Measuring stress signalling responses of stomata in isolated epidermis of graminaceous species.

Our current understanding of guard cell signalling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signalling in the stomata of graminaceous species (including many of the world’s major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signalling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20°C and 40°C

Investigating the control of stomatal development and function in T. aestivum and H. vulgare

Stomata are small pores present on the leaves and shoots of the majority of land plants, which can open and close to allow the plant to balance the uptake of CO2 for photosynthesis with the loss of water vapour by transpiration (Hetherington and Woodward, 2003). There are two broad classes of stomatal guard cells: the kidney-shaped guard cells found in most plant species, such as mosses, ferns, gymnosperms, dicots, and non- graminaceous monocots; and the dumb-bell shaped guard cells found in grasses and most other monocots. The majority of what we know about the control of stomatal development and function has been learnt through study of model plants, including Arabidopsis tha/iana, Commelina communis and Vicia faba. The aim of the work presented in this thesis was to ,~A advance our knowledge regarding the stomata of the agronomicatle- and commercially valuable cereals, Hordeum vu/gare (barley) and Triticum aestivum (wheat). A screen of barley eceriferum mutants identified three lines, BWll1, BW117 and BW119, with aberrant stomatal development; and a screen of barley wilting leaf blade mutants identified one line, WLB2, with a significantly reduced stomatal response to the plant drought hormone abscisic acid (ABA), and a second line, WLB1S, which could potentially be an ABA biosynthesis mutant. Study of stomatal number in a collection of wheat varieties identified a weak negative trend between water use efficiencv and stomatal density on the adaxial leaf surface. Transcriptomic analysis of the wheat guard cell identified several genes which are likely to be involved in stomatal development or function in wheat, and one candidate, PKABAl (likely orthologue of Arabidopsis OSTl) was further studied in a cross-species complementation experiment. The results of this experiment demonstrated that the wheat and barley PKABAl genes were able to partially complement the stomatal response to reductions in atmospheric humidity when transformed into the Arabidopsis ostl-4 mutant. It was shown that the barley PKABAl gene (but not the wheat PKABAl gene) was also able to partially restore the stomatal closure response to ABA in the Arabidopsis ostl-4 mutant. Phenotypic characterisation of two barley eceriferum mutants (BW117 and BW119) demonstrated that they had similar and complex abnormalities in stomatal development and vasculature development; and BW119 showed altered water loss, and potentially altered cuticular wax. Genotypic characterisation of BW117 and BW119 suggested that BW119 is a further backcrossed derivative of BW117; and this analysis allowed detailed syntenic mapping of the mutation. In conclusion, this project has provided evidence that wheat and barley are adequate subjects for stomatal research, and has identified a number of avenues for further investigation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo