Populus endo-beta-mannanase PtrMAN6 plays a role in coordinating cell wall remodeling with suppression of secondary wall thickening through generation of oligosaccharide signals

Authors

  • Yunjun Zhao,

    1. National Key Laboratory of Plant Molecular Genetics/Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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  • Dongliang Song,

    1. National Key Laboratory of Plant Molecular Genetics/Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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  • Jiayan Sun,

    1. National Key Laboratory of Plant Molecular Genetics/Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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  • Laigeng Li

    Corresponding author
    • National Key Laboratory of Plant Molecular Genetics/Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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For correspondence (e-mail lgli@sibs.ac.cn).

Summary

Endo-1,4-β-mannanase is known to able to hydrolyze mannan-type polysaccharides in cell wall remodeling, but its function in regulating wall thickening has been little studied. Here we show that a Populus endo-1,4-β-mannanase gene, named PtrMAN6, suppresses cell wall thickening during xylem differentiation. PtrMAN6 is expressed specifically in xylem tissue and its encoded protein localizes to developing vessel cells. Overexpression of PtrMAN6 enhanced wall loosening as well as suppressed secondary wall thickening, whilst knockdown of its expression promoted secondary wall thickening. Transcriptional analysis revealed that PtrMAN6 overexpression downregulated the transcriptional program of secondary cell wall thickening, whilst PtrMAN6 knockdown upregulated transcriptional activities toward secondary wall formation. Activity of PtrMAN6 hydrolysis resulted in the generation of oligosaccharide compounds from cell wall polysaccharides. Application of the oligosaccharides resulted in cellular and transcriptional changes that were similar to those found in PtrMAN6 overexpressed transgenic plants. Overall, our results demonstrated that PtrMAN6 plays a role in hydrolysis of mannan-type wall polysaccharides to produce oligosaccharides that may serve as signaling molecules to suppress cell wall thickening during wood xylem cell differentiation.

Introduction

In higher plants, cell walls (CWs) make up the bodily structure and stockpile the majority of photosynthesis-fixed carbon and solar energy. CWs can be generally classified into the primary cell wall (PCW) and secondary cell wall (SCW), which are formed through different processes and are regulated via different pathways. The PCW begins to form along with cell plate assembly during cytokinesis and continues to be modified as the cell expands. However, SCW differentiation occurs only in certain types of cells, such as vessel (or tracheary) elements and fiber cells in vascular tissue. Thus, to develop wall-thickened cells, meristematic cells must receive signals that initiate the cell differentiating and wall thickening programs.

Several plant hormones have been reported to regulate the differentiation of wall-thickened vascular tissue. Auxin plays a role in promoting the differentiation of procambial cells into xylem (Milioni et al., 2001; Moyle et al., 2002), and brassinosteroids (BRs) are required for the later stages of tracheary element (TE) differentiation (Iwasaki and Shibaoka, 1991; Yamamoto et al., 2001). In contrast, cytokinins play an important role in maintaining procambial cell identity and preventing protoxylem specification (Mahonen et al., 2006). Gibberellin has also been shown to stimulate xylem expansion in Arabidopsis hypocotyl (Ragni et al., 2011). Some peptides and oligosaccharides have also been reported to function as extracellular signaling molecules that regulate TE differentiation. Oligosaccharides (Roberts et al., 1997) and sulphated pentapeptides (PSK-α; Matsubayashi et al., 1999) promoted TE differentiation in a zinnia (Zinnia elegans L.) culture system. Moreover, xylogen, a proteoglycan-like factor, was found to mediate an inductive cell-cell interaction involved in plant tissue differentiation (Motose et al., 2004). Also in the zinnia xylogenic culture system, a 12-amino acid peptide from the CLAVATA3/ESR-related (CLE) gene family that inhibited TE differentiation was isolated as an extracellular signaling molecule (Ito et al., 2006).

Galactoglucomannan oligosaccharides (GGMOs, with degree of polymerization (DP) 4-8) are considered to be a type of signaling molecule that affects cell differentiation. Exogenous GGMOs affect SCW thickening by regulating the differentiation of protoxylem-like TEs and metaxylem-like TEs in xylogenic cultures of zinnia (Benova-Kakosova et al., 2006). Interactions between GGMOs and auxin have been studied in the process of seed germination in mung bean (Kollarova et al., 2010). GGMOs induced adventitious root growth in the absence of auxin and inhibited adventitious root induction in the presence of auxin (Kollarova et al., 2005). How GGMOs are produced and integrated in the larger signaling network regulating cell differentiating is still unclear.

Mannan-type oligosaccharides are thought to be produced by endo-1,4-β-mannanase (E.C.3.2.1.78) (MAN) that catalyzes the cleavage of β(1–4) bonds in the backbones of mannan polymers (Shallom and Shoham, 2003). Plant MANs are reported to be involved in the seed germination and post-germination process by promoting hydrolysis of mannan-rich endosperm CWs (Bewley et al., 1997; Nonogaki et al., 2000; Gong and Bewley, 2007; Iglesias-Fernandez et al., 2011a,b; Rodriguez-Gacio Mdel et al., 2012). In tomato, LeMAN1 is expressed in the endosperm of germinated seeds and plays a role in hydrolyzing stored polysaccharides to provide germinating seeds with its energy and carbon source needs (Bewley et al., 1997; Nonogaki et al., 2000). Tomato LeMAN2 (Nonogaki et al., 2000) and Arabidopsis AtMAN5, AtMAN6 and AtMAN7 (Iglesias-Fernandez et al., 2011a,b) are involved in the hydrolysis of mannan-rich CWs to allow for radicle emergence and the completion of germination. MAN activity was also detected in CW softening associated with fruit ripening (Bewley et al., 2000; Schroder et al., 2006). Tomato LeMAN4 is expressed mainly in the skin and outer pericarp of the fruit (Bewley et al., 2000; Banik et al., 2001; Bourgault and Bewley, 2002), and probably contributes to the loosening of the CW through mannan hydrolysis (Bewley et al., 2000) or mannan transglycosylase (Schroder et al., 2006) activity. However, it is unclear whether the mannan-type oligosaccharides produced through MAN hydrolysis play a role in regulating plant development.

In this study, we identified a Populus endo-1,4-β-mannanase gene, PtrMAN6, that is specifically expressed in xylem tissue and that regulated the transcriptional program governing secondary wall thickening during xylem differentiation. We also present evidence to show that PtrMAN6 catalyzes the hydrolysis of mannan-type wall polysaccharides to produce GGMOs, which in turn serve as signaling molecules to regulate the transcriptional program of CW thickening.

Results

Expression of MAN genes in Populus trichocarpa

In our previous study, a MAN protein was identified in the plasma membrane of Populus differentiating xylem tissue (Song et al., 2011). Although MAN has been studied for its roles in seed germination and fruit ripening (Bewley et al., 2000; Gong and Bewley, 2007; Iglesias-Fernandez et al., 2011a), the aforementioned result suggests the possibility that MAN plays a yet to be determined role in xylem development.

Eight MANs are predicted in the Populus trichocarpa genome (Yuan et al., 2007) and named PtrMAN1 to PtrMAN8, respectively. The MAN protein that was identified in the differentiating xylem plasma membrane is encoded by the PtrMAN6 gene. When we analyzed the expression of the eight genes, only five gene transcripts (PtrMAN4, PtrMAN5, PtrMAN6, PtrMAN7 and PtrMAN8) were detected in the examined tissues, which included differentiating xylem, differentiating phloem, matured leaf, young leaf and shoot tip. The expression of the five genes was spatially regulated in P. trichocarpa (Figure 1). PtrMAN4, PtrMAN6 and PtrMAN8 were specifically expressed in differentiating xylem tissue that is consistent with the tissue location where the PtrMAN6 protein was detected in our previous study. Meanwhile, PtrMAN5 was expressed in all tissues examined, while PtrMAN7 was mainly expressed in young leaf and shoot tip.

Figure 1.

Expression profile of PtrMANs in various Populus tissues. This experiment was performed three times using different batches of plants. Error bars represent the standard error (SE) of three technical replicates using pooled samples of at least three independent plants. Xy, xylem; Ph, phloem; St, shoot tip; Yl, young leaf; Ml, mature leaf.

Sequence analysis of the five expressed Populus MANs indicated that PtrMAN4 and PtrMAN6 shared 93% sequence identity while other MAN sequences were more divergent, sharing only about 50% sequence identity (Table S1). The highly conserved sequence makes it likely that PtrMAN4 and PtrMAN6 could initiate similar pathways in Populus.

Localization of PtrMAN6

The PtrMAN6 protein is predicted to be a secretory protein that contains a signal peptide at its N terminus (Figure S1a; Petersen et al., 2011). To examine the subcellular localization of PtrMAN6, a construct coding for a PtrMAN6:GFP fusion protein was generated under the control of a cauliflower mosaic virus (CaMV) 35S promoter. The construct was transformed into Arabidopsis and the young roots of the transgenics were used for fluorescence location analysis. Results revealed that PtrMAN6 was specifically localized on the plasma membrane (Figure 2a,b), a finding that was further confirmed by plasmolysis analysis (Figure 2c,d). Western blot analysis detected PtrMAN6 protein in the microsomal but not in the soluble fraction of Populus xylem proteins (Figure 2e). However, PtrMAN6 could be partially washed off from the membrane fraction using 100 mm sodium carbonate. This finding suggests that PtrMAN6 may be bound to the membrane as an integral monotopic protein. Transient expression of PtrMAN6 in onion epidermal cells was carried out to determine which portion of the peptide is responsible for the localization. A full-length PtrMAN6 and the N-terminal sequences of 31 amino acids were both fused with green fluorescent protein (GFP) (Figure S1b). Both the full-length PtrMAN6 and putative signal peptide were able to direct its fused GFP protein onto the plasma membrane (Figure S1c,d) and were compared with the GFP control that showed fluorescent signals in the cytoplasm and nuclei (Figure S1e). Together, these results demonstrate that PtrMAN6 is localized on the plasma membrane by its N-terminal peptide.

Figure 2.

Analysis of PtrMAN6 plasma membrane localization. (a,b) Stable expression of PtrMAN6:GFP fusion protein in Arabidopsis root cells, showing fluorescent signals on the plasma membrane. (c,d) Verification of PtrMAN6:GFP fluorescent signals on the plasma membrane by plasmolysis treatment with 30% sucrose. (a,c) Fluorescent images. (b,d) Images under bright-field microscopy. (e) Western blot showing PtrMAN6 is present in the microsomal (MS), not in the soluble fraction (SF). M, molecular mass marker. Scale bar = 50 μm.

In order to determine the cell-type expression of PtrMAN6, antibodies against two unique peptides from PtrMAN6 protein were produced and the antibody specificity in recognizing PtrMAN6 was confirmed (Figure S2a–c). Immunolocalization was carried out using stem and young petiole, of which the sections were hybridized with specific antibodies against PtrMAN6. Highly specific PtrMAN6 signals was detected in the developing vessel cells of xylem tissue (Figure 3), and this result suggested that PtrMAN6 plays a role specifically in vessel element development during Populus xylem differentiation.

Figure 3.

Immunolocalization of PtrMAN6 in developing vessel cells in Populus. (a, b, f) Cross-sections and (c) longitudinal sections of the stem at the sixth internode and (d, e) cross-sections of young petiole were hybridized with anti-PtrMAN6 antibodies (a–e) or preimmune IgG (f). PtrMAN6 is specially localized in developing vessel. (b,e) High magnification of the frames in (a, d). Xy, xylem; Ph, phloem; Ca, cambium; Pi, pith; Ve, vessel cell; Xf, xylem fiber cell; Ra, ray cell. Scale bars in (a, f) = 200 μm; in (b,c) = 50 μm; in (d) = 100 μm; in (e) = 500 μm.

PtrMAN6 activity, glycosylation and dimerization

As the PtrMAN6 gene was predicted to code for a putative endo-1,4-beta-mannanase, it was expected to be able to digest azurine cross-linked (AZCL)-galactomannan (Schroder et al., 2006). We first produced recombinant proteins of PtrMAN6 in E. coli but its enzymatic activity could not be determined (Figure S2c,d). When we examined both recombinant protein and plant-sourced PtrMAN6 protein by western blot analysis, the latter showed a larger molecular size than the former (Figure S2e). This difference suggests potential post-translational modification of PtrMAN6 that results in a larger molecular size as detected in the western blot. Thus we transformed Populus with a CaMV 35S:PtrMAN6 construct in order to produce a large amount of plant-sourced PtrMAN6 protein from non-PtrMAN6-expressing tissue.

PtrMAN proteins were extracted from the young leaves of transgenic Populus plants that overexpressed PtrMAN6 (PtrMAN6 expression is minimal in wild type; Figure 1) and proteins were used to determine its enzymatic activity. A significant amount of PtrMAN6 hydrolysis activity was detected with the protein from the transgenic leaves, while small levels of activity were observed from the wild type (Figure 4). A detailed characterization of the enzyme properties was conducted using plant-sourced proteins. At 37°C, PtrMAN6 was particularly sensitive to pH conditions and exhibited maximum activity at pH 5 (Figure 4a). At its optimal pH, PtrMAN6 showed a temperature optimum of 50°C (Figure 4b). Thus the conditions of pH 5 and 50°C were applied to subsequent measurements of enzyme activity. Under these conditions, the enzyme was examined for its ability to hydrolyze various polysaccharide substrates. Results demonstrated that PtrMAN6 was able to cleave mannan-type polysaccharides including galacto-glucomannan (GGM), galactomannan, glucomannan and mannan with the highest activity in digestion of GGM polysaccharides (Figure S2g).

Figure 4.

Enzymatic activities of PtrMAN6. Proteins from the leaves of PtrMAN6-overexpressed plants were used for enzyme analysis. WT, wild type plants. (a) Effect of pH on PtrMAN6 activity. (b) Effect of temperature on PtrMAN6 activity. (c) PtrMAN6 treated with endoglycosidase Endo Hf and analyzed by western blot. Lane M, molecular mass standard; Lane 1, untreated protein; Lane 2 and 3, the protein treated with Endo Hf for 30 min and 60 min, respectively. (d) Effect of Endo Hf treatment on PtrMAN6 activity. (e) Native PtrMAN6 protein from Populus was electrophoresed on 10% SDS-PAGE gels under non-reducing (PtrMAN6) or reducing condition (+β-ME: 5% β-mercaptoethanol; +DTT: 10 mm 1,4-dithiothreitol) and detected by immunoblot with anti-PtrMAN6 IgG. Monomeric and dimeric proteins are indicated. Lane M, molecular mass standard. (f) Effect of dimerization on PtrMAN6 activity. Error bars represent standard error (SE) of triplicate sample measurements.

Four glycosylation sites were predicted in the PtrMAN6 amino acid sequence (Figure S2f). PtrMAN6 from Populus xylem tissue displayed a single band with a molecular mass of approximate 58 kDa based on western blot analysis (Figure 4c, Lane 1). After treatment with endoglycosidase H (Endo Hf), PtrMAN6 was detected with a molecular mass of about 50 kDa (Figure 4c, Lane 3), which is consistent with its theoretical molecular mass. The result suggests that plant-sourced PtrMAN6 is modified through N-glycosylation. When plant-sourced PtrMAN6 was treated incompletely with Endo Hf, several bands between 50 and 58 kDa were also observed (Figure 4c; Lane 2), a finding that suggested that PtrMAN6 might contain multiple N-glycosylation sites.

To examine the effect of glycosylation on PtrMAN6 activities, native enzymes extracted from Populus differentiating xylem were treated with Endo Hf. PtrMAN6 activity decreased by about 50% after 2 h of Endo Hf treatment, compared with the untreated controls (Figure 4d). These results suggest that N-glycosylation of PtrMAN6 is required for its enzymatic activities in Populus. Furthermore, when analyzed by SDS-PAGE and immunoblot under reducing conditions, PtrMAN6 proteins migrated as monomeric proteins (Figure S2a). However, under non-reducing conditions, PtrMAN6 proteins migrated with an apparent molecular mass of approximately twice that of the monomers (Figure 4e). When the proteins were treated with different thiol reductants (β-mercaptoethanol (β-ME) and dithiothreitol (DTT) for 2 h, the fraction of PtrMAN6 monomers increased significantly (Figure 4e). The fraction of PtrMAN6 dimers gradually decreased with increase in the reductant concentration (Figure S2h). This finding suggests that native PtrMAN6 tends to form disulfide-linked homodimers. However, prokaryotic recombinant PtrMAN6 in E. coli was not able to dimerize under either non-reducing or reducing conditions (Figure S2i). To examine the effect of dimerization on PtrMAN6 activities, native enzymes were treated with thiol reductants. Compared with the untreated control, PtrMAN6 activity decreased by about 84 and 53% after 2 h of 5% β-ME or 10 mm DTT treatment, respectively (Figure 4f). These results suggest that disulfide-linked dimerization of PtrMAN6 is also required for its enzymatic activities in Populus.

Effects of PtrMAN6 on Populus vascular development

To investigate the genetic function of PtrMAN6, we transformed Populus with constructs that resulted in the overexpression and knockdown of PtrMAN6. At least 30 independent transgenic lines were generated for each construct. Three transgenic lines with high expression of the transgene (Figure S3a) were selected and characterized for morphology, wood anatomy and other characteristics. Compared with wild type (WT), overexpression of PtrMAN6 resulted in softer stems and petioles while the transgenic plants with downregulated expression of PtrMAN6 gene displayed slightly stronger stems (Figure 5a,b).

Figure 5.

Phenotype of PtrMAN6-overexpressed and PtrMAN6-suppressed transgenic Populus. (a) Representative PtrMAN6-overexpressed plant (OE, left), wild type plant (WT, middle) and PtrMAN6-suppressed plant (AS, right). (b) Partial shoots of the plants in (a). Arrow indicates the petiole of the14th leaf from the tip. (c) Cross-sections of the 14th internode stem showing weaker lignin deposition in the xylem cell walls (CWs) of OE plants (left) and stronger lignin deposition in the xylem CWs as well as the pith CWs of the AS plants (right) compared with the WT (middle). (d) High magnification of the framed zones in (c). Arrow indicates vessel cells. (e) Lignin content of wood tissue in 1-year-old plants. (f) Crystalline cellulose content. (g) Size of vessel cells measured in the stem sections at the 12th internode. Scale bars in (a) = 20 cm; in (b) = 3 cm; in (c) = 500 μm; in (d) = 100 μm.

Examination of the stem cross-section revealed that the CW thickening in the vascular cells of transgenic plants differed from that in WT. In PtrMAN6 overexpressed plants, lignin deposition was significantly delayed and reduced in xylem cells. In contrast, lignin deposition occurred earlier and at elevated levels in PtrMAN6 downregulated plants (Figure 5c,d). It appears that PtrMAN6 downregulation also affected the wall thickening process in pith cells and fiber cells (Figure 5c). Determination of lignin content further confirmed that overexpression of PtrMAN6 resulted in lignin reduction (Figure 5e). Crystalline cellulose content in PtrMAN6-overexpressed plants was also decreased (Figure 5f). The size of vascular cells (in diameter) in the 12th internode stem sections were measured under ultraviolet (UV) light view (Figure S3b–d) and showed large differences between transgenic and WT plants (Figure 5g). In addition, morphological differences of adventitious root development in calli were observed in the PtrMAN6-overexpressed plants (Figure S3f) but not in transformed controls (Figure S3e). Together, these results suggest that PtrMAN6, in addition to functioning as an enzyme to digest mannan-type polysaccharides resulting in a relaxation of the CW, is also involved in regulating other biological events during CW thickening.

Monosaccharide composition in the transgenic plants was determined. As shown in Table 1, the neutral sugars content in transgenic plants was changed dramatically. The content of mannose and xylose was significantly lower in PtrMAN6-overexpressed plants. In the examined transgenic lines, mannose content decreased by 33 and 36%; xylose content was reduced by 18 and 24%. These two sugars are the main components of mannan and xylan, which are abundantly localized in the SCWs of dicots (Handford et al., 2003; Scheller and Ulvskov, 2010). In contrast, the levels of other sugars such as fucose, arabinose and rhamnose were increased significantly. These sugars are relatively more abundant in primary wall hemicellulose and pectin. In PtrMAN6-suppressed plants, changes in sugar content that were the opposite of those in PtrMAN6-overexpressed plants were observed. Together, these data suggest that secondary wall formation is repressed in PtrMAN6-overexpressed plants but accelerated in PtrMAN6-suppressed plants.

Table 1. Monosaccharide composition of cell wall residues
ResiduesRhamnoseFucoseArabinoseXyloseMannoseGalactoseGlucose
  1. AIR: Alcohol-insoluble residues. Value: mean (μg mg−1 AIR) ± standard error (SE) of four replicates.

  2. *P < 0.05 and **P < 0.01, respectively, in Student's t-test.

WT6.59 ± 0.131.66 ± 0.0513.35 ± 0.93216.93 ± 7.0117.45 ± 0.8713.34 ± 0.4337.47 ± 1.04
PtrMAN6AS Line 55.60 ± 0.20**1.22 ± 0.04**6.91 ± 0.59**280.55 ± 7.32**19.87 ± 0.76*9.59 ± 0.41**34.69 ± 1.12
PtrMAN6AS Line 65.19 ± 0.19**1.16 ± 0.03**6.56 ± 0.38**288.67 ± 5.23**20.19 ± 0.14**8.85 ± 0.37**34.44 ± 0.66*
PtrMAN6OE Line 19.72 ± 0.13**3.2 ± 0.07**35.96 ± 0.96**165.69 ± 1.83**11.83 ± 0.28**31.53 ± 0.61**45.94 ± 0.87**
PtrMAN6OE Line 28.78 ± 0.15**2.69 ± 0.04**24.56 ± 0.60**178.64 ± 5.29**11.32 ± 0.29**22.20 ± 0.49**38.32 ± 1.12

Transcriptional program regulated by PtrMAN6 expression

It is known that several transcription factors, such as wood-associated NAC domain transcription factors WNDs (WND1A to WND6A and WND1B to WND6B in Populus), MYB3(v-myb avian myeloblastosis viral oncogene homolog), MYB20 and MYB28 are key regulators that dictate the transcriptional program toward xylem cell differentiation, CW thickening and lignin biosynthesis (Zhong and Ye, 2009; McCarthy et al., 2010; Zhong et al., 2010; Ohtani et al., 2011). To examine how PtrMAN6 is involved in the suppression of CW thickening, we examined the expression of the key transcriptional factors in transgenic and WT plants. Overexpression of PtrMAN6 downregulated the expression of transcription factors including WND1A, WND2A, WND3A, WND4A, WND5A, WND6A, MYB3, MYB20 and MYB28 (Figure 6a). Furthermore, several other SCW-related genes, such as CesA8, GT43B, C3H1 and CAD4 genes, in Populus were also downregulated in PtrMAN6-overexpressed plants (Figure 6a). On the other hand, suppression of PtrMAN6 upregulated the transcriptional activities of these genes (Figure 6b). These results indicated that upregulation of PtrMAN6 suppresses the transcriptional program that regulates xylem cell differentiation and CW thickening.

Figure 6.

Expression of secondary wall-associated genes in transgenic plants. (a) Expression of secondary wall-associated genes in PtrMAN6-overexpressed plants. (b) Expression of secondary wall-associated genes in PtrMAN6-suppressed plants. Error bars indicate SE of three technical replicates using pooled samples of three independent plants. This experiment was performed three times using different batches of plants.

Regulation of PtrMAN6 may be mediated by oligosaccharides

To understand how PtrMAN6 hydrolysis is implicated in the regulation of transcriptional activities during vascular cell development, we examined the products from Populus GGM hydrolyzed by PtrMAN6. GGM was extracted from Populus xylem tissue and digested by plant-sourced PtrMAN6 enzyme. The products were examined by high pressure liquid chromotography/quadruple time of flight mass spectrometry (HPLC/QTOF-MS). In digestions of 8–24 h, the same oligosaccharide products were identified (Figure 7; Figure S4), whilst no such products were detected in the reaction with heat-inactivated PtrMAN6 enzymes. The identified GGMOs had a DP range from 2–7. We next investigated how the GGMO molecules affected plant growth and vascular cell differentiation.

Figure 7.

Relative abundance of identified oligosaccharides. DP, degree of polymerization; number: m/z ratio.

First, we treated the Populus stem vascular cells with GGMOs (see experiment description in Experimental Procedures and in Figure S5). Before treatment, the vascular tissue contained approximate 1–7 layers of xylem cells (Figure 8a). After 1 week of treatment, about seven extra layers of new xylem cells developed. Compared with the control (Figure 8b,e), the CW thickening process in the newly formed cells was suppressed and lignification was inhibited after treatment with GGMO (Figure 8c,f; Figure S5d), a finding that suggested that GGMO treatment suppressed CW thickening during xylem development. Meanwhile, we also investigated the transcript levels of genes associated with cell wall thickening and found that GGMO treatment downregulated the expression of several wall thickening-associated transcriptional factors, such as WNDs, MYB3, MYB28, and also genes associated with the lignin biosynthesis pathway (Figure 8d). In this case, GGMO treatment had a similar effect on xylem CW thickening as the changes that occurred in PtrMAN6-overexpressed plants. This finding presents the distinct possibility that the regulatory role of PtrMAN6 may be mediated through its catalytic products, GGMOs, that function as signaling molecules to regulate the transcriptional program of CW thickening.

Figure 8.

Oligosaccharide effect on wall thickening in xylem tissue. (a) Lignin deposition in the 6th internode of the stem before galactoglucomannan oligosaccharide (GGMO) treatment. (b) Lignin deposition in the same internode after 1-week treatment with buffer as control. (c) Lignin deposition in the same internode after 1-week treatment with 0.4 mg ml–1 GGMO solution. (d) Expression of secondary wall-associated genes in the xylem after GGMO treatment. (e,f) High magnification of the framed zones in (b) and (c), showing lignin deposition in xylem before treatment (BT) and after treatment (AT). Arrows in (b,c,e,f) indicate the new xylem zone formed after treatment. Scale bar = 100 μm.

Second, GGMOs were used to treat the leaf discs from WT and PtrMAN6-overexpressed plant for callus induction during transformation (Figure S6). Adventitious root growth, a phenomenon observed during transformation (Figure S3), was recorded following different treatments. At the callus stage, no adventitious root growth was observed in the untreated WT (Figure S6a). Treatment with GGMOs resulted in adventitious root growth in about 50% of the calli, and was similar to the frequency observed with the PtrMAN6-overexpressed lines (Figure S6b–d). Overall, MAN-produced oligosaccharides were sufficient to induce phenotypes similar to those caused by PtrMAN6 overexpression. These results further point to the role that mannan-derived oligosaccharides produced by PtrMAN6 plays as signaling molecules to suppress plant SCW thickening during xylem development.

Discussion

Endo-1,4-beta-mannanase, localized on the plasma membrane, can hydrolyze GGM to produce GGMOs

GGMOs, a group of mannan-derived oligosaccharides, have been reported to act as extracellular signal molecules and regulate xylary cell differentiation (Benova-Kakosova et al., 2006; Richterova-Kucerova et al., 2012). In xylogenic cultures of zinnia, the application of GGMOs is able to increase cell population density and decrease the ratio of protoxylem-like to metaxylem-like TEs (Benova-Kakosova et al., 2006). In mung bean seedlings, GGMOs enhance cell elongation and delay xylem maturation during primary root growth (Richterova-Kucerova et al., 2012). The two studies indicate that GGMOs play a role in regulation of the process of cell growth and differentiation. However, there is little evidence as to how they are produced in plants. Here, we present a body of evidence that supports the role of PtrMAN6, an endo-1,4- beta-mannanase from poplar, in the production of GGMO molecules that act as signals to suppress CW thickening. The results provide new insights into the signaling networks that direct the transcriptional program for SCW formation during xylem differentiation.

Overexpression of PtrMAN6 in Populus leaves produced active proteins that displayed strong hydrolyzing activity toward mannan-type polysaccharides but minor activity toward other polysaccharides. PtrMAN6 enzyme used GGM as a preferred substrate. When the GGM isolated from Populus xylem CWs was hydrolyzed, GGMO molecules were detected and showed biological activity in suppression of cell wall thickening when applied to developing xylem tissue. Further characterization of the oligosaccharide linkage structure would be a next study toward a full elucidation of the mechanisms underlying the GGMO signaling activity.

We found that PtrMAN6 is a glycoprotein and undergoes N-glycosylation that is required for its enzymatic activities. When expressed in a plant system, native PtrMAN6 forms a disulfide-linked homodimer that is essential for its enzymatic activities. However, prokaryotic recombinant PtrMAN6 protein cannot form a dimer under both non-reducing and reducing conditions – a finding that may explain why no enzymatic activity was detected with the prokaryotic recombinant PtrMAN6 protein. In previous studies, MAN activities have been detected using prokaryotic recombination proteins (Bourgault and Bewley, 2002; Schroder et al., 2006). It has not been reported that the N-glycosylation modification and disulfide-linked homodimer are needed for MAN activity in plants (Rodriguez-Gacio Mdel et al., 2012). Our results revealed that PtrMAN6 displays different characteristics when compared with the MAN members in other plants (Rodriguez-Gacio Mdel et al., 2012), suggesting that PtrMAN6 may play a new function during xylem development.

PtrMANs play a role in coordinating cell wall remodeling, with suppression of SCW formation during xylem differentiation

The hydrolysis of CW mannan-type polysaccharides by MAN is believed to be a necessary biochemical step during seed germination (Bewley et al., 1997; Nonogaki et al., 2000; Gong and Bewley, 2007; Ren et al., 2008; Iglesias-Fernandez et al., 2011a,b), fruit ripening (Bewley et al., 2000; Bourgault et al., 2005) and flower development (Filichkin et al., 2004). In those studies, MAN enzyme is localized in CWs where it hydrolyzes mannan-type polysaccharides (Bewley et al., 2000; Rodriguez-Gacio Mdel et al., 2012). PtrMAN6 is localized on the plasma membrane, probably with its catalytic domain on the non-cytosolic side, and digests CW mannan-type polysaccharides to loosen the CW as well as produce oligosaccharide molecules. The different subcellular location could be due to the N-terminal structure as, compared with other reported MAN members, PtrMAN6 has a rather different N-terminal sequence structure that may be responsible for its membrane location.

During seed germination, fruit ripening and flower development, MANs mainly function in wall loosening. However, during xylem development, PtrMANs display rather different characteristics. In addition to wall loosening, here we show that MANs are also involved in xylem differentiation and may play a crucial role in suppressing SCW formation during xylem differentiation.

Overexpression of PtrMAN6 in Populus suppressed lignin deposition, while downregulation of PtrMAN6 accelerated lignin deposition in xylem tissue. Consistent with this result, the PtrMAN6-overexpressed transgenics contained less xylose and mannose, which are the main monosaccharides for SCW hemicellulose (Scheller and Ulvskov, 2010), when compared with WT plants. Conversely, the PtrMAN6-suppressed transgenics were more enriched in secondary wall-related monosaccharides. This evidence suggests that PtrMAN6 not only functions in the digestion of polysaccharides, which allows for the relaxing of CW in the process of cell expansion, but could also suppress the thickening of the SCW in xylem tissue.

Gene expression analysis showed that three of the five PtrMAN genes are highly expressed in xylem tissue, a finding that is consistent with the expression patterns obtained from the PlaNet consortium database (Mutwil et al., 2011). Interestingly, immunolocalization revealed that PtrMAN6 was specifically localized in xylem vessel cell, but not in other xylem cells such as fiber, ray, parenchyma cell, and cambium cells. Whether expression of the other two PtrMANs has different cell-type specificity need to be further investigated. It is possible that the three PtrMANs could be associated with different cell types in xylem tissue. This suggestion. though, does not rule out the potential for different PtrMAN members to carry out a similar biochemical function of digestion to produce oligosaccharides. Actually, the fiber cell wall thickening in the transgenics with PtrMAN6 antisense gene expression was also affected. This result could be due to sequence similarity among PtrMANs (such as PtrMAN 6 and PtrMAN4). The antisense PtrMAN6 might have affected other PtrMAN expression in various types of xylem cells.

To form secondary walls, a set of transcriptional programs needs to be launched in certain type of cells (Cano-Delgado et al., 2010; Ohashi-Ito et al., 2010). In this study, we show that PtrMAN6 is involved in the transcriptional program that regulates SCW formation. Transcription factors regulated by PtrMAN6 include WNDs, which are considered the master switches for SCW formation (Zhong et al., 2010; Ohtani et al., 2011). Overexpression of PtrMAN6 downregulated WND expression, while suppression of PtrMAN6 upregulated the WND transcription factor genes. Several WNDs, MYB3 and MYB20, are considered to be main components in the transcriptional networks that direct SCW formation in poplar (McCarthy et al., 2010). MYB28 is thought to be a switch that regulates lignin biosynthesis (Zhong and Ye, 2009). Expression of these genes involved in lignin, xylan and cellulose biosynthesis is downregulated when PtrMAN6 is overexpressed. Here, the evidence supports the role that MAN plays in negatively regulating SCW formation during xylem development.

The MAN regulatory function is mediated through oligosaccharide molecules

MAN, acting as a hydrolase enzyme, is able to suppress SCW formation. What is the mechanism behind this regulation? In previous studies, GGMOs – the degraded products from mannan-type polysaccharides – have been isolated from wood (Dey, 1978, 1980), kiwifruit (Schroder et al., 2001), and tobacco cell cultures (Sims et al., 1997). As signaling molecules, GGMOs have been shown to play a wide range of biological activities in the regulation of cell differentiation (Auxtova et al., 1995; Benova-Kakosova et al., 2006). In this study, results demonstrated that the active PtrMAN enzyme was able to hydrolyze xylem CW GGM to produce GGMOs, which in turn was active in regulation of SCW thickening of xylem tissue. PtrMAN6 suppression caused early SCW thickening. Overexpression of PtrMAN6, which was able to enhance GGMO production, resulted in delayed SCW thickening during xylem differentiation, and had the same result as the application of exogenous oligosaccharides. Thus, both PtrMAN6 expression regulation and exogenous oligosaccharide application altered the transcriptional activity of secondary wall thickening in a similar manner, a finding that suggested that GGMOs may act as signaling molecules to mediate the regulatory function of PtrMAN6.

Together, this study supports the model presented in Figure 9. The MAN genes are expressed specifically in xylem cells at the stage of cell expansion. The MAN genes display cell-type specificity and may perform similar biochemical function in different cell types. In this study, PtrMAN6 showed vessel cell specificity. MAN protein is found in plasma membranes localized with its catalytic domain to hydrolyze CW mannan-type polysaccharides. The products of GGMOs are able to function as signaling molecules to modulate the transcriptional program of SCW thickening. GGMOs inhibit transcriptional activity of genes such as WNDs, MYB3, MYB20 and MYB28, which are critical players in the transcriptional networks governing SCW thickening. This model provides a new ‘lens’ to understand the regulation of SCW thickening during xylem differentiation. However, further study is needed to decipher the more detailed mechanisms of the model. For example, how is the GGMO signal perceived and transmitted across the plasma membrane? Conversely, precise regulation of MAN expression is required in order to accurately direct SCW thickening. The PtrMAN6 gene contains a target sequence of miRNA159 that is expressed specifically in developing xylem (Lu et al., 2005), therefore could miRNA play a role in regulation of MAN expression in developing xylem? Although further evidence will aid in producing a fuller picture of the process, the results of the current study provide a set of new insights to understand the signaling pathways that suppress the SCW thickening process during xylem cell differentiation.

Figure 9.

A proposed model for the role of endo-1,4-β-mannanase (MAN) in the regulation of secondary cell wall (SCW) formation. Xylem differentiation involves cell expansion and cell wall (CW) thickening, which require many factors to loosen cell walls and commence wall thickening program. MAN enzyme, an integral monotopic protein with its catalytic domain on the non-cytosolic side, plays a function in hydrolyzing cell wall CW galactoglucomannan (GGM). This function would result in cell wall loosening and generation of galactoglucomannan oligosaccharides (GGMOs) that serve as signal molecules to modulate the transcriptional program of cell wall thickening. miRNA159 expression may negatively regulate expression of PtrMAN that contains a miRNA159 target sequence (Lu et al., 2005). MYB, v-myb avian myeloblastosis viral oncogene homolog in Populus; WND, wood-associated NAC domain transcription factor.

Experimental Procedures

Plant material

Populus trees in this study were grown in a phytotron for the first 3 months and then moved to a greenhouse. P. trichocarpa was used for gene cloning and expression analysis. Populus × euramericana cv.'Nanlin895' was used for genetic transformation according to the protocol used in our laboratory (Li et al., 2003). Arabidopsis (Columbia) was grown in a phytotron with a light and dark cycle of 16 h and 8 h at 22°C and used for genetic transformation in accordance wth previously described methods (Clough and Bent, 1998).

Molecular cloning, constructs and expression of PtrMAN genes

PtrMAN sequences were retrieved from the poplar genome database (http://www.phytozome.net/poplar). Total RNA was isolated from various tissues and treated with RNase-free DNase I to remove DNA contamination, in accordance with a previously established protocol (Gui et al., 2011). The full coding sequence of PtrMAN6 was cloned by RT-PCR and deposited into the NCBI database. To explore the genetic effects of PtrMAN6 on Populus plants, PtrMAN6 cDNA was subcloned into a binary pBI121 vector in both sense and antisense orientations under the control of the CaMV 35S promoter. To investigate the subcellular localization of PtrMAN6 in onion epidermal cells, full length and partial cDNA were subcloned into a pA7 vector (Voelker et al., 2006) in frame fusion with GFP under the control of the CaMV 35S promoter. After the sequence of the constructs were confirmed, the above pA7 constructs were bombarded directly into onion epidermal cell and the other constructs were mobilized into Agrobacterium tumefaciens strain GV3101 for transformation of Populus and Arabidopsis. Prokaryotic protein expression was carried out via a previously established protocol (Gui et al., 2011).The sequences of primers used in this study are listed in Table S2. For real-time quantitative PCR measurement, primers (Table S2) were designed to amplify a specific fragment (100–300 bp in length) from the target genes. Gene expression values were normalized using the Populus ACTIN2 gene as a reference.

Antibody production

Two PtrMAN6-specific peptides (EQFKTMVEEVDNH, residues 37–49; ELNDVEEDEWL, residues 61–71), were synthesized and used to raise polyclonal antibody in rabbits (Abmart, Shanghai, China, http://www.ab-mart.com.cn/). Crude antisera were purified using a protein-A Sepharose Cl-4B column. Anti-GFP, anti-His and anti-actin monoclonal antibodies were purchased from Abmart.

MAN activity assay

Samples were ground in liquid nitrogen to a fine power and homogenized at 4°C in 1.5 vol of extraction buffer that contained 1 m sodium acetate buffer (pH 5.0), 10 mm ethylenediaminetetraacetic acid (EDTA), 10 mm sodium azide, and 3 mm phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged at 10 000 g for 30 min at 4°C. The supernatant was further purified and concentrated through a 10 000 Mr cut-off filter. The protein concentration was measured by bicinchoninic acid (BCA) Reagent (Tiangen Biotech, Beijing, China, http://www.tiangen.com/) using bovine serum albumin (BSA) as a standard. MAN activity was determined following the protocol of Iglesias-Fernandez and Matilla (2009) with some modifications. Briefly, 200 μl of reaction mixture that contained 100 μl of 1% (w/v), in 0.1 m sodium acetate buffer (pH 5.0) AZC l-galactomannan (Megazyme, Wicklow, Ireland, http://www.megazyme.com/) and 20 μg of extracted enzyme protein or BSA. After 2 h, the reaction mixture was centrifuged at 12 000 g for 5 min and measured at an absorbance of 590 nm. The enzyme activity of samples was calculated using commercial endo-β-mannanase (E-BMANN, Megazyme) as a reference according to the manufacturer's instructions. PtrMAN6 activity was examined with various substrates including ivory nut mannan (Megazyme), konjac glucomannan (Megazyme), carob galactomannan (low viscosity, Megazyme), Populus galactoglucomannan (extracted GGM), tamarind seed xyloglucan (amyloid, Megazyme), birchwood xylan (Sigma-Aldrich, www.sigmaaldrich.com/) and carboxyl methyl cellulose (Sigma-Aldrich). Next, 20 μg of extracted enzyme protein was incubated with 0.5 ml substrates at 2 g L–1 in 0.1 m sodium acetate buffer at pH 5.0. After 2-h incubation at 50°C, the reaction was stopped by the addition of equal volume of 3,5-dinitrosalicylic acid (DNS) reagent (Miller, 1959). One unit of mannan endo-1,4-β -mannanase activity was defined as the amount of enzyme that released 1 μ mole of mannose equivalents in 2 h under the condition.

Western blot, immunolocalization and PtrMAN glycosylation and dimerization analysis

Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. For non-reducing conditions, samples were mixed with an equal volume of 2 × loading buffer (0.1 m Tris–HCl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol). For reducing conditions, samples were mixed with equal volume of 2 × loading buffer supplemented with 0.5–5% β-mercaptoethanol (β-ME) or 10 mm 1,4-dithiothreitol (DTT). Unless noted, all SDS-PAGE were performed under reducing conditions. All samples were boiled for 5 min prior to electrophoresis. Western blot analysis and immunolocalization were performed according to methods detailed in previous studies (Song et al., 2010). Sodium carbonate treatment was performed as described previously (Fujiki et al., 1982). For PtrMAN glycosylation analysis, extracted PtrMAN protein was first denatured at 100°C for 10 min. Endoglycosidase Hf (Endo Hf, New England Biolabs, http://www.neb-china.com/) was incubated with the denatured proteins at 37°C for 30 min or 1 h according to the manufacturer's instructions. Then, the molecular size of the proteins was estimated by western blot. Native proteins treated by Endo Hf at 37°C for 2 h were analyzed directly for enzyme activity. The same proteins incubated without Endo Hf in the same Endo Hf buffer for the same period were used as a control. For PtrMAN6 dimerization analysis, native proteins incubated with/without 5% β-ME or 10 mm DTT at 37°C for 2 h were analyzed for enzyme activity.

CW composition and vessel size analysis

Wood tissue from 1-year-old Populus was used to prepare alcohol-insoluble residues (AIRs) of the CWs. Analysis of monosaccharide composition and crystalline cellulose content was conducted as described previously (Xiong et al., 2010). Lignin content was determined as Foster et al. (2010) and lignin deposition was stained with 1% phloroglucinol (w/v) in 12% HCl for 5 min and immediately observed under a light microscope. For measurement of vessel size, the 12th internode stem was fixed and cross-sectioned as described (Hong et al., 2010). Sections were observed under a UV fluorescence microscope equipped with the Image J program for area measurement. Data from 10 sections in each of three line plants were collected and analyzed statistically using Student's t-test.

GGMO analysis

Crude galactoglucomannan (GGM) was extracted from Populus xylem tissue as described previously (Auxtova et al., 1995). The GGM (1 ml of 0.2% (w/v) in sodium acetate–acetic acid (NaAc–HAc) buffer, pH 5.0) was then treated with the extracted MAN protein (5 μg) at 50°C for various time periods (8, 16 and 24 h). After centrifugation at 13 000 g for 10 min, the supernatant was passed through a 10 000 Mr cut-off filter and dried in a vacuum evaporator. Then the hydrolyzed products were derivatized with 0.5 m 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP, Sigma-Aldrich) as described (Honda et al., 1989).

The derivatives were analyzed using an Agilent 6520 series LC 1200 MS 6520 QTOF system (Agilent, http://www.home.agilent.com/agilent/home.jspx?cmpid=4542&lc=chi&cc=CN) packed with a Zorbax Extend-C18 column (3.0 × 50 mm, 1.8 μm, Agilent). Next, 3 μl of analyte was injected with a constant mobile phase flow rate of 0.3 ml min–1. The mobile phase consisted of 10 mm ammonium acetate in H2O (A) and 20 mm ammonium acetate in acetonitrile (B) using a gradient elution of 22, 30 and 80% buffer B by a linear increase from 0, 5 and 10 min. The diode array detector (DAD) was set at 214 nm for monitoring and the TOF mass spectrometer was set as scan range from 150–3000 at 160 V and radio frequency (RF) at 750 V in positive scan mode at 4 GHz resolution. The temperature of dry gas of electrospray ionization (ESI) was set at 350°C with holding flow at 9 L min–1. Relative quantification of oligosaccharides was calculated by integrating the peak area of m/z 673.27, 835.32, 997.38, 1159.48, 1321.48 and 1483.54 as qualifiers that are derivatives from GGMOs and PMP.

GGMO effect assay

Crude GGM (20 mg) was digested with plant-sourced PtrMAN6 protein at 50°C for 12 h. The reaction mixture was passed through a 10 000 Mr cut-off filter to remove proteins and polysaccharides. GGMOs content was determined according to the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959) and diluted to 0.4 mg ml–1 in 0.1 m NaAc buffer. To investigate the GGMO effect on xylem CW thickening, 3-month-old Populus trees were selected and treated with 0.4 mg ml−1 GGMOs in 0.1 m NaAc buffer or 0.1 m NaAc buffer only (control). Treatment of the developing xylem tissue was carried out as follows: the bark of the stem at the 6th internode from the top was gently peeled back about 0.5 cm. A small cotton ball soaked with GGMO solution was inserted in and sealed with Parafilm. After 1 week, the treated developing xylem tissue was examined for cell thickening morphology and gene expression. At least three trees were used for each treatment. In addition, filter-sterilized GGMOs were applied to the solid medium at 2-week subculture intervals to investigate the effect of the treatment on tissue culture. Three groups of experiments (leaf disc explants from WT cultured in the medium with or without 50 mg L–1, leaf dics from PtrMAN6 overexpressed plants cultured in the medium without GGMOs) were carried out for tissue culture observation.

Sequence information

The GenBank accession numbers for the poplar genes studied in this article are PtrMAN4 (XM_002309155), PtrMAN5 (XM_002310780), PtrMAN6 (XM_002323644, JX840449), PtrMAN7 (XM_002327649), PtrMAN8 (XM_002330651), C3H1 (XM_002-308824), CAD4 (EU603306), CesA8 (XM_002316779), GT43B (JF518935), WND1A (HQ215847, XM_002317023), WND2A (HQ215849), WND3A (XM_002322362), WND4A (XM_002329829), WND5A (XM_002310261), WND6A (XM_002327206), MYB3 (XM_002299908), MYB20 (XM_002313267), MYB28 (XM_002-307154), ACT2 (XM_002298674).

Acknowledgements

We thank Dr Hongxuan Lin for assistance with microtome sectioning, Dr Yining Liu for LC-QTOF-MS analysis, Dr Yihua Zhou for CW composition analysis, and Mr Xiaoshu Gao for confocal laser scanning microscopy. This work was supported by the National Key Basic Research Program of China (2012CB114502), the National Natural Science Foundation of China (31130012) and Shanghai Science and Technology Commission (11XD1405900) to LL.

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