A microscopy study of specialized cell walls in the roots of orchids of the genus Miltoniopsis.
Thesis DisciplineBiological Sciences
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Plant cell walls are essential in providing shape to the many different cell types required to form the tissues and organs of a plant, form the interface between adjacent cells, hence facilitating intercellular communication, and the gatekeeper in plant-microorganism interaction, including as defence against potential pathogen. As such, much interest has been given to studies into understanding the cell wall function, structure and biosynthesis in various plant species. Orchid roots showcase multiple specialized cell walls in the root with the formation of adaptive secondary cell wall thickenings such as the phi thickenings in the cortical cells and the velamen layer surrounding the root. Cortical cells also exhibit a beneficial interaction with fungus, the orchid mycorrhizae which would require specialized changes in its cell walls. Using a combination of histological and immunocytochemical techniques via confocal microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray microtomography (μCT), this study investigated secondary cell wall development in phi thickenings and the velamen, and looked at changes in cell wall composition due to orchid mycorrhiza in the roots of Miltoniopsis. Chapter 2 shows that during phi thickening formation, microtubules align lengthwise along the thickening during early and intermediate stages of development, and callose is deposited within the thickening in a pattern similar to the microtubules. Double labelling of microtubules and actin show some coordination between actin and secondary wall deposition. These developing thickenings also label with the fluorescently-tagged lectin wheat germ agglutinin (WGA). Phi thickenings function is discussed in Chapter 3. As the movement of fluorescent tracers through the apoplast was not blocked by phi thickenings, and as phi thickenings developed in the roots of sterile cultures in the absence of fungus and did not prevent fungal colonization of cortical cells, the phi thickenings in Miltoniopsis do not function as a barrier. However, as phi thickenings were found to be induced by water stress, it is possible that they may hinder cell collapse during dehydration. In roots dried to conduct X-ray microtomography, some evidence was observed for phi thickenings preventing cortical cell collapse, although this was not conclusive. In Chapter 4, different labelling patterns were observed in the interfacial matrix that surrounds the pelotons in living and degrading pelotons, signifying either a change in cell wall composition or a change in epitope accessibility. These changes included increased labelling with antibodies against pectin and with using wheat germ agglutinin (WGA) which were only found in degraded pelotons. An in vitro infection system was also developed in which whole roots grown under sterile conditions could be infected with a pure fungal strain, thought to be a Tulasnella species, was established. Finally, Chapter 5 showed that the development of the helical-like thickenings in the velamen begins with microtubule-dependant cellulose deposition with the structure subsequently undergoing lignification. As secondary wall ridges develop, parallel microtubule bound the side of the ridge. The actin cytoskeleton was also observed to associate with secondary thickening formation. Fluorescent tracer experiments demonstrated that the mature velamen retained a primary cell wall that was capable of blocking uptake of large molecules. Cellulose organisation in the velamen was investigated using pontamine staining which showed bifluorescence of the cellulose strands. Overall, orchid roots provide an exceptional system that showcases the links between intracellular functions such as the cytoskeleton and cell wall production, and the visible cell wall and an entire plant’s physiology.