In muro feruloylation and oxidative coupling in monocots

Abstract

Recently we have suggested that (glucurono)arabinoxylans [(G)AX] feruloylation and oxidative coupling occur both intra-protoplasmically and, extra-protoplasmically, in the plant cell wall. In this work we illustrate a model of two possible mechanisms of polysaccharide feruloylation and oxidative coupling in plants. Moreover, we take into consideration the possible role of in muro feruloylation as a rapid defense mechanism against potential plant pathogen and parasite infections.

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Oxidative burst is one of the fastest defense responses activated by plants in order to resist to pathogen and parasite attacks. It consists in the production of reactive oxygen species (ROS), mainly hydrogen peroxide (H₂O₂), at the first site of pathogen invasion: the plant cell wall. In the presence of ROS, a rapid formation of intermolecular cross-links among various polymers of the cell wall occurs with consequent increase of the resistance to pathogen penetration and spread. These events take place before the transcription-dependent defense mechanisms are activated.1–4

The hydroxycinnamic acids, in particular ferulic acid, ester-linked to arabinose and galactose of pectic and hemicellulosic polysaccharides, are thought to be the main cause of cross-link formation among cell wall structural polymers. It has been well established that oxidative coupling of polysaccharides through their ester-linked feruloyl groups occurs both within the protoplast and after secretion into the apoplast. This process is catalyzed by multiple cell wall bound and, possibly, Golgi resident isoforms of peroxidases which use hydrogen peroxide as substrate.5,6 The formation of different isomers of intra- and inter-polysaccharidic dehydrodimers and oligomers of ferulate is thought to contribute to wall assembly7 and to plant defense responses by restricting cell wall accessibility to lytic enzymes.3 The site, or sites, of the transfer of feruloyl groups on to polysaccharides is, in contrast, still controversial. Although many pieces of evidence suggest that feruloylation occurs mainly intra-protoplasmically, during the de novo synthesis of matrix polysaccharides at the Golgi apparatus level,7–10 by comparing the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall of wheat seedling root apical segments, we have recently suggested that an additional and more rapid feruloylation reaction may take place extra-protoplasmically on pre-existing polysaccharides.11

Here we propose a graphic model (Fig. 1) summarizing the two possible mechanisms of attachment of a feruloyl group to a polysaccharide chain and the oxidative coupling of feruloyl residues to form bridges. It comprises: (i) a mechanism by which feruloylation and feruloyl coupling occur within the protoplast, most likely in the Golgi apparatus;7–10 (ii) a mechanism that occurs in muro.11–13 In this model, the endogenous cinnamate synthesized in the cytoplasm from phenylalanine by the action of phenylalanine ammonia lyase (PAL), a key enzyme in the phenolic metabolism of plants, is rapidly converted to feruloyl-CoA thorough the phenylpropanoid pathway. The same conversion also occurs to the exogenously supplied [¹⁴C] cinnamate, which, at physiological pH, rapidly permeates through the plasma membrane in absence of a specific carrier. The feruloyl-CoA represents the major donor substrate from which feruloyl groups are transferred on to the arabinose residues of (glucurono) arabinoxylans [(G)AX] during their synthesis into the Golgi apparatus. As previously described, feruloyl-(G)AX can be oxidatively coupled within this compartment by forming ferulate dimers or larger coupling products. Feruloyl-polysaccharides and their coupled derivatives are transported into the wall by the secretion pathway, passing through the trans-Golgi network (TGN). This pathway is sensitive to Brefeldin A, a well-known inhibitor of secretion, and requires at least 5 minutes of transit time before the newly synthesized feruloyl-polysaccharides reach the cell wall.11

Alternative feruloyl donors could be the glycosidic esters of ferulate, e.g., 1-O-feruloyl-β-D-glucose (β-Glc-Fer), or other still unknown activated feruloyl precursors (AFP). These compounds may be synthesized in the cytoplasm where they can act as a “reserve” pool of feruloyl units that may be gradually recruited for intracellular polysaccharide feruloylation, either directly9 or via a small and rapid turning-over pool of feruloyl-CoA14 and/or, in part, sequestered as vacuolar conjugates.15 Besides, these low-M_r activated metabolites may be released from the protoplasm into the apoplast with a rapid (>2 min) and BFA insensitive mechanism. In cultured wheat cells, Obel et al.9 have reported the release of a high proportion of the total β-Glc-Fer into the medium. In the apoplast these compounds could act as potential donor substrates of feruloyl residues for in muro O-esterification reactions of arabinosyl groups of pre-existing (G)AX catalyzed by new hypothetical extracellular feruloyl transferases.

Since it has been proposed that the majority of feruloyl groups that were suitably positioned to be capable of coupling had already dimerised intra-protoplasmically,7 the rapid addition of feruloyl groups in muro could give new substrates for dimers formation to strengthen the plant cell wall during the first phases of pathogen infection. In the light of this hypothesis, we incubated 3-day-old wheat seedlings (Triticum durum Desf., cv. Capeiti) for 3 h in the presence of the H₂O₂-generating system glucose (0.5 mM)/glucose oxidase (0.5 Uml⁻¹) (G/GO) that determine a steady-state accumulation of ∼6 µM H₂O₂ for a time longer than 3 h, closely mimicking the oxidative burst induced by a pathogen attack.16 After 1 h of incubation in the presence or absence of G/GO, trans-[U-¹⁴C]cinnamic acid was added as a tracer. As previously reported,11 the trans-[U-¹⁴C] cinnamic acid was rapidly taken up by the seedlings and metabolized in a series of intermediate molecules, including feruloyl-CoA and, probably, β-Glc-Fer that are used as substrate for the synthesis of feruloyl-polysaccharides. All the analyses were performed on 1 cm long apical root segments (1 cm) excised from the seedlings at the end of the incubation time as previously described.11 Oxidative stress did not influence the uptake, but it led to a significant increase in the incorporation of ¹⁴C into the cell wall polymeric material (Table 1). Subjecting the destarched and partially deproteinated cell walls to saponification with diluted alkali, the ester-linkages were hydrolyzed liberating monomers of ¹⁴C-ferulic acid but also ¹⁴C-dimers, ¹⁴C-trimers and ¹⁴C-oligomers. These compounds were acidified, partitioned against ethyl acetate and partially resolved by TLC on Merck silica-gel F₂₅₄ plates (plastic-backed, with fluorescent indicator) (Merck Sharp & Dohme S.p.A., Rome, Italy) in benzene/acetic acid (9;1, v/v; BzA) for 2.5 h as described by Fry et al.14 By counting the radioactivity associated to each compound, we confirmed that ¹⁴C-trimers and products with higher molecular weight (R_F 0.00 on TLC) are the major oxidative coupling products released by saponification of the wall fraction either in the presence or absence of G/GO, suggesting that they are the most involved in polysaccharide cross-linking and cell wall stiffening. In addition we found that, although a 100% increase in the cell wall polysaccharides ester-linked ¹⁴C-diferulates, ¹⁴C-triferulates and ¹⁴C-ferulic acid oligomers was clearly determined by the G/GO treatment, the amount of ester-linked ferulic acid monomers did not significantly decrease (Table 2). If polysaccharide feruloylation occurred exclusively inside the protoplast, a decrease of feruloyl monomers in favor of an increase of coupling products could be expected when the in muro oxidative coupling is stimulated by the extracellular production and accumulation of H₂O₂. The stable content of feruloyl monomers, even in condition of oxidative stress, is easily explicable considering the presence of an in muro addition of feruloyl groups as proposed by our model. The in muro feruloylation would increase polysaccharide degree of substitution and hence their capacity to dimerize. The amount of dehydrodimers is, in fact, proportional to that of esterified ferulic acid.10 The in muro addition of feruloyl residues keeps the amount of monomers of ferulate ester-linked to cell wall polysaccharides stable. In conclusion, the rapid increase of the number of ferulic acid residues linked to the pre-existing polymeric components of the cell wall and on the level of cross-linking between the feruloyl-polysaccharides, which happens in muro to strengthen the wall, could represent a rapid defense response against potential plant pathogen infection.

Figures and Tables

Figure 1 Potential routes of (G)AX feruloylation and oxidative coupling. $\cdots \text{X-}\text{X}\text{|}\text{A}\text{-X}\cdots$, arabinoxylan; F, feruloyl group; TGN, trans-Golgi network; BFA, Brefeldin A; AFP, Activated feruloyl precursor.

Table 1 Distribution of the radioactivity incorporated from trans-[U-¹⁴C]cinnamic acid into the polymers of the purified cell walls, defined by their extractability into pronase and 0.1 M NaOH

Purified cell wall fractions	Radioactivity (%)
	Control	G/G0
Pronase extract	9.1	5.8
NaOH 0.1 M extract
acetylic phase	71.6	72.8
aqueous phase	10.4	10.6
NaOH-insoluble residue	8.8	10.6
Total radioactivity (Bq)	768 ± 50	1030 ± 71

The saponified cell wall material was fractioned into saponifiable phenols (acetylic phase), material failing to partition into ethyl acetate after acidification (aqueous phase) and NaOH-insoluble residue. Cell walls were isolated from 1-cm long apical root segments excised from 3-day-old wheat seedlings incubated for 3 h in the presence or absence of G/G₀. Radioactivity is expressed as % of the total radioactivity incorporated into partially purified cell walls. Data are mean values of triplicate experiment ± SD.

Table 2 Quantitative and qualitative composition of ¹⁴C-feruloyl derivatives ester linked to destarched and partially deproteinated purified cell walls

Ferulic acid derivatives	Radioactivity (Bq)		Stimulation (%)
	Control	G/G0
Ferulate	330 ± 20	300 ± 19	−9
Diferulates	70 ± 6	140 ± 15	+100
Ferulic acid oligomers	150 ± 13	310 ± 21	+107
Total	550 ± 39	750 ± 55	+36

Data are mean values of triplicate experiment ± SD.

Acknowledgements

This work was financed by MIUR, project AGRO-GEN.

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