou’], which is mainly cultivated in Fujian province of China, and its bud mutants ‘Hongroumiyou’ [Citrus maxima (Burm.) Merr. ‘Hongroumiyou’] and ‘Sanhongmiyou’ [Citrus maxima (Burm.) Merr. ‘Sanhongmiyou’] are highly affected by juice sac granulation. This ailment drastically reduces their nutritive qualities and commodity values. Several studies assessing this abnormality have been published; however, the chemical mechanism underlying juice sac granulation remains largely unknown. Previously, we have demonstrated that lignin metabolism plays an important role in juice sac granulation, because lignin contents were increased during the process.3 In addition, key genes involved in lignin metabolism are expressed specifically in granulated juice sacs.4 Lignin, one of the most abundant structural polymers in plant cell walls,5 protects plants from biotic and abiotic stresses.6 It also provides mechanical support and enables water and solutes to transport through the vascular system.7 As a complex heteropolymer, lignin is polymerized mainly from three p-hydroxycinnamoyl alcohol monomers, namely, pcoumaryl, coniferyl, and sinapyl alcohols, which are derived from three main lignin metabolic pathways, respectively, and differ from each other in the degree of methoxylation.5 After integration into the lignin polymer, the three monolignols become p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively.8−10 During lignin polymerization, the βposition is favored by the radical coupling, and after coupling © 2014 American Chemical Society

Received: Revised: Accepted: Published: 12082

August 29, 2014 November 11, 2014 November 24, 2014 November 24, 2014 dx.doi.org/10.1021/jf5041349 | J. Agric. Food Chem. 2014, 62, 12082−12089

Journal of Agricultural and Food Chemistry

Article

proposed, including milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL),15 they are not suitable for pummelo fruits due to low yield and contamination.11,16,17 The organic solvent extraction method is an effective lignin extraction technique that produces less contaminant, does not alter lignin structure,18−21 and is useful for isolating lignins from pummelo fruits. Multiple studies assessing lignin have mainly focused on its application in bioenergy22,23 and pulping;17,24 however, the effect of lignin on pummelo fruit quality has been rarely studied. Here, we compare the composition and structure linkages of lignin between granulated juice sacs and three other tissues, in three severely granulated cultivars of pummelo. Our findings provide useful information about the main lignin synthesis pathway and specific lignin deposition in juice sacs, which is important for the further investigation of the relationship between lignin metabolism and juice sac granulation. To our knowledge, this is the first report evaluating the composition and structure of specific lignins accumulated in juice sacs of pummelo fruits.

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MATERIALS AND METHODS

Plant Materials. Plant materials used in this study included the granulated juice sacs, segment membranes, stems, and leaves. Granulated juice sacs were obtained from three cultivars, ‘Hongroumiyou’, ‘Guanximiyou’, and ‘Sanhongmiyou’, abbreviated HR, GX, and SH, respectively (Figure 1). Segment membranes, stems, and leaves were all from cultivar HR. The main leaf veins were removed before lignin extraction. Lignin Isolation. Soluble substances in samples were washed out according to the modified method of Müse et al.25 A total of 50 g (segment membranes, stems, and leaves) or 100 g (granulated juice sacs) of plant material was homogenized in 150 mL of homogenization buffer (50 mM Tris-HCl, 5 mL/L Triton X-100, 1 M NaCl; pH 8.3) using a juice extractor. After transfer to an 80 mL Beckman tube with screw cap, the homogenate was thoroughly vortexed and centrifuged (8000g, 10 min). The sediments were washed twice with the homogenization buffer, twice with 80% acetone, and twice with pure acetone. The washing procedure was repeated once for stem samples, twice for those from segment membranes and leaves, and three times for granulated juice sac specimens. The sediments from each tissue were combined and lyophilized. After lyophilization, the sediment samples were extracted with toluene/ethanol (2:1, v/v) for >7 h with a Soxhlet extractor and dried. Crude lignin was extracted and purified according to the method of Wu et al.,19 with the following modifications. Sediment samples (8 g), 4 g/L nitric acid (16 mL), and a mixture of formic acid/acetic acid/H2O at the ratio of 11:5:4 were placed into a 250 mL three-necked flask. The solid to liquid material ratio was 1:20. After extraction of the crude lignin for 3 h at 110 °C, the liquid part was dried using a rotary evaporator. The sediments were washed with double-distilled water several times and dried by desiccation. The crude lignin was purified according to the method of Wu et al.19 Preliminary Characterization of Lignin by UV. Lignin was preliminarily characterized by UV−vis spectroscopy after solubilization in dioxane/H2O (9:1, v/v) according to the method described by Wu et al.19 with minor modifications. The scanning spectrum was from 200 to 400 nm. HSQC NMR Sample Preparation. Lignin acetylation was performed according to previous studies,13,26 with minor changes. Purified lignin (20 mg) or segment membrane lignin (8 mg) was dissolved in 2 mL of pyridine/acetic anhydride (1:1, v/v) at room temperature in the dark for 72 h. After the sample had been transferred into an 80 mL Beckman tube, 30 mL of ethyl ether was added and the mixture centrifuged (15000g, 7 min). The sediments were washed with ethyl ether until disappearance of the pyridine odor. The sediments

Figure 1. Cultivars and juice sacs of pummelo used in this study: (A) GX segment with white juice sacs and segment membranes; (B) HR segment with red juice sacs and segment membranes; (C) SH segment with red juice sacs and segment membranes; (D) comparison of normal juice sacs (I) with the different degrees of juice sac granulation (II and III) in cultivar HR. The granulation was more severe in III than in II. were then dissolved in dimethyl-d6 sulfoxide (DMSO-d6, containing 0.03% TMS) and used for HSQC NMR analysis. HSQC NMR Procedures. All NMR experiments were conducted on a Bruker AVANCE III 500 instrument equipped with a z-gradient BBO probe at a temperature of 298 K. The Larmor frequencies of 1H and 13C were 500.0 and 125.7 MHz, respectively. The spectral widths in HSQC experiments were 5000 and 20833 Hz for the 1H and 13C dimensions, respectively. The number of complex points collected was 1024 for the 1H dimension; the acquisition time was 0.1 s with a recycle delay of 2 s. The number of transients was 24 (except for the sample of segment membranes for which 96 transients were accumulated to achieve a reasonable signal-to-noise ratio), and 256 time increments were recorded in the 13C dimension. The experiments were optimized for the 1 JCH of 145 Hz. Prior to Fourier transformation, the data matrices were zero filled to 1024 points in the 13C dimension. A squared cosine-bell apodization function was applied in both dimensions. The central solvent (DMSO-d6) peak was used as an internal chemical shift reference point (δC/δH 40.1/2.50). Semiquantitative analysis of different linkages and basic compositions of lignins was carried out according to previous reports.15,27 In the aliphatic oxygenated region, the relative abundances of interunit linkages were estimated from Cα−Hα cross-signals. In the aromatic region, C2−H2 cross-signals from G′ units were also incorporated into G units in the calculation. C2,6−H2,6 cross-signals from S units and 12083

dx.doi.org/10.1021/jf5041349 | J. Agric. Food Chem. 2014, 62, 12082−12089


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Figure 2. UV spectra of lignins isolated from different tissues: (A) lignin from HR leaves; (B) lignin from HR stems; (C) lignin from HR segment membranes; (D) lignin from HR granulated juice sacs; (E) lignin from GX granulated juice sacs; (F) lignin from SH granulated juice sacs. doubles of C2−H2 cross-signals from G and G′ units were used to estimate the G/S ratio.

prominent signals corresponding to the β-O-4′ substructures. The Cα−Hα cross-signals in β-O-4′ substructures (A and A′) were observed at δC/δH 73.81/5.88 (for erythro) and 74.05/ 5.96 ppm (for threo), whereas the Cβ−Hβ cross-signals were observed at δC/δH 78.96/4.86 and 80.64/4.57 for substructures linked to G and S units, respectively. The Cγ−Hγ cross-signals of substructure A were seen at δC/δH 62.37/3.96−4.29, whereas the γ-acylated β-ether units A′ were identified at δC/δH 63.60/ 3.90−4.26, partially overlapping with other signals. Strong signals from β-β′ substructures (B) were observed only in the spectrum of lignin samples from stems, with their Cα−Hα, Cβ− Hβ, and the two Cγ−Hγ cross-signals at δC/δH 85.35/4.71, 54.19/3.07, and 71.77/3.86 and 4.21, respectively. Phenylcoumaran (β-5′) substructures (C) were found in lignins of all samples except for leaves; their Cα−Hα and Cβ−Hβ crosssignals were observed at δC/δH 87.29/5.53 and 49.80/3.78, respectively, and the Cγ−Hγ correlation signal overlapped with other signals around δC/δH 64.63/4.38. The abundance of these three linkages varied among the tested tissues. Leaf lignin only had β-O-4′ substructures, whereas stem lignin contained all three linkages with a predominance of β-O-4′ units (A and A′, 76.48% of total side chains) followed by β-β′ substructures (B, 14.88%) and lower amounts of phenylcoumaran (β-5′) substructures (C, 8.64%). In the lignin of fruits (segment membranes and granulated juice sacs), β-O-4′ substructures (A and A′) were predominant with abundances between 78.76 and 87.65% and certain amounts of β-5′ substructures (B, 12.35−21.24%) were also observed; however, β-β′ substructures (C) were not detected. Analysis of Lignin Units. The main lignin 13C−1H crosssignals in the aromatic region of HSQC spectra correspond to the aromatic rings of various lignin units.11 According to our results, the three main lignin units from the monolignol metabolic pathway were clearly observed. Syringyl (S) units showed a prominent signal for C2,6−H2,6 correlation at δC/δH 103.92/6.68, whereas guaiacyl (G) units displayed different cross-signals for C2−H2, C5−H5, and C6−H6 at δC/δH 111.83/ 6.98, 117.13/7.01, and 120.06/6.93, respectively. In addition, signals corresponding to C2−H2 and C6−H6 cross-signals of Cα-oxidized G units (G′) at δC/δH 112.11/7.16 and 123.10/

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RESULTS Lignin Extraction and Characterization. Because the lignin contents of the four tissues studied were very low compared with those of woody plants, and no previous work was available, we established a method appropriate to analyze the lignin content and purity of each sample. The organic solvent extraction method was good enough for us to obtain relatively pure lignin suitable for NMR analysis. The preliminary characteristics of lignins from different tissues are shown in Figure 2. Lignins of all tested tissues displayed a wide absorption peak at 240−300 nm and a characteristic absorption peak at 249 nm. In addition, stem lignin showed a characteristic absorption at 282 nm, whereas that from segment membranes and granulated juice sacs in all three cultivars had an adsorption peak at 286 nm. The lignin from leaves did not have a characteristic peak, but adsorbed between 275 and 282 nm. Because lignins have a specific absorption at 200−300 nm,28 we concluded that lignin might be present in our samples. These samples were then used for subsequent NMR analysis. HSQC NMR Analysis of Isolated Lignin. The lignin samples from leaves, stems, and segment membranes in HR, and from granulated juice sacs in HR, GX, and SH were analyzed by HSQC NMR. Before measurement, the lignin samples were acetylated to ensure complete solubility in the NMR solvent29 and acquisition of unique and well-resolved cross-signals.15 Because the lignin content of segment membranes was low, only 8 mg was used for analysis, but the results were not affected. The HSQC spectra revealing the key information regarding lignin structures are shown in Figures 3 and 4. Lignin contours in the HSQC spectra were assigned by comparison with data from previous studies.15,26,30−33 The assignments and relative abundances of main lignin 13C−1H cross-signals are listed in Tables 1 and 2. The main lignin substructures identified in HSQC spectra are presented in Figure 5. Analysis of Lignin Interunit Linkages. In the side-chain region of HSQC spectra, the main lignin interunit linkages β-O4′, β-β′, and β-5′ were distinctly observed. All spectra showed 12084



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Figure 3. Side-chain regions of 13C−1H cross-signals in HSQC spectra of acetylated lignins: (A) lignin from leaves (HR); (B) lignin from stems (HR); (C) lignin from segment membranes (HR); (D) lignin from granulated juice sacs (HR); (E) lignin from granulated juice sacs (GX); (F) lignin from granulated juice sacs (SH). The cross-peaks are color coded according to the structures shown in Figure 5.

assigned to C4−H4, C2,6−H2,6, and C3,5−H3,5 of the X structure, respectively. The X structure was none of the three basic lignin units and was therefore excluded from abundance calculations. The composition of S, G, and H units presented big differences among tissues. The lignin of leaves was predominantly composed of H (55.81%) and S (44.19%) units, whereas that of stems contained all three units with a large amount of S units (58.57%), followed by a lower amount of G units (38.73%) and traces of H units (2.7%). The G units were predominant in the lignin from segment membranes and granulated juice sacs, with the abundances of 92.45 and 91.13− 96.82%, respectively. Of note, S and H units were not detected

7.06, respectively, were observed. Despite the weak signals of phydroxyphenyl (H) units, we were able to verify the structures using heteronuclear multiple-bond correlation (HMBC) and heteronuclear single-quantum coherence−total correlation spectroscopy (HSQC-TOCSY) NMR (data not shown). Finally, the cross-signals at δC/δH 121.6/6.97 and 130.50/ 7.24 of C3,5−H3,5 and C2,6−H2,6 were assigned to H units, respectively. The 13C−1H cross-signals at δC/δH 126.63/7.16, 128.34/7.22, and 129.54/7.22 could not be clearly identitied. The chemical shifts and HMBC and HSQC-TOCSY data appeared to suggest a phenyl connected to alkyl chains (X) as a potential candidate structure. The three cross-signals were 12085



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Figure 4. Aromatic regions of 13C−1H cross-signals in HSQC spectra of acetylated lignins: (A) lignin from leaves (HR); (B) lignin from stems (HR); (C) lignin from segment membranes (HR); (D) lignin from granulated juice sacs (HR); (E) lignin from granulated juice sacs (GX); (F) lignin from granulated juice sacs (SH). The cross-peaks are color coded according to the structures shown in Figure 5.

Although MWL is a common method used in lignin extraction, it was not adopted in this research due to its low yield, modifications during milling, and potential “contaminating” compounds such as lignin-linked carbohydrates (LLC).11,16 The improved organic solvent extraction method used in this work was suitable for the isolation of lignin from pummelo fruits: the resulting products were relatively pure, and yields were high enough for various analyses. According to the NMR results, the signals of LLC linkages and other carbohydrates were not detected, unlike data obtained after MWL extraction, in which lignin signals are often overlapping with those of carbohydrates.11,15,26,30 Without the overlapping carbohydrate signals, we were able to distinguish the various

in the lignin from segment membranes and HR, respectively, and barely observed in other tissues. The G/S ratio was high in the lignin from granulated juice sacs but varied among cultivars: ratios of 30.45, 18.19, and 66.94% were obtained for HR, GX, and SH, respectively.

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DISCUSSION Because lignin metabolism is closely associated with the juice sac granulation of pummelo fruits, it deserves careful attention and thorough analysis. To our knowledge, no study concerning lignin metabolism in juice sac granulation has been published. Therefore, it is of particular importance to carry out studies that reveal the mechanisms underlying juice sac granulation. 12086



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Table 1. Assignments of Main Lignin 13C−1H Cross-Signals in HSQC Spectra Shown in Figures 3 and 4 label

δC/δH

assignment

Cβ Bβ OMe Aγ Aγ

49.80/3.78 54.19/3.07 56.17/3.73 62.37/3.96−4.29 63.60/3.90−4.26

Cγ Bγ Bγ Aα Aα Aβ(G) Aβ(S) Bα Cα S2,6 G2 G′2 G5 G6 H3,5 G′6 H2,6

64.63/4.38 71.77/3.86 71.77/4.21 73.81/5.88 74.05/5.96 78.96/4.86 80.64/4.57 85.35/4.71 87.29/5.53 103.92/6.68 111.83/6.98 112.11/7.16 117.13/7.01 120.06/6.93 121.60/6.97 123.10/7.06 130.50/7.24

Cβ−Hβ in β-5′ substructures (C) Cβ−Hβ in β-β′ substructures (B) C−H in methoxyls (OMe) Cγ−Hγ in β-O-4′ substructures (A) Cγ−Hγ in γ-acylated β-O-4′ substructures (A′) Cγ−Hγ in β-5′ substructures (C) Cγ−Hγ in β-β′ substructures (B) Cγ−Hγ in β-β′ substructures (B) Cα−Hα in β-O-4′ substructures (A) (erythro) Cα-Hα in β-O-4′ substructures (A) (threo) Cβ−Hβ in β-O-4′ linked to G (A) Cβ−Hβ in β-O-4′ linked to S (A) Cα−Hα in β-β′ substructures (B) Cα−Hα in phenylcoumaran (C) C2,6−H2,6 in syringyl units (S) C2−H2 in guaiacyl units (G) C2−H2 in oxidized G units (G′) C5−H5 in guaiacyl units (G) C6−H6 in guaiacyl units (G) C3,5−H3,5 in H units (H) C6−H6 in oxidized G units (G′) C2,6−H2,6 in H units (H)

interunit linkages depend largely on changes in monolignol availability during the polymerization process of lignin. Lignins composed mainly of G units contain more β-5′ linkages (C) than those made of S units, which show an abundance of β-β′ linkages (B). This view is in line with our results. As G units increased in the lignin samples from leaves, stems, to segment membranes and juice sacs, the β-5′ linkages (C) increased despite the variations observed in juice sac samples from different cultivars. Meanwhile, β-β′ linkages (B) were detected only in lignin samples from stems, in which the S units were more abundant than in other tissues. In addition, the G/S ratio was found to reflect the degree and nature of polymeric crosslinking. The high G content is known to yield more highly cross-linked lignins characterized by a great proportion of biphenyls and other carbon−carbon bonds, whereas S units are typically linked by more labile ether bonds. Therefore, G-rich lignins depolymerize with more difficulty compared with those rich in S units.38,39 In addition, plant cell walls rich in guaiacyl lignins thicken more rapidly than those rich in syringyl lignins.40 The high G/S ratio obtained in the lignin from granulated juice sacs indicated that the specific lignin accumulated may be tightly integrated and not easily degradable; in addition, the cell walls in granulated juice sacs may thicken more quickly. Other linkages and units reported in previous studies16,31,33,35,41−43 were not detected in our samples; conversely, certain linkages and units found here have not been detected by other groups. This might be due to the insufficient time for NMR procedures and/or their low abundances in the tested tissues. The cause of lignin formation and accumulation in juice sac granulation is unknown. According to previous studies, lignin deposition not only follows a developmental process but can also be triggered by several biotic and abiotic stresses.5 As shown above, the lignin deposited in granulated juice sacs was different from that present in other tissues. However, the composition of lignin linkages and interunits did not change significantly among cultivars. This observation suggests a similar mechanism for juice sac granulation among cultivars. The particularly high G/S ratio of the lignin in SH showed that the lignified juice sacs in this cultivar were stiffer than those in GX and HR; therefore, the juice sac granulation occurring in SH might be more severe. Consequently, cultivar SH could be used as an important material for assessing juice sac granulation. Besides, the G/S ratio of the lignin in GX was much lower than those obtained for HR and SH, which had darker fruits (Figure 1). This suggests that juice sac granulation might be associated with fruit pigmentation. In addition, the extremely high abundances of G units in the lignin of

types of lignin structures and determine the lignin composition accurately. As numerous lignin isolation methods have focused mainly on plant samples, especially woody plants that contain high levels of lignins,17,22,24,34−36 the method described here might be useful for studies assessing lignin from plant materials with relatively low lignin levels. In addition, as an aromatic polymer, lignin has a strong ultraviolet absorption; the shape and absorption coefficient of the peak are determined by the type and content of lignin units and its functional groups.13,28,37 Although different lignin contents were obtained for the tested tissues, the ultraviolet absorption data have provided useful information for the characterization of lignins in various tissues. Indeed, the different absorption peaks among tissues might indicate the distinct lignin types that they contain. However, for a given tissue, the similar lignin absorption peaks for different cultivars might indicate similar lignin composition. These findings were consistent with the HSQC NMR data. As expected, the β-O-4′ linkage (A) was the most frequent interunit linkage in our samples. This linkage is the easiest to chemically cleave compared with other bonds.9 According to previous papers,9,11 the relative abundances of different

Table 2. Relative Abundances of Main Linkages and Units of Lignin from Pummelo characteristic linkages (% side chains involved) β-O-4′ ary ether (A, A′) β-β′ resinols (B) β-5′ phenylcoumarans (C) lignin units (%) H G S G/S ratio a

leaf

stem

segment membrane

HR

GX

SH

100 nda nd

76.48 14.88 8.64

81.04 nd 18.96

78.76 nd 21.24

81.76 nd 18.24

87.65 nd 12.35

55.81 nd 44.19 0

2.7 38.73 58.57 0.66

7.55 92.45 nd 100

nd 96.82 3.18 30.45

3.86 91.13 5.01 18.19

2.84 95.73 1.43 66.94

nd, not detected. 12087



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Figure 5. Main structures identified in Figures 3 and 4: (A) β-O-4′ linkages; (A′) γ-acetylated β-O-4′ substructures; (B) resinol substructures formed by β-β′/α-O-γ′/γ-O-α′ linkages; (C) phenylcoumaran substructures formed by β-5′/α-O-4′ linkages; (H) p-hydroxyphenyl unit; (G) guaiacyl unit; (G′) α-oxidized guaiacyl unit; (S) syringyl unit; (X) phenyl connected to alkyl chains.

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ABBREVIATIONS USED 2D-NMR, two-dimensional nuclear magnetic resonance; TA, thioacidolysis; Py-GC-MS, pyrolysis−gas chromatography− mass spectrometry; DFRC, derivatization followed by reductive cleavage; UV−vis, ultraviolet−visible; FT-IR, Fourier transform infrared; G, guaiacyl; H, p-hydroxyphenyl; S, syringyl; HSQC, heteronuclear single quantum coherence; MWL, milled wood lignin; CEL, cellulolytic enzyme lignin; DMSO-d6, dimethyl-d6 sulfoxide; TMS, tetramethylsilane; HMBC, heteronuclear multiple-bond correlation; HSQC-TOCSY, heteronuclear single-quantum coherence-total correlation spectroscopy; LLC, lignin-linked carbohydrates

granulated juice sacs indicated that lignins might have polymerized mainly from coniferyl alcohol. No evidence of factors inducing lignin formation was obtained. We propose that regulators or triggers that participate in the synthesis of G units might be the key factors for abnormal lignin accumulation in granulated juice sacs. This hypothesis remains to be tested in subsequent studies. In this study, we established a method to isolate lignins from pummelo fruits. The lignin isolated from leaves, stems, and segment membranes of HR were compared with those from granulated juice sacs of HR, GX, and SH. To our knowledge, this represents the first report describing the specificity of lignins in granulated juice sacs and the possible metabolic pathway for lignin accumulation in pummelo fruits. Our results provide a new insight for further investigations of the mechanism of juice sac granulation in citrus fruits.

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REFERENCES

(1) Shomer, I.; Chalutz, E.; Vasiliver, R.; Lomaniec, E.; Berman, M. Scierification of juice sacs in pummelo (Citrus grandis) fruit. Can. J. Bot. 1989, 67, 625−632. (2) Pan, D. M.; Zheng, G. H.; Chen, G. X.; She, W. Q.; Guo, Z. X.; Shi, M. T.; Lin, H. Y. Analysis of the reasons caused granulation of juice sacs in Guanximiyou pomelo variety. J. Fruit Sci. 1999, 16, 202− 209. (3) She, W. Q. An analysis on physiological changes and gene differential expressions in the process of pummelo fruit [Citrus grandis (L.) Osbeck] juice sac granulation. Thesis, Fujian Agriculture and Forestry University, Fuzhou, China, 2009. (4) Xu, Y. Cloning and expression of lignin genes in Citrus maxima (Burm.) Merr. Thesis, Fujian Agriculture and Forestry University, Fuzhou, China, 2014. (5) Cesarino, I.; Araujo, P.; Domingues, A. P.; Mazzafera, P. An overview of lignin metabolism and its effect on biomass recalcitrance. Braz. J. Bot. 2012, 35, 303−311. (6) Vogt, T. Phenylpropanoid biosynthesis. Mol. Plant 2010, 3, 2−20. (7) Vanholme, R.; Morreel, K.; Ralph, J.; Boerjan, W. Lignin engineering. Curr. Opin. Plant Biol. 2008, 11, 278−285. (8) Baucher, M.; Halpin, C.; Petit-Conil, M.; Boerjan, W. Lignin: genetic engineering and impact on pulping. Crit. Rev. Biochem. Mol. Biol. 2003, 38, 305−350. (9) Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. Plant Biol. 2003, 54, 519−546. (10) Weng, J. K.; Chapple, C. The origin and evolution of lignin biosynthesis. New Phytol. 2010, 187, 273−285.

AUTHOR INFORMATION

Corresponding Authors

*(D.-M.P.) Phone: 0591-83787126. Fax: 0591-83735681. Email: [email protected]. *(T.-F.P.) Phone: 0591-83789281. Fax: 0591-83735681. Email: [email protected]. Funding

This work was supported by the National Key Technology R&D Program of China (2007BAD07B01), the China Spark Program (KH1400530), the Natural Science Foundation of Fujian Province of China (2012J05041), and the Special Foundation for Young Scientists of the Horticultural College of Fujian Agriculture and Forestry University (FAFU2012YYPY02) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Zong-Lie Hong from the University of Idaho and Jian-Jun Chen from the University of Florida for reviewing the manuscript and Yun Lin from the University of Fuzhou for helping with figure preparation. 12088



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