Different sources of silicon precursors influencing on surface characteristics and pore morphologies of mesoporous silica nanoparticles

The Plant Journal (2008) 54, 993–1003

doi: 10.1111/j.1365-313X.2008.03456.x

Sequential cell wall transformations in response to the induction of a pedicel abscission event in Euphorbia pulcherrima (poinsettia) YeonKyeong Lee1, Paul Derbyshire2, J. Paul Knox3 and Anne Kathrine Hvoslef-Eide1,* Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, 1432 Aas, Norway, 2 Department of Metabolic Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK, and 3 Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK

1

Received 25 November 2007; revised 10 February 2008; accepted 14 February 2008; published online 21 April 2008. * For correspondence (fax +47 64965615; e-mail: [email protected]).

Summary Alterations in the detection of cell wall polysaccharides during an induced abscission event in the pedicel of Euphorbia pulcherrima (poinsettia) have been determined using monoclonal antibodies and Fourier transform infrared (FT-IR) microspectroscopy. Concurrent with the appearance of a morphologically distinct abscission zone (AZ) on day 5 after induction, a reduction in the detection of the LM5 (1 fi 4)-b-D-galactan and LM6 (1 fi 5)-a-L-arabinan epitopes in AZ cell walls was observed. Prior to AZ activation, a loss of the (1 fi 4)-b-Dgalactan and (1 fi 5)-a-L-arabinan epitopes was detected in cell walls distal to the AZ, i.e. in the to-be-shed organ. The earliest detected change, on day 2 after induction, was a specific loss of the LM5 (1 fi 4)-b-Dgalactan epitope from epidermal cells distal to the region where the AZ would form. Such alteration in the cell walls was an early, pre-AZ activation event. An AZ-associated de-esterification of homogalacturonan (HG) was detected in the AZ and distal area on day 7 after induction. The FT-IR analysis indicated that lignin and xylan were abundant in the AZ and that lower levels of cellulose, arabinose and pectin were present. Xylan and xyloglucan epitopes were detected in the cell walls of both the AZ and also the primary cell walls of the distal region at a late stage of the abscission process, on day 7 after induction. These observations indicate that the induction of an abscission event results in a temporal sequence of cell wall modifications involving the spatially regulated loss, appearance and/or remodelling of distinct sets of cell wall polymers. Keywords: flower abscission, cell wall alteration, cell wall polysaccharides, immunolabelling, FT-IR.

Introduction Abscission is the shedding of organs such as leaves, branches, flowers and fruits from plants, effecting the dispersal of reproductive organs, saving metabolic energy by the removal of unwanted organs, and protecting against biotic or abiotic stresses. It can be influenced by environmental factors including temperature, light, disease, water stress, and nutrition (Addicott, 1982). Abscission is a natural process that occurs during plant development, but premature abscission can result in severe post-harvest losses in the horticultural and agricultural industries. The developmental programme of abscission is likely to be a highly coordinated process, but the precise mechanisms of abscission and their control are not well understood (Addicott, 1982; Patterson, 2001; Roberts et al., 2000, 2002). Abscission involves the ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd

breakdown of cell walls at discrete regions of organs and at specific stages of development. However, the spatial and temporal regulation of the dissolution of primary cell wall polymers and middle lamella during abscission processes has not been studied systematically, although the loss of middle lamella pectin to promote cell separation is known to be involved in abscission events (Clements and Atkins, 2001; Patterson, 2001; Roberts et al., 2000, 2002; Uheda and Nakamura, 2000). A network of cellulose microfibrils tethered by crosslinking glycans (CLGs or hemicelluloses) is a major component of cell walls and contributes to the mechanical properties of cell walls. A structurally complex pectic matrix is co-extensive with the cellulose–CLG network in primary 993

994 YeonKyeong Lee et al. cell walls and is abundant in the intercellular matrices of middle lamellae. The pectic network is composed of sets of heterogeneous polysaccharides in which galacturonic acid (GalA) and rhamnose (Rha) are common sugars (Ridley et al., 2001; Willats et al., 2001). Pectic polysaccharides can be split into two major classes: homogalacturonan (HG) with an associated rhamnogalacturonan-II (RG-II) domain and rhamnogalacturonan-I (RG-I). Homogalacturonan is composed of unbranched a-1,4-linked GalA residues that can be methyl- or acetyl-esterified and can be cross-linked through calcium ions as well as through borate diesters by means of the RG-II domain. Rhamnogalacturonan-I has a backbone of a-1,4-linked GalA residues alternating with a-1,2-linked Rha. Rhamnogalacturonan-I is a highly variable set of polymers that often has (1 fi 5)-a-L-arabinan and (1 fi 4)-b-D-galactan as structural motifs of its side chains (Albersheim et al., 1996; Mohnen, 1999). Euphorbia pulcherrima (poinsettia or Christmas star) is an important ornamental plant worldwide, especially during the Christmas season, but the premature abscission of flowers leads to severe losses in crop value. Usually, abscission of flower buds in poinsettia occurs over approximately 2–3 weeks. Here we report a controlled in planta abscission system in poinsettia in which an abscission event can be induced by flower decapitation (Munster, 2006). Cutting off a flower bud at a specific site, leads to a temporally and spatially defined abscission event in the flowerless pedicel. Decapitation of the flower pedicel results in a disruption to a balance of hormones to promote flower abscission. This provides a tractable synchronized system for studying the abscission process. The abscission region of poinsettia pedicel can be divided into three defined groups of cells. One group is the abscission zone (AZ) itself, including the separation layer. Another is the distal area belonging to the organ to be shed. The third region is the proximal area belonging to the mother plant from which the organ is shed. Our inducible abscission system in the poinsettia flower pedicel is similar to that described previously for natural or induced abscission in many plant species (Bornman et al., 1967; Hashim et al., 1980; Kuang et al., 1992; Oberholster et al., 1991; Webster, 1970). Using this poinsettia-inducible abscission system we have identified abscission-specific cell wall modifications in the AZ and adjacent parenchyma systems, using a range of monoclonal antibodies and Fourier transform infrared (FT-IR) microspectroscopy. Results Cellular morphogenesis during induced pedicel abscission in E. pulcherrima To investigate the abscission process, poinsettia flower buds (cyathia) were decapitated and the development of the

AZ was studied. The poinsettia flower AZ had a relatively large AZ and showed distinct differentiation in distal and proximal areas. Up to day 4 after induction it was not possible to see an AZ on the flower pedicel. At day 5 after induction (D5) the AZ became visible on the flower pedicel base, and the pedicel distal to the AZ became yellow–green (Figure 1a). This region of yellow and senescing pedicel eventually collapsed and abscised after 7–10 days (Figure 1e). Longitudinal sections of the AZ region indicated active cell divisions within six to eight cell layers centred on the AZ at day 5 (Figure 1b). Cells that make up the AZ can be readily distinguished immediately prior to cell and organ separation. The AZ comprises several cell layers that are generally smaller (as a result of cell division) than adjacent non-separating cells and are more densely protoplasmic. As abscission progressed, cells in the AZ enlarged, rounded up and separated from each other so as to present the abscission plane as seen in longitudinal sections through the AZ (Figure 1f). By day 5, cell walls immediately distal to the potential fracture plane displayed UV-induced autofluorescence (Figure 1c), whereas at day 4 there was no sign of the AZ or the autofluorescence in the flower pedicel (data not shown). At day 7, the adjacent cell walls both distal and proximal to the AZ and separation layer showed high levels of autofluorescence (Figure 1g). Electron micrographs indicated the irregular cell ultrastructure and cell separation in the AZ at day 7 (Figure 1d). Breakdown of the middle lamellae was accompanied by distortion and dissolution of cell walls along the abscission plane (Figure 1d,h). Epitope mapping indicates spatial alterations to pectic polysaccharides in relation to AZ activation To investigate any alterations to cell wall pectic polysaccharides during the abscission process, extracts of the AZ, the adjacent distal and proximal pedicel regions, and from control pedicels were assessed for the presence of HG (JIM5, JIM7, LM7 and PAM1), (1 fi 4)-b-D-galactan (LM5), feruloylated (1 fi 4)-b-D-galactan (LM9), (1 fi 5)-a-L-arabinan (LM6) and xylogalacturonan (LM8) epitopes using immunodot assays (IDAs). The antibodies are described in Table 1. LM7, LM8, LM9 and PAM1 did not bind to any of the extracts (data not shown). The AZ and distal area at day 5 showed a relatively reduced labelling intensity while the proximal area retained intense labelling with JIM5, JIM7, LM5 and LM6 antibodies (Figure 2a). A densitometric comparison of the IDAs at 100-fold dilution of the extracts is shown in Figure 2(b). The AZ and the distal area both showed a large decrease in the content of (1 fi 4)-b-D-galactan (LM5) and (1 fi 5)-a-L-arabinan (LM6) epitopes whilst the JIM5 and JIM7 HG epitopes showed smaller reductions in occurrence. These observations indicate that pectic HG and RG-I-related polymers were being remodelled or removed from AZ cell walls and also in cell walls in the region distal to the AZ.

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Cell wall remodelling in flower abscission 995

Figure 1. Development of the abscission zone (AZ) in the Euphorbia pulcherrima flower pedicel. The AZ appears as a yellow–green ring on the pedicel as shown for day 5 (D5) (a). At day 7 (D7), cells are beginning to separate from the epidermis (e). Empty arrows indicate the AZ on the pedicel. Micrographs of longitudinal sections (1 lm thick) of the AZ show active cell division at day 5 (b) and cell separation at day 7 (f). By day 5 autofluorescence was observed in the cell walls of the AZ and distal area (c, g). The abscising cells are disintegrated (d) and middle lamellae are dissolved (h). Square brackets indicate the AZ. Bars: 50 lm (b, c, f, g); 20 lm (d); 5 lm (h).

Table 1 Cell wall polysaccharide-directed monoclonal antibodies (MAb) used in this study

MAb

Cell wall polymer/epitope recognized

Reference

LM5 LM6 JIM5 JIM7 LM7 LM8 LM9 PAM1 LM10 LM11 LM15

(1 fi 4)-b-D-galactan (1 fi 5)-a-L-arabinan Partially methyl-esterified/unesterified HG Partially methyl-esterified HG Non-block-wise partially methyl-esterified HG Xylogalacturonan (detaching cell-specific epitope) Feruloylated (1 fi 4)-b-D-galactan Unesterified HG (1 fi 4)-b-D-xylan (1 fi 4)-b-D-xylan/arabinoxylan Xyloglucan (XXXG motif)

Jones et al. (1997) Willats et al. (1998) Clausen et al. (2003) Clausen et al. (2003) Clausen et al. (2003) Willats et al. (2004) Clausen et al. (2004) Manfield et al. (2005) McCartney et al. (2005) McCartney et al. (2005) S.E. Marcus and JPK, unpublished

Differential distribution of pectic polysaccharide epitopes in walls of AZ, AZ adjacent cells and the non-abscising control pedicel of E. pulcherrima To study the spatial and temporal distribution of pectic polysaccharides in situ, indirect immunofluorescence labelling was performed on longitudinal sections of flower pedicels using the same set of antibodies. (1 fi 4)-b-Dgalactan, (1 fi 5)-a-L-arabinan and JIM7 HG (indicative of methyl-esterified HG) epitopes were abundant in control (D0) flower pedicels and located evenly throughout the cell walls (Figure 3a,b,d), whereas the JIM5 HG epitope (indicative of low/no methyl-esterified HG) had a more restricted

occurrence and was often detected only in middle lamellae or at corners of the intercellular spaces (Figure 3c). The same labelling patterns and intensities were retained by day 4 (Figure 3e–h); however, distinct changes were observed in the AZ and distal area at day 5. By this stage, a marked reduction in the (1 fi 4)-b-D-galactan epitope within the five to six cell layers of the AZ was observed (Figure 3i). At day 7, this epitope was absent in these regions (Figure 3m). The (1 fi 5)-a-L-arabinan epitope was also reduced in the AZ at day 5 (Figure 3j) and as the AZ developed (day 7) became absent from the AZ and the distal zone (Figure 3n). In contrast, proximal areas retained the (1 fi 4)-b-D-galactan and (1 fi 5)-a-L-arabinan epitopes during the abscission process

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996 YeonKyeong Lee et al. epidermal and cortical cell walls of the proximal area both retained the (1 fi 4)-b-D-galactan epitope (Figure 4b). By day 4, distal cortical cell walls showed a clear reduction of the (1 fi 4)-b-D-galactan epitope (Figure 4c). A reduction in the occurrence of the (1 fi 5)-a-L-arabinan LM6 epitope in far distal parenchyma (about 300 lm away from the AZ) was detectable a day earlier, by day 3 (Figure 4d). These two epitopes, often associated with RG-I sidechains, were almost absent in the distal area by day 7 (data not shown). A reduction in the occurrence of the JIM7 HG epitope in distal cell walls was first detected at day 4 (Figure 4f) after the changes to the RG-I related epitopes. Location of HG in the AZ and adjacent areas

Figure 2. Immunodot assays of isolated cell wall polymers from the AZ, distal and proximal areas at day 5 and control flower pedicel of Euphorbia pulcherrima using pectic monoclonal antibodies. Plant extracts were applied to a nitrocellulose membrane in dilution series at the loading levels indicated (a) and dot intensity at a 100-fold dilution is compared in the densitogram (b). NC represents negative controls performed by omitting the primary antibody resulting in no binding.

(data not shown). The JIM5 epitope indicating no or low methylester HG had a distinct temporal and spatial pattern of occurrence. At day 5, the epitope was confined to discrete regions of the AZ that were weakly labelled (Figure 3k). However, by day 7 the JIM5 HG epitope was detected abundantly in AZ cell walls and at cell corners (Figure 3o). Moreover, by day 7 the JIM5 epitope was abundant throughout cell walls of the AZ and not restricted to regions near intercellular spaces. The JIM7 epitope indicating HG with greater methylesterification was considerably reduced in the AZ by day 5 and 7 (Figure 3l,p), while the proximal region did not show any change in the extent of labelling by this antibody. Changes in the occurrence of (1 fi 4)-b-D-galactan and (1 fi 5)-a-L-arabinan epitopes in the distal area of the pedicel (approximately 300 lm away from the AZ) were observed prior to day 4 and are shown in Figure 4. The first distinct change observed was in the epidermis, when at day 2 the (1 fi 4)-b-D-galactan epitope was observed to be absent in epidermal cell walls of the distal area but remained abundant in the walls of the cortical cells (Figure 4a). In contrast,

An interesting dynamic in relation to HG methylesterification patterns related to AZ activation was shown by detection of the JIM5 and JIM7 HG epitopes. A more detailed study using both immunofluorescence and immunoelectron microscopy (IEM) demonstrated that, at day 7, the JIM5 HG epitope was abundant in the AZ and least abundant in the proximal zone (Figure 5a). Immunoelectron microscopy revealed that the JIM5 HG epitope was mostly associated with the loosening cell wall and middle lamellae regions (Figure 5b). In contrast, the JIM7 HG epitope occurred less in the AZ and distal area (Figure 5c,d), but remained abundant in the proximal region (Figure 5c). These patterns of occurrence indicate that a reduction in HG methylesterification occurs in cell walls of the AZ and distal area during the abscission process. To see the extent of de-esterification, longitudinal sections taken from pedicels at day 7 were incubated with Na2CO3, a treatment that removes methylester groups from HG. Abundant JIM5 HG epitope was observed in the AZ and distal regions in both control and Na2CO3-treated sections (Figure 6). In contrast, cell walls of the proximal region had an increase in JIM5 epitope after alkali treatment (Figure 6c,f) indicating the presence of highly methylesterified HG. Together, these data indicate that highly methylesterified HG is de-esterified in the AZ and distal regions during the abscission process. Cell wall analysis by FT-IR microspectroscopy To ascertain any differences in cell wall composition between abscission and non-abscission zones, we used FTIR microspectroscopy to detect specific cell wall macromolecules by their absorbance of IR radiation (McCann et al., 1993). Longitudinal sections were taken from pedicels to include abscission and proximal area. For each section 30 spectra were obtained from each zone and compared by exploratory principal components analysis (PCA; Kemsley, 1998), a statistical method that reduces the dimensionality of the data from more than a hundred variates (one every

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Cell wall remodelling in flower abscission 997

Figure 3. Micrographs of indirect immunofluorescence detection of pectic epitopes in a control flower pedicel and in the abscission zone (AZ). Longitudinal sections (1 lm thick) were probed with monoclonal antibodies LM5 (a, e, i, m), LM6 (b, f, j, n), JIM5 (c, g, k, o) and JIM7 (d, h, l, p): (a–d) are control (D0) flower pedicel; (e–h) are at day 4 after induction (D4); (i–l) are at day 5 after induction (D5); (m–p) are at day 7 after induction (D7). Square brackets indicate the AZ. Bars: 50 lm.

8 cm)1 from 1800 to 800 cm)1) to only a few principal components (PCs). The PCs are ordered in terms of decreasing variance. Each observation (spectrum) has a corresponding set of PC scores, which describes the variance of that spectrum relative to the mean of the population for each PC. The PC scores of the spectra can then be plotted against one another to reveal patterns or structure in the data (Kemsley, 1998). It is possible to mathematically derive a ‘spectrum’ (called a PC loading) from a PC to identify molecular factors responsible for the separation of groups of spectra (Chen et al., 1998; Kemsley, 1998). The analysis showed that spectra collected from the AZ can be separated

from spectra of the proximal area by PC2, accounting for 8% of the total variance in the combined populations (Figure 7a). The loading for PC2 depicted in Figure 7(b) showed characteristics of arabinose, pectin and cellulose in fingerprint regions (peaks at 1065, 1103, and 1169 cm)1, respectively; Cael et al., 1975; Coimbra et al., 1999; Kacˇura´kova´ et al., 2000), carboxylate regions of pectin (1613 cm)1; Se´ne´ et al., 1994; Wilson et al., 2000), xylan (988 cm)1) and lignin (1509 cm)1; Stewart and Morrison, 1992). The cellulose/ arabinose/pectin peaks are negatively correlated with xylan and lignin. Because the PC scores of spectra of the AZ cell walls were negative relative to the mean, the data suggest

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998 YeonKyeong Lee et al.

Figure 4. Micrographs of indirect immunofluorescence detection of pectic epitopes in proximal and distal regions of Euphorbia pulcherrima flower pedicels during the early stages of abscission. Longitudinal sections of flower pedicel were labelled with LM5, LM6, JIM5 and JIM7. Epidermal and sub-epidermal cortical cells in distal area (a) and proximal area (b) at day 2 (D2) and labelled with LM5. Cortical cells in the distal area of the flower pedicel at day 3 (d) and day 4 (c, e, f): (c) LM5; (d) LM6; (e) JIM5; (f) JIM7. White arrows in (a) and (b) indicate epidermal cells. Bars: 50 lm.

Figure 6. Micrographs of indirect immunofluorescence detection of the JIM5 homogalacturonan (HG) epitope in the abscission zone (AZ) (b, e), distal (a, d) and proximal (c, f) areas of Euphorbia pulcherrima flower pedicels at day 7. (a–c) Longitudinal sections of flower pedicel immunolabelled with JIM5. (d–f) Prior to labelling, sections were treated with 0.05 M Na2CO3 to de-esterify HG. Bars: 50 lm.

that the cell walls of the AZ are relatively richer in xylan and lignin and poorer in cellulose, arabinose, and pectin when compared with proximal area cell walls. Differential distribution of xylan and xyloglucan epitopes and lignin accumulation in cell walls of the AZ, regions adjacent to the AZ and non-abscising control pedicel of E. pulcherrima

Figure 5. Micrographs of indirect immunofluorescence (a, c) and immunoelectron detection of homogalacturonan (HG) epitopes (b, d) of the abscission zone (AZ) and adjacent area in Euphorbia pulcherrima flower pedicels at day 7. Longitudinal sections stained with JIM5 (a, b) and JIM7 (c, d). Square brackets indicate the AZ. Arrows indicate gold particles. Bars: 50 lm (a, c); 1 lm (b, d).

The FT-IR analysis indicated that xylan and lignin were abundant components in the cell walls of the AZ. To extend these observations, we used a recently developed anti-xyloglucan monoclonal antibody LM15 directed to tamarind xyloglucan (S. E. Marcus and JPK, University of Leeds, UK, unpublished), anti-xylan monoclonal antibodies (LM10 and LM11) and lignin-specific stains to assess cell wall components. The induction of abscission led to the appearance of the LM15 xyloglucan epitope in the AZ and distal region at day 7 (Figure 8a–c) while the proximal area and control (D0) showed much less labelling (Figure 8d,e). There was only a

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Cell wall remodelling in flower abscission 999

Figure 7. Fourier transform-infrared (FT-IR) analysis of cell walls in the Euphorbia pulcherrima flower abscission zone (AZ) and proximal area at day 7. Exploratory principal components analysis (PCA) was performed using 30 FT-IR spectra from each zone. Principal component 2 (PC2) clearly discriminates AZ spectra from proximal area spectra (a). Axes represent the mean values for the population. (b) The corresponding loading has features characteristic of arabinose, cellulose, pectin, xylan and lignin (peaks of interest are marked). Pectin, arabinose and cellulose peaks are negatively correlated with xylan and lignin, indicating that the AZ is pectin-, arabinose- and cellulose-deficient and xylan- and lignin-rich relative to the proximal area.

Figure 8. Micrographs showing indirect immunofluorescence detection of xyloglucan and xylan epitopes in cell walls of a control flower pedicel and the abscission zone (AZ). Longitudinal sections of flower pedicel at day 7 were labelled with anti-xyloglucan (LM15) (a–d). An overview of the AZ of a pedicel at day 7 (a) shows dense labelling in the AZ (b) and distal area (c). The proximal area at day 7 (d) and the control (D0) (e) have less xyloglucan. A xylan epitope (LM11) is restricted to secondary cell walls of xylem vessels at day 0 (f) but occurs in primary cell walls of a distal area at day 7 (g). Phloroglucinol-HCl staining for lignin indicates no lignin in the control pedicel (h) but high lignin accumulation in the AZ (i). Square brackets indicate the AZ. The arrow indicates the separation area. Bars: 50 lm.

very limited detection of this xyloglucan epitope in cell walls at day 0 (Figure 8). In non-abscising control organs and the early stages of AZ activation xylan epitopes were only identified in the secondary cell walls of the xylem vessels and not in primary cell walls of the parenchyma system (Figure 8f). However, in the later stages of AZ development at day 7, both xylan epitopes became detectable in the primary cell walls of the distal region (Figure 8g). At day 0, there was no lignin accumulation (Figure 8h) but lignin staining was observed in a broad area that covered the AZ and adjacent cells including the proximal and distal regions close to the AZ at day 7 (Figure 8i).

Discussion Cell wall epitope and FT-IR analysis have been used to document cell wall changes in relation to an induced abscission event in poinsettia pedicels. Morphological changes and cell divisions indicative of AZ activation were first seen on day 5 after induction. In many plants, cell division is not a requirement for abscission. Unlike in poinsettia, cell division may be absent prior to abscission in Arabidopsis, tobacco, tomato and Begonia flower (van Doorn and Stead, 1997). These plants have a recognizable abscission zone long before abscission is initiated

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1000 YeonKyeong Lee et al. whereas the poinsettia pedicel does not have a recognizable AZ prior to a cell division stage. The first cell wall changes to be detected at the AZ were concurrent with this. On day 5 cell walls of the AZ displayed autofluorescence and a reduction in RG-I related LM5 and LM6 epitopes. Ultraviolet-induced autofluorescence may demonstrate accumulation of polyphenolics such as lignin (Barsberg and Nielsen, 2003; De Micco and Aronne, 2007) and the phloroglucinol-HCl staining supports distribution/ accumulation of lignin in the AZ. By day 7, when the abscission plane is clearly observed, AZ HG was largely unesterified and xylan, xyloglucan and lignin were detected in the cell walls of the AZ. However, the earliest changes to cell walls as a result of the induction of abscission were in the distal region six to eight cells away from the AZ. These involved loss of the LM5 epitope from the pedicel epidermal cell walls (day 2), and loss of LM6 and LM5 epitopes from cortical cell walls on day 3 and day 4, respectively, i.e. RG-I related polymers are being removed or remodelled in the organ to be lost prior to the formation of a visibly recognizable AZ and its associated cell wall changes. HG remodelling and alterations to RG-I epitopes A cell wall change that would directly function in the activation of the abscission planes and the separation process would appear to be the de-esterification of HG in cells at the AZ, which was observed on day 7. Pectic HG is a major component of primary cell walls and middle lamellae. It has long been implicated in cell adhesion and cell separation processes although we have only a rudimentary knowledge in molecular terms of what happens to pectins during cell separation (Leslie et al., 2007; Roberts et al., 2002). Homogalacturonan is generally highly methylesterified in most primary cell walls (as shown here) and in muro de-esterification by pectin methylesterases (PMEs) resulting in de-esterified HG presents sites for the action of polygalacturonases (PGs) to cleave HG chains. Considerable evidence supports a role for polygalacturonases in abscission events (Tucker et al., 2007), although it is somewhat surprising that pectin methylesterase genes or proteins have not been identified to be associated with AZ as the action of pectin methylesterase would appear to be required to produce HG sensitive to polygalacturonase cleavage (Arancibia and Motsenbocker, 2006; Leslie et al., 2007; Roberts et al., 2002). The significance of the loss of the LM5 and LM6 epitopes concurrent with AZ activation and prior to HG de-esterification is not yet clear but may be part of cell wall matrix alterations that allow access of HGremodelling enzymes to their sites of action. The loss/ alteration of RG-I domains could modulate other, as yet unidentified, properties of the pectic matrix in preparation for AZ activation.

The loss of RG-I related epitopes in cell walls distal to the site of future AZ activation might reflect remodelling or dismantling of cell wall components and the retrieval of cell wall molecules to the plant. These observations indicate that alterations to cell walls are an early, pre-AZ, activation event throughout the organ to be abscised. Up to now, many studies have emphasized putative hydrolytic enzymes that might be involved in the loss of wall components during abscission (Atkinson et al., 2002; Orozco-Cardenas and Ryan, 2003; Torki et al., 2000; Trainotti et al., 2006; Tucker et al., 2002; Wu and Burns, 2004). The loss of LM5 and LM6 epitopes may possibly result from the action of enzymes such as galactanases and arabinanases and less-esterified JIM5 HG may implicate the possible involvement of PME and PG. Oligosaccharides released by the action of PG can induce plant defence responses such as accumulation of reactive oxygen species, biosynthesis of phytoalexin, accumulation of PR protein, lignin and polyphenols in plants (Coˆte´ and Hahn, 1994). In the distal area, the rapid reduction of pectic polysaccharides may suggest a role for hydroxyl ions (•OH) produced by peroxidase (Cosgrove, 2005; Fry, 1998; Liszkay et al., 2003). •OH can cleave wall polysaccharides by non-enzymatically removing a hydrogen atom from polysaccharides, and endogenous •OH can be produced by wall peroxidases (Liszkay et al., 2003) from superoxide anion and hydrogen peroxide. Indeed, debalding bean leaves showed an increase in peroxidase (Poovaiah and Rasmussen, 1973), and in our system decapitated poinsettia flower pedicels also revealed peroxidase in the distal area (data not shown). Such peroxidases can produce high amounts of •OH, leading to pectic polysaccharide cleavage, and may be involved in cell wall alterations in the distal cells. Appearance of xylan, xyloglucan and lignin in primary cell walls The lignification of cells at both sides of an AZ has been reported before (Clements and Atkins, 2001; van Doorn and Stead, 1997; Henderson et al., 2001). The appearance of xyloglucan and xylan epitopes at the AZ and in distal cells was more unexpected. Xylans are abundant in secondary cell walls of dicotyledons, as indicated by binding of LM10 and LM11 antibodies in a range of species (Carafa et al., 2005; McCartney et al., 2005). In this system the epitopes were detected in primary cell walls in response to induction of the abscission event. Xyloglucan is known to be abundant in primary cell walls of dicotyledons and the reason for absence in control cell walls is not clear. There are two broad possibilities to explain these observed dynamics. The first is that these polymers are placed into cell walls as part of the remodelling process and the second is that the epitopes appear due to remodelling and loss of pectic components that allows the CLG/hemicellulose epitopes to be detected, i.e. the epitopes are unmasked. In addition to PGs, two

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Cell wall remodelling in flower abscission 1001 further classes of cell wall proteins that are associated with AZs are endo-b-1,4-glucanases and expansins (Belfield et al., 2005; Leslie et al., 2007; Trainotti et al., 2006) and these may impact upon xyloglucan structures. It is possible that the action of one or both of these protein classes could lead to increased detection of xyloglucan epitopes. In summary, RG-I pectic polymers (inferred by the loss of LM5 and LM6 epitopes) are altered prior to HG remodelling at the AZ. In addition, two sets of cell wall changes have been identified to be associated with abscission in addition to cell wall transformation events at the AZ itself. The first is an alteration, prior to formation of a visibly recognizable AZ, of RG-I related epitopes in distal tissues, i.e. cells that will be shed. The second is the appearance, at a late stage of abscission, of CLG/hemicellulose epitopes along with lignin around the AZ and in distal tissues. Further work will be required to determine how representative this induced abscission system is of natural abscission. Experimental procedures Plant materials Euphorbia pulcherrima Willd. ex Klotzsch ‘Lilo’ plants were grown under greenhouse conditions at 23C and long-day conditions (20/ 4 h photoperiod at 150 lmol m)2 sec)1). Five-centimetre-long cuttings with two-half expanded leaves were taken and planted in moist Jiffy7 pellets (Jiffy International AS; http://www.jiffypot.com, 38 mm diameter). Cuttings were maintained in 100% humidity and 50% shade and after 3 weeks the rooted cuttings were transplanted into 12-cm pots in a growing medium containing 1:3 (v/v) coarse perlite (Norwegian Perlite Group): fertilized and limed Norwegian peat (Norwegian Floralux veksttorv; http://www. nittedal-torvindustri.no). Plantlets were grown on for a further 3 weeks under long-day conditions after which flowering was induced using short-day conditions (10/14 h photoperiod) at 20C. Plants started anthesis of the third-order flower approximately 2 months after transfer to short-day conditions.

Induction of abscission in the flower pedicel When cyathia (flowers) of the third order began to open, the cyathia were decapitated with a razor blade just below the floral organs, with the floral bottom still intact. The cyathia were then left to develop AZs under short-day conditions.

Plant tissue preparation and examination For structural studies and immunolabelling, plant materials were embedded in LR-White resin (London Resin Company, http:// www.londonresin.com/). Small pieces (2–3 mm thick) of the plants were immediately fixed in 1% formaldehyde, 0.025% glutaraldehyde, and 0.1% (v/v) Tween 20 in 0.01 M sodium phosphate buffer, pH 7.2 and vacuum infiltrated for 1 h. Fixed and infiltrated tissues were placed at 4C overnight. The fixed samples were washed twice with sodium phosphate buffer for 4 h. Washed samples were then dehydrated in a graded ethanol series. Infiltration was performed with a progressively increasing ratio of LR White resin to ethanol. At

the end of the infiltration process, the specimens were transferred into an embedding mould and polymerized at 50C for 24 h. Plant materials embedded in LR White blocks were sectioned with a diamond knife (Diatome, http://www.diatome.ch/) on an ultramicrotome (Leica, http://www.leica.com/). Sections (1 lm thick) were placed on Vectabond (Vector Laboratories, http://www.vectorlabs. com/) coated glass slides and heated to 55C on a warm plate to adhere the sections to the slide. For cytochemical staining, sections were stained with Calcofluor (Sigma, http://www.sigmaaldrich. com/), rinsed with distilled water and mounted with Citifluor AF1 (EMS, http://www.emsdiasum.com/) before observation. The stained sections were observed using a Leitz microscope equipped with epifluorescence (Leica; http://www.leica.com). For the examination of ultrastructure, sections (60–70 nm-thick) were taken as described above and mounted on Formvar coated copper slot grids (EMS). Grids were stained with a saturated solution of uranyl acetate (1%), followed by Reynold’s lead citrate. Sections were examined using a Philips CM100 transmission electron microscope (Philips, http://www.philips.com/). To examine the lignin accumulation, the plant materials were embedded in Paraplast. For Paraplast embedding the flower buds of E. pulcherrima were cut into small pieces (2–3 mm thick) and immediately fixed in 4% paraformaldehyde in sodium phosphate buffer and 0.1% (v/v) Tween 20 under vacuum for 1 h, and left overnight at 4C. After fixation, samples were washed in 0.85% (w/v) saline, dehydrated through a graded alcohol series and embedded in Paraplast (Sakura; http://www.sakura.com) using a Tissue-Tek VIP jr. automatic embedding machine (Sakura). The plant materials embedded in Paraplast sections (10 lm thick) were collected on poly-L-lysine coated slides, deparaffinized using Histoclear (CellPath, http://www.cellpath.co.uk/), rehydrated with an ethanol series, and washed with water. Dewaxed and rehydrated sections were stained with 10% (w/v) phloroglucinol in 95% ethanol for 10 min to examine lignin accumulation in the plant tissues. An equal volume of concentrated HCl was added and the sections left for 2–3 min. The sections were rinsed thoroughly with distilled water, air dried, and mounted with Depex (BDH; http://www.vwr.com). The stained sections were examined using a Leitz microscope.

Immunoanalyses of cell wall polysaccharides The rat monoclonal antibodies used in this study are described in Table 1. For IDAs, at day 5 after induction plant material was harvested from the AZ, distal, proximal area and controls, and frozen in liquid nitrogen. Control plant material was harvested immediately after decapitation of the flower bud (third order). Material was ground in a mortar and pestle and extracted with equivalent volumes per fresh weight of plant materials of 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-HCl buffer (pH. 7.2) containing 50 mM trans-1,2-diaminocyclohexane-N,N,N¢,N¢,-tetraacetic acid (CDTA) and 25 mM dithiothreitol. Extracted materials were spotted onto a nitrocellulose membrane (Schleicher & Schuell; http:// www.whatman.com) as a replicated dilution series and probed as described previously (Willats et al., 1999a,b). For immunodot assays, 1-ll aliquots of adjusted supernatants were applied to a nitrocellulose membrane and dried for 1 h. Nitrocellulose membranes were blocked with 3% (w/v) milk protein in phosphatebuffered saline (MP/PBS) for 1 h followed by incubation with the anti-pectin monoclonal primary antibodies (1:10 dilution) for 1.5 h at 20C. After washing extensively with water, membranes were incubated in secondary antibody solutions (1:1000 dilution). For the secondary antibody, anti-rat antibody conjugated with alkaline phosphatase (Sigma) was used for 1.5 h at 20C. Membranes were

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 993–1003

1002 YeonKyeong Lee et al. washed as described above prior to development in substrate solution nitro-blue tetrazolium chloride/5-bromo-4-chloro-3¢-indolyphosphate p-toluidine salt (NBT/BCIP) in the dark. Colour development was stopped by washing the membrane with tap water. The intensities of the immunodots were calculated with Quantity One (Bio-Rad, http://www.bio-rad.com/) and the values were shown as a densitogram. For indirect immunofluorescence labelling, semi-thin sections (1 lm thick) of LR White-embedded material were obtained as described above. The sections were incubated in MP/PBS, pH 7.2, for 30 min to block non-specific binding sites. Sections were then incubated with primary rat monoclonal antibody diluted 1:10 in MP/PBS for at least 1 h at 20C or overnight at 4C. The sections were washed with several changes of PBS prior to incubation with the secondary antibody, anti-rat-IgG (whole molecule) linked to fluorescein isothiocyanate (FITC; Sigma) diluted 1:100 in MP/PBS for 1 h at 20C. The sections were washed in PBS buffer as described above before mounting with Citifluor AF1 (EMS) and examined with a Leitz microscope equipped with epifluorescence. Saponification (including de-esterification of HG) of sections was carried out by incubation in 0.05 M Na2CO3 solutions for 30 min prior to blocking in MP/PBS. For immunoelectron microscopy (IEM), ultrathin sections (60–70 nm thick) were cut using a diamond knife on an ultramicrotome (Leica). Sections were mounted on Formvar and carbon-coated nickel grids (100-mesh; EMS). The grids were blocked in 3% (w/v) bovine serum albumin in phosphate-buffered saline (BSA/PBS) by floating the EM grid section-side down on a droplet (at least 20 ll) on Parafilm for 30 min. The grids were transferred to a droplet of primary antibody diluted 1:10 in BSA/PBS and incubated for 1 h at 20C. Grids were then washed a minimum of three times in PBS and incubated in secondary antibody solution containing anti-rat IgG coupled to 10 nm gold (Sigma) diluted 1:20 in BSA/PBS for 1 h at room temperature. The grids were rinsed with PBS and distilled water. Staining of the grids was done with a saturated solution of uranyl acetate (1%), followed by Reynold’s lead citrate. Sections were examined with a Philips CM100 transmission electron microscope.

Fourier transform infrared microspectroscopy analysis Micro-thin sections (10 lm thick) of pedicels covering the AZ adjacent to the proximal area on day 7 were collected onto a film of distilled water and washed to remove most of the cytoplasmic components and then transferred onto barium fluoride windows (Crystran, http://www.crystran.co.uk/) and dried at 37C for 1 h. The windows were supported on the stage of a UMA500 microscopy accessory for a Bio-Rad FTS175c FT-IR spectrometer equipped with a liquid nitrogen-cooled mercury cadmium telluride detector. An area of cells (100 · 100 lm) was selected for spectral collection in transmission mode. One hundred and twenty-eight interferograms were collected with 8 cm)1 resolution and co-added to improve the signal-to-noise ratio for each sample. For each section, 10 spectra were collected from different areas of the AZ and proximal area and repeated in triplicate. All data sets were baseline corrected and area normalized before statistical methods were applied. Exploratory PCA was carried out using Win-Discrim software (Kemsley, 1998).

Acknowledgements We thank Kari Borger and Hilde Raanaas Kolstad for technical help and the Norwegian Research Council (project number 158872) for funding.

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