“Ultrastructure and cytochemistry of bryophytes in the context of land plant phylogeny”
Research area: “Management, conservation and restoration of environmental resources”
The bryophytes is a highly diversified and extremely primitive group of land plants that traditionally includes the
anthocerotes, liverworts and mosses. We have been working on two distinct lines of research, the first concerning
cellular interactions in liverwort-fungi symbioses, the second the evolution of vascular tissues.
Cellular interactions in liverwort-fungus symbioses
Our research activity on this line of enquiry during 2002 has produced the results detailed below.
In three species of the genus Treubia sampled during a recent expedition to New Zealand (South Island) partially
supported by CNR, we have discovered a novel type of fungal association hitherto unreported in liverworts. This
association is characterized by the presence of a glomalean fungus both in mucilage-filled intercellular spaces and
within the cells in a specific area of the thallus. The intracellular fungal infection involves host cytoplasm proliferation
and the formation of typical arbuscules.
The comparative analysis of four members of the monotypic order Haplomitriales (Haplomitrium blumei, H.
hookeri, H. gibbsiae, H. ovalifolium) has revealed that this primitive group of liverworts if regularly infected by
glomalean fungi. These colonize the apical part of the leafless underground axes (rhizomes) typical of Haplomitrium
and is rapidly destroyed by intracellular lysis in more mature areas. Unlike other liverworts, where the fungus colonizes
the internal parenchima of the thallus, in Haplomitrium only the epidermal cells are infected, with the formation of
typical arbuscules. The infection may extend to the underlying layer of parenchyma cells but here the fungus is
immediately destroyed and sequestered in the vacuole in the form of masses of collapsed hyphae.
An immunocytochemical screening of the two marchantialean liverworts Marchantia polymorpha and
Conocephalum conicum with a battery of monoclonal antibodies against polysaccharide and glycoprotein components
of the cell wall in higher plants has shown a positive response to CCRC-M1 (specific to the fucosylated side group
present in xyloglucan and, to a lesser extent, in rhamnogalacturonan I) and JIM 7 (specific to partially methyl-esterified
homogalacturonan). In both species JIM 7 labels the whole wall of parenchyma cells, except the expanded middle
lamella areas at the cell corners, with no difference between infected and uninfected cells. By contrast, CCRC-M1
specifically labels a thin wall layer close to the plasmalemma. The labelling with this antibody is very weak in
uninfected cells whist it is relatively abundant in cells harbouring fungal hyphae. Examination of hyphal penetration
points has revealed that, while entering a host cell, the fungus induces the lysis of the part of the host wall that is not
labelled with CCRC-M1 whilst the inner labelled layer invaginates and expands along with the fungus, to form a
continuous coat around the intracellular hyphae. The labelling pattern of CCRC-M1 suggests that fungal penetration
induces an increase of the fucosylated xyloglucan in the innermost wall layer of host cells. This probably gives this
layer the plasticity necessary to expand and accomodate the whole intracellular fungal system. The absence of labelling
with an antibody for callose (a polysaccharide produced by plants as a response to wounding or attack by pathogens)
reflects the high cellular compatibility between the liverworts and fungal symbionts.
Conducting tissues and land plant phylogeny
The adaptation of plants to life on land has demanded the evolution of vascular tissues specialized for long-
distance translocation of water and nutrients. Alongside the highly specialized vascular tissues occurring in the
tracheophytes (xylem and phloem), water- and food-conducting cells may also occur in bryophytes. The evolutionary
relationships between the bryophytes and tracheophytes date back to very early stages of land plant evolution and are
highly controversial. It is the aim of this line of research to gain insights into the early phylogeny of land plants through
a comparative ultrastructural and immunocytochemical study of conducting tissues in bryophytes and primitive
tracheophytes.
In 2002 we have completed an immunocytochemical investigation of water-conducting cells in a range of
liverwort and moss species selected as representatives of all bryophyte taxa known to possess water-conducting tissue.
The tests have revealed that bryophyte water-conducting cells display distinctive immunocytochemical properties
relative to other tissues in the same species. Immunocytochemical differences between major taxa are consistent with
the hypothesis of a multiple origin of these cells.
Research group:
Roberto Ligrone – Professor of Plant Biology
Anna Carafa – Ph-D student
External collaborations:
Prof. J.G. Duckett (School of Biological Sciences, Queen Mary College, University
of London, UK)
Prof. Karen Sue Renzaglia (Department of Biology, Southern Illinois University,
Carbondale, IL, USA)
Prof. D. Read (Department of Animal and Plant Science, University of Sheffield,
UK)
Dr. Kevin C. Vaughn (US Department of Agriculture, Southern Weed Science
Research Unit, Stoneville, MS, USA)
Prof. J. Paul Knox (Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT,
UK )
Prospective developments:
In 2003 the research will concentrate on the following aspects:
Cellular interactions in liverwort-fungus symbioses:
Further ultrastructual characterization of liverwort-fungus associations.
Cellular interactions between host plants and fungal endophytes. We will use immunocytochemical techniques to
analyse the interfacies between the symbionts.
Effects of reconstituted soils on the development of arbuscular mycorrhyzas. We will investigate how composts
obtained from different kinds of organic wastes affect the establishment and development of mycorrhizas in
plants inoculated with specific strains of glomalean fungi.
Conducting tissues and land plant phylogeny:
The development of conducting tissues in bryophytes and primitive members of the tracheophytes (Lycopodium and
primitive ferns), with emphasis on cell wall modifications.
Publications:
Ligrone R., Vaughn K.C., Renzaglia K.S., Knox J.P., Duckett J.G. (2002). Diversity in the distribution of
polysaccharide and glycoprotein epitopes in the cell walls of bryophytes: new evidence for the multiple
evolution of water-conducting cells. New Phytologist 156: 491-508.
Aliotta G., Ciniglia C., Ligrone R., Pinto G., Pollio A., Stanzione M. (2002). Application of microscopic techniques to
the study of seeds and microalgae under olive oil wastewater stress. In: F.A. Macias, J.C.G. Galindo, J.M.G.
Molinillo and H.G. Cutler (eds), Recent Advances in Allelopathy, vol II., in press.
Carafa A., Duckett J.G., Ligrone R. (2003). The placenta in Monoclea forsteri Hook. and Treubia lacunosa (Colenso)
Prosk: insights into placental evolution in liverworts. Annals of Botany, in press.