RG2 in non-flowering plants

The occurence of RG-II in the cell wall of bryophytes, lycophytes and pteridophytes

Summary

The results of our recent studies (1) have shown that comparable amounts of borate cross-linked RG-II are present in the walls of lycophytes, pteridophytes, gymnosperms, and angiosperms. By contrast, bryophyte walls contain little if any of this pectic polysaccharide. Thus, the appearance of borate cross-linked RG-II in the wall or a dramatic increase in the amounts of this polysaccharide in the wall may have been one of the major changes in wall structure associated with the bryophyte-lycopodiophyte transition (1, 2).

Cell walls were isolated from a range bryophytes, liverworts, hornworts, lycophytes, and pteridophytes:

Bryophytes	Lycophytes	Pteridophytes
Bryopsida Dicranodontium denudatum Hypnum oldhamii Loeskeobryum cavifolium Platyhypnidium riparoides Pogonatum japonicum Polytrichum juniperinum Sphagnum palustre Takkakia ceratophylla Hepaticopsida Bazzania pompeana Conocephalum conicum Marchantia polymorpha Pallavicinia subciliata Anthocerotopsida Phaeoceros carolinianus	Lycopodiales Huperzia lucidula Lycopodium nummularifolium Lycopodium scariosum Lycopodium tristachyum Selaginellales Selaginella kraussiana Selaginella tamarriscina	Psilopsida Psilotum nudum Equisetopsida Equisetum hyemale Equisetum variegatum Filicopsida Angiopteris lygodiifolia Ceratopteris thalictroides Ceratopteris richardii Cyrtomium falcatum Lacosteopsis orienatlis Matteuccia sturthiopteris Platycerium bifurcatum Sceptridium sp Salvinia molesta

RG-II is a component of lycophyte and pteridophyte walls

Several of the glycosyl residues that are characteristic of RG-II (MeFuc, MeXyl, Api, and AceA) were readily detected in the walls of all the pteridophytes and lycophytes. However, these glycoses were barely detectable in bryophyte walls.

To confirm that lycophytes and pteridophytes do synthesize RG-II and to determine if this RG-II is cross-linked by borate the walls isolated from these plants were treated with endopolygalacturonase (EPG).

Previous studies have established that RG-II together with RG-I and a mixture of oligogalacturonides are solubilized by EPG treatment of flowering plant walls.

The material solubilized by EPG was fractionated by size-exclusion chromatography (SEC) using a Superdex-75 HR10/30 column and refractive index detection (see figure on right). Peaks that elute in the region for the borate cross linked RG-II dimer and the RG-II monomer are clearly present.

No peaks corresponding to the RG-II monomer or dimer were detected when the material solubilized by EPG treatment of the bryophyte walls was analyzed by SEC-RI.

The material in the peaks corresponding to RG-II from several different lycophytes and pteridophytes was collected and analyzed in detail.

Glycosyl residue and glycosyl-linkage composition analyses, together with ¹¹B-NMR spectroscopy, confirmed that RG-II was present in these walls as a borate cross linked dimer.

It is clear from our results (1) that RG-II is indeed present in the walls of non-flowering vascular plants that include lycophytes, psilopsids, horsetails and ferns.

3-O-methyl rhamnose is present in the RG-IIs synthesized by some lycophytes and pteridophytes

Somewhat unexpectedly, several of the RG-IIs isolated from lycophytes and pteridophytes contained 3-O-methyl rhamnosyl residues. 3-O-Me Rha is not present in any of the RG-IIs that have been isolated from the walls of flowering plants. This glycose (common name acofriose) has, however, been reported to be present in the primary walls of charophytes, bryophytes, and homosporus lycopodiophytes (3), and is present in complex carbohydrates synthesized by Chlorella vulgaris, and by Campylobacter and Mycobacterium.

We established that the 3-O-Me Rha residue is located on side chain B of the RG-IIs (see figure on left) using a combination of glycosyl-linkage composition analyses and electrospray-ionization mass spectromerty.

Bryophyte walls contain small amounts of a RG-II-like polysaccharide

The combined results of glycosyl residue composition analyses and EPG treatment of bryophyte walls suggested that these walls contain little if any RG-II. However, small amounts of an RG-II-like molecule were detected when the material solubilized by Drisealse treatment of bryophyte walls was analyzed by size-exclusion chromatography in combination with inductively coupled plasma mass spectrometry (SEC-ICP-MS).

SEC-ICP-MS is a analytical method where the SEC column is interfaced directly to the ICP source of the mass spectrometer thereby allowing the ¹¹B content of the eluent to be determined.

The Driselase digests of the lycophyte and pteridophyte walls all gave strong signals for ¹¹B in the region expected for RG-II (see figure on right).

By contrast, only weak signals for ¹¹B were detected in the Driselase digests of the bryophyte walls (see figure on right - note: the scale of the ¹¹B ion intensity for the bryophyte profiles [bottom four] is 40 times more senstive than the scale for the lycophytes and pteridophytes).

The amounts of RG-II in the various walls was calculated from the amount of ¹¹B that eluted in the region for RG-II and by assuming that the boron content of RG-II is 1 mg/g.

The amounts of borate cross-linked RG-II in the walls of various land plant groups

Land Plant Group	Average amount of borate cross-linked RG-II	Range of the amount of borate cross-linked RG-II
	mg/g dry wt cell wall ±SD	mg/g dry wt cell wall
Bryophytes^a Lycophytes Pteridophytes Dicotyledons and non-graminaceous monocotyledons^b Gramineae ^b	0.1 ± 0.06 7.4 ± 6.2 9.3 ± 4.9 22.8 ± 8.5 3.8 ± 1.0	0.04 - 0.25 2.0 - 19.1 1.9 - 19.9 5.0 - 36.0 3.0 - 5.0

^a The presence of RG-II in bryophytes is based soley on the of boron contents of the walls obtained by SEC-ICP-MS analyses. No RG-II has been isolated from bryophyte walls and structurally characterized (1). ^b Amounts calculated from 4

References

1.Matsunaga et al (2004). Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants. Plant Physiol. 134, 339-351.

2.Popper et al (2003). α-D-glucuronosyl-(1,3)-L-galactose, an unusual disaccharide from polysaccharides of the hornwort Anthoceros caucasicus. Phytochemistry 64, 325-335.

3.Popper and Fry (2003). Primary cell wall composition of bryophytes and charophytes. Annals Bot. 91, 1-12.

4. Matoh et al (1996). Ubiquity of a borate-rhamnogalacturonan II complex in the cell walls of higher plants. Plant Cell Physiol. 37, 636-640.

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