Changes in Cinnamic Acid Derivatives Associated with the Habituation of Maize Cells to Dichlobenil

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1 Molecular Plant Volume 4 Number 5 Pages September 2011 RESEARCH ARTICLE Changes in Cinnamic Acid Derivatives Associated with the Habituation of Maize Cells to Dichlobenil Hugo Mélida a,1, Jesús Álvarez a, José Luis Acebes a, Antonio Encina a and Stephen C. Fry b a Área de Fisiología Vegetal, Facultad de CC Biológicas y Ambientales, Universidad de León, E León, Spain b Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Daniel Rutherford Building, The King s Buildings, Edinburgh, EH9 3JH, UK ABSTRACT The habituation of cell cultures to cellulose biosynthesis inhibitors such as dichlobenil (DCB) represents a valuable tool to improve our knowledge of the mechanisms involved in plant cell wall structural plasticity. Maize cell lines habituated to lethal concentrations of DCB were able to grow through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of arabinoxylans. The aim of this work was to investigate the phenolic metabolism of non-habituated and DCB-habituated maize cell cultures. Maize cell cultures were fed [ 14 C]cinnamate and the fate of the radioactivity in different intra-protoplasmic and wall-localized fractions throughout the culture cycle was analyzed by autoradiography and scintillation counting. Non-habituated and habituated cultures did not markedly differ in their ability to uptake exogenous [ 14 C]cinnamic acid. However, interesting differences were found in the radiolabeling of low- and high-m r metabolites. Habituated cultures displayed a higher number and amount of radiolabeled low-m r compounds, which could act as reserves later used for polysaccharide feruloylation. DCB-habituated cultures were highly enriched in esterified [ 14 C]dehydrodiferulates and larger coupling products. In conclusion, an extensive and early cross-linking of hydroxycinnamates was observed in DCB-habituated cultures, probably strengthening their cellulose-deficient walls. Key words: Cell wall; Zea mays; maize; dichlobenil; DCB; dehydrodiferulate; ferulate; cinnamic acid. INTRODUCTION Primary cell wall of the Poales (grasses, cereals, and related plants) is composed of a framework of cellulose microfibrils embedded in a hemicellulosic matrix (arabinoxylans, xyloglucan, and, in some cases, mixed-linked glucans) and smaller amounts of pectins and glycoproteins (Carpita and Gibeaut, 1993). Poalean cell walls are also characterized by the presence of cell wall phenolics, mainly the hydroxycinnamates ferulic and p-coumaric acid, which are found as substituents linked to arabinoxylans by ester bonds to a-l-arabinosyl residues (Smith and Hartley, 1983; Kato and Nevins, 1985). Hydroxycinnamates are susceptible to oxidative coupling when exposed to hydrogen peroxide plus peroxidase (Geissmann and Neukom, 1971) and it has been widely suggested that the ester-linked dehydrodiferulates formed can cross-link cell wall polysaccharides, contributing to wall assembly (Fry, 2004; Parker et al., 2005). The first discovered dimer was 5 5 -dehydrodiferulate and, after this, several other dimers have been obtained by alkaline hydrolysis of plant cell wall polysaccharides (Ralph et al., 1994; Waldron et al., 1996). In at least some tissues, trimers and larger oligoferulates quantitatively predominate over dimers (Fry et al., 2000), and certain trimers (Bunzel et al., 2003; Rouau et al., 2003; Funk et al., 2005) and tetramers (Bunzel et al., 2006) have been characterized. The variety and quantity of the complex structures observed suggest that these oligomers of hydroxycinnamates may play an important role in the architecture of the cell wall. However, we still lack definitive evidence that dehydrodiferulate acts as a genuine (i.e. inter-polymeric) cross-link between polysaccharide chains as distinct from an intra-polymers loop (Lindsay and Fry, 2007). Recently, Burr and Fry (2009) demonstrated that, besides ester-linked diferulates, some alkali-stable (ether-like) bonds are formed and may also contribute to polysaccharide cross-linking. Phenolic coupling can occur intra-protoplasmically, before or during Golgi vesicular transit to cell membrane, and/or wall-localized, just following polysaccharide secretion or after wall integration 1 To whom correspondence should be addressed. h.melida@unileon.es, tel ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: /mp/ssr038, Advance Access publication 13 May 2011 Received 25 February 2011; accepted 5 April 2011

2 870 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells (Fry et al., 2000; Obel et al., 2003; Mastrangelo et al., 2009). Once polysaccharide-bound ferulate residues have been oxidatively coupled extra-protoplasmically, the cell wall may undergo a significant alteration in a number of properties such as growth cessation, resistance to pathogens and insects, and cell wall degradability (Buanafina, 2009). Intra-protoplasmic coupling of feruloyl residues, on the other hand, probably helps to maintain high wall extensibility by consolidating the contents of each Golgi vesicle as a coherent entity ( coagulum ) with a restricted surface area and thus a reduced ability to bond to previously or subsequently wall-deposited feruloyl-arabinoxylans (Fry et al., 2000). Owing to the key role of cellulose in the cell wall structure, cellulose biosynthesis inhibitors have become valuable tools for the analysis of cell wall structure and biogenesis (Sabba and Vaughn, 1999; Vaughn, 2002; Acebes et al., 2010). Although cellulose biosynthesis inhibitors are highly specific and potent herbicides, cell cultures of several species have been habituated to grow in the presence of these by incremental exposure over many culturing cycles. The first cellulose biosynthesis inhibitor for which this was achieved was dichlobenil (2,6-dichlorobenzonitrile, DCB; Shedletzky et al., 1990), which specifically inhibits the polymerization of glucose into b-1,4-linked glucan (Delmer, 1987). The habituation of different cell cultures to DCB (Shedletzky et al., 1990; Encina et al., 2001, 2002; Alonso-Simón et al., 2010) reflects the ability of plant cells to modify cell wall structure and composition in order to cope with abiotic stresses, and therefore represents a valuable tool for improving our knowledge of the mechanisms involved in plant cell wall plasticity and ability to maintain cell wall integrity. We obtained maize cell lines habituated (H) to lethal concentrations of DCB (Mélida et al., 2009), which, in spite of showing a 75% reduction in cellulose content, were able to grow through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of arabinoxylans. Further characterization (by HPLC and RT PCR) of phenolic contribution in maintaining the functionality of a cellulose impoverished cell wall showed an overall enrichment in cell wall-esterified hydroxycinnamates and changes in the expression of several phenylpropanoid-related genes in H cells (Mélida et al., 2010a, 2010b). The advantages of radiolabeling and using cell cultures to track and study polysaccharide feruloylation and related items have been widely demonstrated in recent years (Fry et al., 2000; Kerr and Fry, 2003; Obel et al., 2003; Kerr and Fry, 2004; Encina and Fry, 2005; Lindsay and Fry, 2008; Burr and Fry, 2009). Experiments involving [ 14 C]ferulate-feeding (Obel et al., 2003) do not necessarily trace the major natural pathway to dehydrodiferulate production because extracellular free [ 14 C]ferulic acid may undergo oxidative coupling prior to its incorporation into polysaccharide chains; however, [ 14 C]cinnamate, which is metabolically inert in the apoplast and begins to be metabolized only when taken up by the cells, does trace the major natural pathway (Lindsay and Fry, 2008). The aim of this work was to investigate the phenolic metabolism in DCB-habituated maize cells, studying the fate of [ 14 C]cinnamate derivatives in different intra-protoplasmic and wall-localized fractions throughout the culture cycle of non-habituated and DCB-habituated maize cells. RESULTS Uptake of [ 14 C]Cinnamic Acid from the Medium Non-habituated cells (NH) and H maize cell-suspension cultures did not markedly differ in their ability to uptake [ 14 C]cinnamic from the culture medium (Supplemental Figure 1). In most cases, and independently of the cell line, the net uptake of 14 C ceased after 30 min, reaching 60 80% of total radioactivity added. Calculated on the basis of the radioactivity remaining in the medium after a 15-min incubation, [ 14 C]cinnamic acid consumption rates were within the range % min 1 for both cell lines, depending on the culture age. Thethin-layerchromatography(TLC)analysisofthe 14 C-labeled compounds present in the cell culture medium (Supplemental Figure 2) showed that both NH and H maize cells had completely metabolized the [ 14 C]cinnamic acid by 120-min incubation. Essentially, all the 14 C-labeled compounds found in the culture medium at 120 min had R F = 0, indicating polymeric and/or highly hydrophilic material. 14 C-Radiolabeling of Cellular Metabolites along the Culture Cycle of Maize Cells: Qualitative Analysis Low-M r (alcohol-soluble fraction, ASF) and high-m r (0.5Malcohol-insoluble residue (AIR) and 6M-AIR) cellular metabolites were assayed throughout the culture cycle of NH and H maize cell cultures (Figure 1). Samples were taken after 120 min [ 14 C]cinnamic acid feeding and thus provide snapshots of the biosynthetic picture in each differently aged culture. ASF of NH (Figure 1A) did not contain many different chromatographically mobile compounds, just some low-r F ones, and the amount of these diminished along the cycle. The opposite trend was observed for H cells (Figure 1B), where the number and the amount of compounds increased when the culture cycle advanced, especially after day 9. Some free p-[ 14 C]coumarate was present in these cultures, especially from day 7 after sub-culturing. The cellular polymers (AIR fraction) were subjected to a sequential fractionation procedure that distinguishes between alkali-labile (ester-linked units) and alkali-stable (ether-linked units) material. After 0.5M-NaOH saponification of AIR (Figure 1C and 1D), ester-bonded 14 C-labeled metabolites were expected to be released. Based on TLC results, the total amount and diversity of ester-linked 14 C-compounds were higher in H cell cultures than in NH ones. Ester-linked [ 14 C]ferulate and p-[ 14 C]coumarate were detected in both cell lines 1 d after sub-culturing. Quantitative and qualitative differences between H and NH cells were found in the region where putative dehydrodiferulic acids (diferulic acids) (DFAs) (named as D1 D6) migrate.

3 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells 871 D1: 8 5B -DFA (benzofuran form) D2: 8-O-4 -DFA D3: 5 5 -DFA D4: 8 5 -DFA D5: 8 8 -DFA D6: 8 8A -DFA (aryl form) In NH cells, D1 (followed by D5) was the major [ 14 C]DFA. A strong accumulation of putative 8 5B -[ 14 C]DFA was observed in15-day-oldcultures. D1 D6wereconsistentlydetectedthroughout the culture cycle of H cells and, from 7 d after sub-culturing, a substantial increase in radiolabeled DFAs was detected. In any case, the radiolabeling intensity of D1 D6 compounds was higher in H than in NH throughout the cell culture cycle. 6M-NaOH saponification (Figure 1E and 1F) released compounds held by relatively alkali-stable (ether-like) bonds. While only p-[ 14 C]coumarate was detected after this treatment in NH cells (from day 7), [ 14 C]ferulate, p-[ 14 C]coumarate and traces of low-r F compounds were detected in H cultures from day 3 after sub-culturing. Interestingly, radioactive material at R F 0 that was synthesized abundantly only in aged (especially 15-day-old) NH cultures became radioactive much earlier in H ones (3 d after sub-culturing). Figure 1. Radiolabelling of Cellular Metabolites along the Culture Cycle Autoradiography of TLCs of Alcohol-Soluble Fractions (ASF; Low-M r 14 C-Compounds) (A, B), polymer-esterified [ 14 C]cinnamate-derivatives (released by 0.5M-NaOH treatment at 20 C) (C, D) and putatively polymer-etherified [ 14 C]cinnamate-derivatives (released by 6M-NaOH treatment at 37 C) (E, F) from non-habituated (NH; (A, C, E)) and DCB-habituated (H; (B, D, F)) maize cell cultures. [ 14 C]Cinnamic acid (2.7 kbq) was fed to 500-ll cultures aged 1, 3, 5,...15 d after sub-culturing. Samples were taken 120 min after feeding. Fractions were chromatographed in benzene/acetic acid (9:1) and then autoradiographed. The positions of the origin (Ori) and external standards are indicated (Cin, cinnamic acid; Fer, ferulic acid; Cou, p-coumaric acid; 5,5, 5 5 -diferulic acid). D1 D6 in (C, D) indicate putative [ 14 C]dehydrodiferulates. Spot D3 is identified as 5 5 -dehydrodiferulic acid. Results shown are representative of two independent experiments. On the basis of the TLC R F of authentic 5 5 -DFA, 8 5 -DFA, 8 8 -DFA, and 8 8A -DFA, C 18 -HPLC retention times (Waldron et al., 1996; Mélida et al., 2010b), and R F values on silica-gel TLC in CHCl 3 /HOAc (9:1) (C.T. Brett, Glasgow University, personal communication), we suggest that the spots were, in order of R F : Kinetics of Radiolabeling between Different Pools along the Culture Cycle: Quantitative Analysis To achieve an overall picture of the flux of carbon from [ 14 C]cinnamate into the major pools during 120-min pulses administered at different time-points throughout the culture cycle of NH and H maize cells, we assayed quantitatively the 14 C in the fractions (cell-free medium (CFM), ASF, 0.5M-AIR, 6M- AIR, and the final residue) obtained in the experiment described above (Figure 2). In the case of NH cultures, a gradual increase in CFM labeling was observed throughout the cell culture cycle up to 11 d, but a decrease was observed in 13- and 15-day-old cultures. However, the CFM fraction was labeled steadily or even decreasingly as H cell cultures aged. On average, the CFM fraction accounted for a slightly lower percentage of the radioactivity in H cells than in NH ones throughout the cell culture cycle. The kinetics of 14 C partitioning into ASF fraction varied between cell lines. In NH cell line, a gradual reduction in the rate of labeling of this pool was observed as cultures aged. In the case of H cells, the rate of incorporation of 14 C into the ASF kept steady throughout the cell culture cycle. In NH maize cells, the proportion of radioactivity incorporated into total AIR (= 0.5M-NaOH + 6M-NaOH + alkali-stable residue) during a 120-min pulse increased as the cultures aged. A particularly sharp increase in radioactivity incorporation into this fraction (up to 64%) was detected 15 d after subculturing. When the kinetics of radiolabeling of the AIR fraction for H cells was assayed, a peak of incorporation was observed 7 9 d after sub-culturing. With the exception of the oldest cultures, the proportion of radioactivity incorporated into AIR fraction was higher in H cell cultures than in NH ones when cultures of the same age were compared.

4 872 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells Figure 2. Distribution of Radioactivity between Major Pools. Fractions from the experiment reported in Figures 1 and 3 were assayed for 14 C. Cell-free medium (CFM) and cellular material from non-habituated (NH) and DCB-habituated (H) maize cell cultures were analyzed. Cellular material was fractionated into alcohol-soluble fraction (ASF) and alcohol-insoluble residue (AIR). The AIR was sequentially fractionated into 0.5M-NaOH and 6M-NaOH extractable material. All these fractions and the final residue (after the 6M-NaOH extraction) were assayed for 14 C. Results shown are mean of two independent experiments. Most of the radioactivity incorporated into AIR was found in the most alkali-labile fraction (0.5M-NaOH extractable material). Further, a variable proportion of AIR labeling needed strong alkali (6M-NaOH extractable material) to be extracted or remained in the final residue (6M-NaOH residue), being regarded as alkali-stable 14 C-compounds (Figure 2; after 6M). In NH cultures, a gradual increment was observed in the proportion of radioactivity in this residue as cultures aged. Both alkali-labile and alkali-stable 14 C-compounds were more abundant in H cells that in NH. Radioactivity Incorporation into Low-Molecular-Weight Fraction of 7- and 15-Day-Old Maize Cultures Seven- and 15-day-old maize cultures were analyzed in more depth as the radioactivity was tracked into different pools during a 180-min pulse of [ 14 C]cinnamate (H 2 O 2 being added after 120 min). After approximately 10 (NH) or 30 (H) min, intracellular free [ 14 C]cinnamate had largely disappeared (Figure 4) and been replaced by other low-m r (Figure 4) and polymer-bound (Figures 5 and 6) 14 C-metabolites. Up to 26% of the 14 Cadded remained as ethanol-soluble 14 C-metabolites (ASF) in both 7- and 15-day-old cultures (Figure 2) 120 min after feeding. In contrast to NH cultures in which free p-[ 14 C]coumarate was detected only in traces and for,5 min (see 15-day-old NH cultures; Figure 4B), free p-[ 14 C]coumarate was clearly detected in 7- and 15-day-old H cultures up to 90 min after 14 C feeding (Figure 4C and 4D). Free [ 14 C]ferulate, in contrast, was undetectable in all cultures. All cultures showed rapid labeling (starting 1min after [ 14 C]cinnamic acid feeding) of highly polar and slightly polar 14 C-compounds (R F 0 and low-r F material, respectively). The slightly polar compounds were stable for at least Figure 3. Kinetics of Incorporation of 14 C into the Alcohol-Insoluble Residue (AIR) of Non-Habituated (NH), and DCB-Habituated (H) Maize Cell Cultures. [ 14 C]Cinnamate was fed (1.6 kbq) to replicate cultures, which were harvested at the time-points indicated on the x-axis. After 120 min, H 2 O 2 (final concentration 1 mm) was added to further cultures, which were harvested after an additional 60 min. AIR was assayed for radioactivity. 120 min and partially disappeared from ASF after H 2 O 2 addition. Quantitative Analysis of Radioactivity Incorporation into High-M r Compounds: Different Requirements of H 2 O 2 Interesting differences were observed in the kinetics of 14 C incorporation (Figure 3) into the AIR of NH and H cells during 180-min pulses (with H 2 O 2 added at 120 min).

5 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells 873 Figure 4. Radioactivity Incorporation into Low-Molecular-Weight Fractions Autoradiography of TLCs of ASFs (low-m 14 r C-compounds) from 7- (A, C) and 15-day-old (B, D) maize cultures; non-habituated (NH; (A, B)) and DCB-habituated (H; (C, D)). [ 14 C]Cinnamate was fed (1.6 kbq) to 300-ll cultures, which were harvested at 0, 1, 2, min, as indicated. After 120 min, H 2 O 2 (final concentration 1 mm) was added to further cultures, which were harvested after an additional period of incubation (+1, +10, and +60 min). ASFs were chromatographed in benzene/acetic acid (9:1) and then autoradiographed. The positions of the origin and external standards are indicated (key as in Figure 1). Seven-day-old NH cultures slowly (during the first 120 min) incorporated into the AIR 18% of the total radioactivity added, and the addition of H 2 O 2 stimulated this incorporation up to 44%. Although H cultures (7- and 15-day-old) incorporated higher amounts of radioactivity (30%) during the first 60 min in contrast to 7-day-old NH cultures, similar trends between both cell lines were observed upon the addition of H 2 O 2,whichagainstimulatedincorporationuptohighlevels (more than 50%). On the other hand, 15-day-old NH cultures continuously incorporated radioactivity at higher rates than the other cultures and, clearly in this case, the requirement of H 2 O 2 was not a limiting factor, since its addition did not change substantially the radioactivity incorporation into the AIR. Qualitative Analysis of Polymer-Esterified Derivatives Carbon incorporation from [ 14 C]cinnamate into polymer-esterified [ 14 C]ferulate and p-[ 14 C]coumarate began very early during the 120-min pulses in both NH and H cells (Figure 5). As expected from the known metabolic pathway, radiolabeling kinetics indicated that p-[ 14 C]coumaroyl preceded [ 14 C]feruloyl groups. In the case of 7-day-old NH, radiolabeled material at R F 0 increased strongly after 30 min [ 14 C]cinnamate feeding. In these same cell Figure 5. Radiolabelling of Polymer-Esterified Derivatives Autoradiography of TLCs of polymer-esterified [ 14 C]cinnamatederivatives (material released from AIR by 0.5M-NaOH treatment at 20 C) synthesized in 7 (A, C) and 15-day-old (B, D) maize cultures; non-habituated (NH; (A, B)) and DCB-habituated (H; (C, D)). The cultures were incubated with [ 14 C]cinnamate for min, as indicated, followed by an additional incubation period after treatment with H 2 O 2 as in Figure C-compounds were chromatographed in benzene/acetic acid (9:1) and then autoradiographed. The positions of the origin and external standards are indicated (key as in Figure 4). cultures, the addition of H 2 O 2 highly stimulated the radiolabeling of esterified p-coumarate, DFAs, and R F 0 compounds. In fact, no putative DFAs were detected before H 2 O 2 addition. In 15-day-old NH cultures, radiolabeled material at R F 0 rapidly accumulated after 3 min [ 14 C]cinnamate feeding, probably indicating an increased degree of intra-protoplasmic coupling as NH cells aged. Moreover, the levels of 14 C incorporation into AIR-esterified p-[ 14 C]coumarate and [ 14 C]ferulate were higher than in younger NH cultures (Figure 5). p-[ 14 C]Coumarate levels reached a plateau 3 min after [ 14 C]cinnamic acid feeding, and kept steady for 120 min. In aged NH cultures, putative 8 5B -DFA (spot D1) appeared 15 min after [ 14 C]cinnamic feeding. In addition, slow migrating compounds became radiolabeled earlier than in 7-day-old NH cultures. In the case of aged NH cultures, the requirement of H 2 O 2 was not a limiting factor and its addition substantially changed neither the amount of radiolabeling of AIR (see Figure 3 also) nor the type of radiolabeled compound (Figure 5). A similar pattern of AIR-radiolabeling was observed for H cells, although these (both 7 and 15 d old) incorporated higher amounts or radioactivity (see Figure 3 also) than NH cultures of the same age. The addition of H 2 O 2 had a marked effect on the increase of dimerization in aged H cultures. Polymer-Etherified Derivatives Under the conditions used for alkaline hydrolysis (0.5M-NaOH, ph 13.7 at room temperature), feruloyl ester bonds are

6 874 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells expected be cleaved with a half-life in the order of 1 min (second-order rate-constant for reaction of methyl ferulate with NaOH M 1 s 1 ; calculated from Fry, 1982). Therefore, esterified ferulate should be essentially completely released by 0.5M-NaOH within the 18 h employed and, if the subsequently employed 6.0M-NaOH at 37 C releases additional hydroxycinnamates, then these may be assumed to have been held by non-ester bonds, such as ethers. The possibility remains, however, that a small proportion of the ferulate released from ester-linkage was carried over from the mild to severe alkaline samples. The faint spots of [ 14 C]ferulate observed by TLC of the 6.0M-NaOH products (Figure 6) are in proportion to those seen in the 0.5M-NaOH samples (Figure 5), supporting the carry-over interpretation. Against this comparison, however, the 6 M samples were relatively rich in p-[ 14 C]coumarate and especially in low-r F radiolabeled compounds (dimers, trimers, or oligomers), suggesting ether-like bonding to the polysaccharides. The kinetics of radiolabeling of material at R F 0 in the 6.0M- NaOH fraction (Figure 6) resembled those of the 0.5M-NaOH fraction (Figure 5) and the spots were of similar intensity, suggesting that this material included roughly equal amounts of cinnamate-derived oligomers linked by ether (and possibly also ester) bonds and of oligomers linked by only ester bonds. The R F 0 spot was more intense in H cells than in NH ones, especially in min radiolabeled samples. In all cases, H 2 O 2 addition increased the amount of R F 0 cell wall etherified compounds. Moreover, in the case of aged H cells, H 2 O 2 addition produced low-r F radiolabeled compounds (dimers, trimers, or Figure 6. Autoradiography of TLCs of Putatively Polymer-Etherified [ 14 C]Cinnamate-Derivatives(Material ReleasedfromAIRby 6M-NaOH at 37 C). Other details as in Figure 5. oligomers), which were almost undetectable in the other three cultures. DISCUSSION It has previously been suggested that feruloyl arabinoxylan cross-linking has consequences in the control of cell expansion and in the response of plant cells to stresses as a defense mechanism (Bolwell et al., 1998; Fry et al., 2000; Buanafina, 2009). Maize cells habituated to what are usually lethal concentrations of the cellulose biosynthesis inhibitor, DCB, exhibited a modified cell wall composition and architecture (Mélida et al., 2009, 2010b) that allowed cells to cope with this toxic compound. Based on previous work, we hypothesized that the phenolic components of the cell wall have a special importance in DCB habituation. In this work, by using pulse chase experiments with [ 14 C]cinnamic acid, and tracking radiolabeled compounds through different pools, we attempted to describe changes in the phenolic metabolism of a cellulose-impoverished cell wall. The fate of [ 14 C]cinnamic acid taken up by cultured maize cells has recently been reported (Lindsay and Fry, 2008) and is comparable to that shown in this work. As cinnamic acid is a lipophilic weak acid, it penetrates the plasma membrane easily and was completely removed from the medium and trapped inside the cells in the form of more polar metabolites, in a few minutes. NH and H cells did not markedly differ in their ability to take up [ 14 C]cinnamic acid from the culture medium. However, large differences in the 14 C-radiolabeled products present in different cellular pools from NH and H cells were found. TLC of CFM showed a single peak of radioactivity at R F 0, corresponding to polymeric and/or highly hydrophilic material released by the cells, probably mainly [ 14 C]feruloylarabinoxylans (Fry et al., 2000; Kerr and Fry, 2003, 2004; Lindsay and Fry, 2008). We suggest that the rapid uptake of [ 14 C]cinnamic acid was followed by a reappearance of some of the radioactivity in the medium owing to the release of those 14 C-labeled hemicelluloses that missed their chance to integrate in the cell wall immediately after secretion and were therefore not retained within the wall. In the case of NH cultures, after a gradual increase in CFM fraction throughout the cell culture cycle, a decrease took place in aged NH cell cultures. This would be explained by phenolic cross-linking of extracellular [ 14 C]feruloyl-arabinoxylans into the cell wall. Therefore, a second chance for integration into the cell wall seemed to be available to old cultures. On average, the CFM fraction accounted for less radioactivity in H cell cultures throughout the cell culture cycle. This may indicate a more efficient integration of newly synthesized 14 C-labeled hemicelluloses into the wall of DCB-habituated cells, implying that such integration may play an important role in wall assembly (Kerr and Fry, 2003). Tracking the radiolabeling of low- and high-m r cellular metabolites during the culture cycle, we found interesting differences between non-habituated and DCB-habituated cells. Some polar low-m r compounds, probably including [ 14 C]hydroxycinnamoyl

7 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells 875 conjugates of sugars or other hydrophilic substances, such as b-glucosyl esters (Harborne and Corner, 1961; Fry et al., 2000; Obel et al., 2003), were detected in both cell lines. However, habituated cells displayed a higher number and radiolabeling of these compounds and, interestingly, an increasing accumulation along the culture cycle could also be observed. These compounds could act as reserves of [ 14 C]hydroxycinnamoyl units, later used for polysaccharide feruloylation either directly (Obel et al., 2003) or more probably indirectly from feruloyl-coa (Fry, 1984; Fry et al., 2000; Lindsay and Fry, 2008). Habituated cells are proposed to require higher levels of these reserves. Short-time feeding experiments of 7- and 15-day-old maize cultures gave results in line with these ideas. Interestingly, most of the low-m r intracellular compounds disappeared when H 2 O 2 was added, confirming that ROS stress can quickly evoke defensive responses. Another rapid response of cultured cells to ROS stress is the secretion of an antioxidant, ascorbate (Parsons and Fry, 2010). The AIR fraction (cellular polymers) includes mature polysaccharides already deposited in the cell wall, but also newly synthesized polysaccharides in the Golgi bodies or being transported in vesicles (Lindsay and Fry, 2008). NH cells increased the proportion of radioactivity incorporated into AIR as cultures aged. In other words, the older the NH cultures, the more the [ 14 C]hydroxycinnamates incorporated into the cell wall, and therefore the more phenolic side-chains available to cross-link adjacent polysaccharides. This result is in line with several studies that have indicated a positive correlation between phenolic cross-linking and growth cessation in various monocot species (Kamisaka et al., 1990; MacAdam and Grabber, 2002; Azuma et al., 2005). Habituated cells incorporated a high proportion of radioactivity into AIR, even in the early growth phases. Moreover, with the exception of very aged cultures, the proportion of radioactivity recovered in AIR fraction was higher in H cell culturesthaninnhoneswhencultureswiththesameagewere compared. On the basis of these results, a higher amount of cell wall hydroxycinnamates and a higher degree of phenolic crosslinking would be expected for H cells. Most of the radioactivity incorporated into AIR was found in a mild-alkali-labile (i.e. ester-linked) fraction. With regard to cell wall-esterified hydroxycinnamates, [ 14 C]ferulate and p-[ 14 C]coumarate were present in both cell lines throughout the culture cycle and were attached to a polysaccharide chain very quickly, as these compounds became detectably radioactive within 1 min, as previously observed (Fry et al., 2000). In accordance with previous results (Mélida et al., 2010b), quantitative and qualitative differences in cell wall esterified [ 14 C]DFAs were found between NH and H cell lines. Habituated cells were highly enriched in [ 14 C]DFAs, supporting the idea that a more cross-linked network of arabinoxylans was acting as a mechanism to compensate, at least partially, for the reduction of cellulose in H cells. According to the rapid formation of [ 14 C]DFAs at short-time, ferulic acid dimerization began earlier in H cells, although aged NH cells also showed high levels of DFAs. While cross-linking seems to be related with growth cessation in NH cells, in the case of H cells, this mechanism may be a wall-strengthening strategy during early cell growth phases. The possibility of benzyl ether bonds contributing to hemicellulose cross-linking in primary cell walls was suggested some time ago (Kerr and Fry, 2004). Recently, it was demonstrated that after oxidative coupling, strong benzyl sugar ether bonds are formed, probably via quinone-methide intermediates (Burr and Fry, 2009). In this paper, we provide results pointing to the presence of these alkali-stable-bonded polysaccharides both in NH and H cell walls. [ 14 C]Ferulate, p-[ 14 C]coumarate, and R F 0 compounds were detected in NH and H cultures. As expected from the known pathways, p-[ 14 C]coumarate began to be incorporated slightly earlier than [ 14 C]ferulate. p-[ 14 C]Coumarate, and possibly also traces of [ 14 C]ferulate, released after a strong alkali treatment could represent a population of ether-linked hydroxycinnamates forming bridges between polysaccharides and lignin-like compounds (Lam et al., 2001). The presence of that sort of linkage has been proposed to occur during primary cell wall assembly in grasses (Iiyama et al., 1990; Lam et al., 1994; Hatfield and Marita, 2010; Ralph, 2010). Considering that not much difference was found between NH and H cells in the amount and diversity of ether-linked hydroxycinnamates, a minor role in DCB habituation is proposed for this sort of very strong phenolic-mediated linkages between polysaccharides. Ferulate cross-linking varies with the physiological condition of cells during the cell culture cycle (Burr and Fry, 2009). The main factor controlling ferulate cross-linking is peroxidase action rather than peroxidase activity, action being dependent on therateofh 2 O 2 production and the presence/absence of low- M r inhibitors as described by Encina and Fry (2005) whereas activity is an enzymological concept measured (in katals) under optimized conditions in vitro. Here, we report that, throughout the cell culture cycle, cross-linking activity started earlier in DCBhabituated cells as a strong accumulation of [ 14 C]DFAs and larger coupling products was obtained from cellular polymers of very young (1-day-old) H cultures. Moreover, the sudden increase in this sort of 14 C-labeled compounds observed for 7-day-old H cultures (same effect is observed in very aged NH cultures) may suggest a burst of H 2 O 2 production and/or the disappearance of an inhibitor of phenolic cross-linking early in the cell culture cycle. In line with this, we show that exogenous H 2 O 2 stimulated dimerization in H cells in all cases, but not in the oldest NH cultures, where dimerization seems already to have taken place such that no substrates for peroxidase would be available. So, although habituated cells showed higher dimerization levels, H 2 O 2 was still a limiting factor for even more extensive feruloylation. Further studies of low-m r inhibitors as described by Encina and Fry (2005) in habituated cells will help to elucidate the role of these compounds controlling peroxidase action. In sum, by using pulse chase experiments with [ 14 C]cinnamate, we have shown that DCB habituation is associated with a modification of cytosolic phenolic metabolism that supplies increased levels of precursor for polysaccharide feruloylation.

8 876 Mélida et al. d Cinnamic Acid Derivatives in Dichlobenil-Habituated Maize Cells Moreover, a more efficient wall integration of newly synthesized hemicelluloses together with an extensive and premature cross-linking of hydroxycinnamates was observed in DCB-habituated cells as expected for the reinforcement of a cellulose-deficient cell wall. METHODS Cell Cultures Maize callus cultures (Zea mays L., Black Mexican sweetcorn) from immature embryos were grown in Murashige and Skoog media (Murashige and Skoog, 1962) supplemented with 9 lm 2,4-dichlorophenoxyacetic acid and 8% agar at 25 C inthelight (Lorences and Fry, 1991) and sub-cultured fortnightly. Calluses were habituated to growth in different DCB (Fluka) concentrations by stepwise transfers with gradual increments, beginning at 2 lm (Mélida et al., 2009); DCB-habituated liquid cultured cells were obtained by transfer to medium supplemented with DCB but without agar (final concentration: 6 lm). Chemicals [ 14 C]Cinnamic acid was prepared from L-[U- 14 C]phenylalanine (17 MBq lmol 1 ; Amersham) as described by Lindsay and Fry (2008), and stored at 20 C. Chromatography TLC was carried out on plastic-backed silica-gel plates with a fluorescent indicator (Merck) in benzene/acetic acid (9:1, v/v). During development, TLC plates were exposed to 366- nm UV, which keeps hydroxycinnamates as rapidly interconverting single spots of cis/trans isomers. Authentic markers were located under 254-nm UV radiation. Based on HPLC retention times (Waldron et al., 1996), TLC R F -values (Encina, personal communication) and previously published data (Lindsay and Fry, 2008), six putative dehydrodiferulates (DFAs D1 D6) were tentatively identified. D3 co-migrated with authentic 5 5 -DFA. Radiolabeling of Liquid Cultures with [ 14 C]Cinnamate and Assay of Radioactivity Aliquots (500 ll) of NH and H maize cell-suspension cultures (1, 3, 5, 7, 9, 11, 13, and 15 d after sub-culturing) were transferred into flat-bottomed vials (i.d. 11 mm) loosely capped with aluminum foil, where they were then left for 1 h to acclimatize to their new environment (25 C and shaking, 150 rpm). [ 14 C]Cinnamic acid (2.7 kbq), in 10 ll H 2 O, was then added to each vial. Samples of medium (10 ll) were removed at each time point (up to 2 h) and assayed by scintillation counting. After 2 h, the cultures were filtered to give cell-free medium (CFM) and cells. Cells were washed and incubated (18 h) with 500 ll of 75% ethanol on a rotating wheel. The alcohol-soluble fraction (ASF) and dried alcohol-insoluble residue (AIR) were collected. AIR was saponified with 0.5M-NaOH for 18 h at about 20 C, which releases esterified hydroxycinnamates, acidified by addition of trifluoroacetic acid (TFA), and partitioned against ethyl acetate (32). The ethyl acetate phases were vacuum-dried and re-dissolved in propan-1-ol (named 0.5M-AIR). The residue after 0.5M-NaOH saponification was then treated with 6M-NaOH for 18 h at 37 C, which breaks mild alkali-stable (ether-like) bonds (as shown by Enoki et al., 1983), acidified, partitioned, and vacuum-dried (named 6M-AIR). Portions of all the fractions (including the final residue after 6M-NaOH treatment) were assayed by scintillation counting. Aliquots (15 ll) of CFM, ASF, 0.5M-AIR, and 6M-AIR were subjected to TLC. The TLC plates were autoradiographed on Kodak film and, in some cases, were cut into 1-cm strips, which were assayed for radioactivity. Aqueous or ethanolic solutions were mixed with 10 volumes of OptiPhase (Wallac Oy), and dry samples were moistened in 2 ml of OptiScint (Wallac Oy). Further experiments were carried out with 300-ll aliquots of NH and H maize cell cultures (7 and 15 d after sub-culturing), which were transferred into flat-bottomed vials (i.d. 7.5 mm) and incubated as indicated previously. [ 14 C]Cinnamic acid (1.6 kbq), in 10 ll H 2 O, was then added to each vial and, after 120 min, H 2 O 2 was added to some vials to give a final concentration of 1 mm. At selected time-points (up to 3 h), 750 ll of 100% ethanol containing 7% formic acid was added to each culture vial, capped, and left on a rotating wheel for 18 h. ASF and AIR were obtained and assayed as before. SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online. FUNDING This work was supported by grants from the Junta de Castilla y León (LE 044A10-2) and the Spanish Ministry of Science and Innovation program (CGL ). S.C.F. thanks the UK Biotechnology and Biological Sciences Research Council for funding. H.M. was financed by a PhD grant from the Spanish Ministry of Science and Innovation s FPU Program. ACKNOWLEDGMENTS We are indebted to Mrs Janice Miller for excellent technical assistance and to John Ralph, Fachuang Lu, and Hoom Kim of the US Dairy Forage Research Center, USDA-Agricultural Research Service, for their kind gifts of DFAs standards. No conflict of interest declared. REFERENCES Acebes, J.L., Encina, A., García-Angulo, P., Alonso-Simón, A., Mélida, H., and Álvarez, J.M. (2010). Cellulose biosynthesis inhibitors: their uses as potential herbicides and as tools in cellulose and cell wall structural plasticity research. In Cellulose: Structure and Properties, Derivatives and Industrial Uses, Lejeune, A., and Deprez T., eds (New York: Nova Publishers), pp

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