Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET)

Abstract

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan–enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0–6.5), K_m for the nonasaccharide XLLGol (Gal₂.Xyl₃.Glc₃.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher K_m values for XLLGol (70–130 μM) than the four mung bean isoenzymes (16–35 μM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.

Introduction

Xyloglucan is a major structural polysaccharide of the primary cell walls of higher plants and as such is thought to play a central role in determining many of the biologically relevant properties of the wall (Fry, 1989a, Hayashi, 1989). It appears to hydrogen-bond to the cellulose microfibrils and possibly to tether them, thus limiting cell expansion (Fry, 1989b, McCann et al., 1990), and a proportion of the wall’s xyloglucan is covalently bonded to pectic polysaccharides (Thompson and Fry, 2000). Enzyme activities that cleave xyloglucan are thus of considerable interest as potential wall-loosening agents which could be important in the mechanism of cell expansion growth, fruit softening during ripening and cell separation during abscission. One enzyme that cleaves xyloglucan is xyloglucan endotransglycosylase (XET; EC 2.4.1.207) (Baydoun and Fry, 1989, Smith and Fry, 1991, Fry et al., 1992, Nishitani and Tominaga, 1992). XET cleaves a glycosidic linkage in the β-glucan backbone of xyloglucan (= donor substrate) with the concomitant formation of a xyloglucan–XET covalent complex (Sulová et al., 1998); this is followed some time later by the transfer of the xyloglucan portion from this complex on to the non-reducing terminus of an acceptor molecule, which can be either another xyloglucan chain or an oligosaccharide thereof.

XET activity and XET gene expression are found in most, if not all, expanding plant cells (Fry et al., 1992, Pritchard et al., 1993, Nishitani and Tominaga, 1992, Xu et al., 1995, Antosiewicz et al., 1997, Smith et al., 1996, Oh et al., 1998). The occurrence of XET protein in primary cell walls has been detected immunologically (Antosiewicz et al., 1997); in addition, it has been shown that active XET and accessible donor substrate chains co-occur in primary cell walls (Ito and Nishitani, 1999, Vissenberg et al., 2000). Often XET activity appears to correlate with rapid cell expansion, suggesting a causal role in this process. Although moderate XET activity often remains detectable in tissues that have stopped expanding (Campbell and Braam, 1999a), this observation does not argue against a role for XET in cell expansion since it is likely that other factors e.g. phenolic cross-linking (Müsel et al., 1997, Fry et al., 2000) over-ride the ability of XET to loosen the wall in mature tissues.

Ammonium sulphate precipitation of proteins from cauliflower florets revealed the existence of several discrete classes of XET activity (Steele and Fry, 1999), and isoelectric focusing of similar extracts resolved at least eight XET activity bands differing widely in isoelectric point (Iannetta and Fry, 1999). The Arabidopsis genome encodes at least 21 XET-related proteins (XTRs, defined as sequences resembling known XETs, whether or not the translation product has yet been shown to possess XET activity) (Xu et al., 1996, Nishitani, 1997, Campbell and Braam, 1999a).

One of the most extensively studied XETs is TCH4 of Arabidopsis. Accumulation of TCH4 mRNA is promoted during mechanical stimulation (e.g. by wind) of the Arabidopsis plant (Braam and Davis, 1990). However, a TCH4::GUS transgene is also expressed in expanding tissues in the absence of deliberate mechanical stimulation (Xu et al., 1995). In addition, an antibody raised against TCH4 (but probably also recognising other XETs) labelled not only Arabidopsis meristems but also developing pith parenchyma and mesophyll cells at the positions of future cell junctions (Antosiewicz et al., 1997). The involvement of XETs in the re-modelling of cell wall contacts during the production of air spaces is also supported by a correlation between XET expression and the production of aerenchyma in flooded maize roots (Saab and Sachs, 1996).

The various XTR genes other than TCH4 are expressed in a tissue-specific manner and in response to different stimuli, e.g. heat-shock, cold, hormone treatments (Aubert and Herzog, 1996, Clouse, 1996) and mechanical stimulation (Purugganan et al., 1997, Nishitani, 1997). However, little is known of the catalytic differences, if any, between the various XTRs, and it is therefore difficult to comment on their individual biological roles.

Potential sources of the variation between native XETs isolated from diverse plant tissues include (a) the genetically defined amino acid sequence and (b) the co- or post-translationally determined degree of glycosylation. The N-glycan is important for enzyme activity in TCH4 but not in two other Arabidopsis XETs — EXGT and XTR9 (Campbell and Braam, 1998, Campbell and Braam, 1999b).

One potential catalytic difference between isoenzymes is in their substrate affinity. The K_m of TCH4 for a low-M_r acceptor substrate (XLLGol; for nomenclature, Section 4.1 and Fry et al., 1993) is 73 μM, whereas its K_m for high-M_r xyloglucan as acceptor substrate is ∼0.3 μM (Purugganan et al., 1997), indicating a much higher affinity for the polysaccharide. This suggests that TCH4 preferentially catalyses interpolymeric rather than polysaccharide-to-oligosaccharide transglycosylation. Comparison of XETs with respect to their K_m values is complicated by the fact that different laboratories have used different oligosaccharides as acceptor substrates. XET from ripening kiwi fruit has a K_m of 100 μM for XXXGol (Schröder et al., 1998). Unpurified XET activity (probably mixed isoenzymes) from pea stems has apparent K_m values of 19, 50 and 33 μM for XLLG, XXFG and XXXG, respectively (Fry et al., 1992) and of ∼300 μM for the pentasaccharide XXG (Lorences and Fry, 1993). Unpurified XET activity (again probably mixed isoenzymes) from the medium of suspension-cultured poplar cells has K_m values of 320 μM for the Glc₄-based heptasaccharide, XXXGol; 230 μM for the Glc₈-based tetradecasaccharide, XXXGXXXGol; and higher values for larger acceptors. No transglycosylase activity was detected with oligosaccharides having backbones of Glc₁₆ or longer (Takeda et al., 1996). In view of the diverse assay conditions used by different authors, a systematic study was required to determine the affinities of different XETs to a standardised substrate.

Another parameter that might vary between XET isoenzymes is temperature-dependence. TCH4 is surprisingly cold-tolerant: its optimum temperature is ca. 12° and at −5° it still exhibits 55% of its maximal catalytic rate (Purugganan et al., 1997), suggesting a role in the adaptation of growing Arabidopsis to cold conditions. TCH4 is exceptional in its cold-tolerance: total XET activity (mixed isoenzymes) from Arabidopsis has a temperature optimum of ca. 30°. More studies were therefore needed of the temperature-dependence of diverse XETs.

All XETs characterised to date have pH optima in keeping with their proposed location in the cell wall. Unpurified XETs from pea stems have an average pH optimum of ca. 5.5 (Fry et al., 1992). Purified TCH4 has an optimum of pH 6.0–6.5 (Purugganan et al., 1997), mung bean stem XET of 5.8–6.0 (Tabuchi et al., 1997), and ripening kiwi fruit XET of 5.5–5.8 (Schröder et al., 1998).

Although many XET and XTR sequences have been identified and their expression patterns documented, at least at the mRNA level, information on the catalytic properties of this large group of cell wall enzymes is not so plentiful. The aim of the present study was therefore to perform a concerted set of enzymatic analyses on a group of pure, native, XET isoenzymes from cauliflower florets and shooting mung beans. Both these tissues have high XET activity but they form an interesting contrast: cauliflower florets are rich in small, densely cytoplasmic cells, where wall assembly predominates; on the other hand, mung bean hypocotyls are rich in rapidly expanding, vacuolated cells, where the re-structuring of existing wall material may predominate. The results highlight catalytic differences between these isoenzymes in their kinetic properties with respect to pH optima, K_m for XLLGol as acceptor substrate, and ability to utilise xyloglucan-derived oligosaccharides of various sizes.