The mechanics of shape in plants

Lead Research Organisation: University of Sheffield
Department Name: Animal and Plant Sciences

Abstract

Stomata are microscopic pores on plant leaves that regulate gas flow into and out of the leaf. The pores are flanked by two specialised epidermal cells called guard cells. Guard cells inflate and deflate, changing their shape and in doing so control the aperture of the pore to regulate the amount of water loss and CO2 uptake of the plant; they do this in response to a variety of extrinsic and intrinsic signals, such as atmospheric CO2 concentration, light intensity and plant hormones. Whilst a lot is known about the signals on which this process depends, and about the development of the stomatal complex, less is known about the shape change of the guard cells and how the guard cell walls permit this. This project sets out to determine the extent to which the mechanical properties of the cell wall influences cell shape and therefore stomatal function. I will do this by combining several disciplines and techniques. Firstly, various molecular biology techniques will be used to screen various mutants of Arabidopsis thaliana with mutations in cell wall genes. Mutants that display interesting phenotypes regarding the guard cell wall will then be imaged using light sheet fluorescence microscopy (LSFM), an imaging technique that is only just beginning to be used to capture plant cells and structures. I hope to generate 3D images of stomatal shape change and also time lapse videos of this process. This visual data will then be combined with force data from AFM (atomic force microscopy), a technique mainly used in physics to determine physical properties of a material. With data on stomatal shape change from LSFM and cell wall mechanical properties from AFM, I hope to link the two and see whether the mechanical properties of stomata cell wall affects shape change, and vice versa. If successful, results from this project could be used to select for, or engineer, crop plants to have more effective or efficient stomata that have a higher intake of CO2 and/or reduced water loss from the plant. This could then improve yield, reduce drought stress, or induce many other long-term effects that could improve crop food sustainability.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1657456 Studentship BB/M011151/1 30/09/2015 29/09/2019 Eleanor Grace Healicon
 
Description • Light sheet fluorescent microscopy can be used to image Arabidopsis guard cells, however for detailed shape change analysis such as the method described in this thesis, confocal microscopy is more appropriate.
• On average, paired guard cells are equal in volume and surface area.
• From closed to open, guard cells increase in volume by 45% and in surface area by 27%. They also undergo significant lengthening along the arc of the cell.
• From closed to open, guard cell cross section shape changes significantly too, with significant tilting and twisting during shape change. This is likely due to a combination of mechanical constraints provided by the cell wall, and the effects of adjacent epidermal cells, although further work into this is necessary. This 'twisting', as opposed to lateral movement of guard cells, to open the stomatal pore may improve efficiency of opening by reducing the energy expended on pushing back against the surrounding epidermal cells.
• arp3 knockout mutants display a significant guard cell shape phenotype after treatment with low CO2. The guard cells swell and bow outwards and a circular, hyper-opened "doughnut"-shaped pore occurs in approximately 25% of stomata.
• Some experiments suggest that arp3 mutants achieve an opened pore with a weaker stimulation threshold i.e. they are more sensitive to opening stimuli and are quick to form the doughnut pore.
• From immunolabelling experiments, there is no evidence to suggest that there are any differences between arp3 and Col-0 in pectin distribution in the leaf cell walls contributing to the unusual guard cell shape phenotype.
• At a whole-plant level, arp3 exhibits significant phenotypic defects when compared to wildtype, such as a reduced rosette area and a lower plant water use efficiency. However, more work is needed to investigate whether these differences are due to aberrant arp3 stomatal function, or whether there are broader effects of the arp3 gene mutation throughout the plant.
• Expansins are a likely candidate for short-term modification of the guard cell wall. Two expansin genes, EXPA4 and EXPA16 are highly expressed in guard cells.
• In extreme conditions, expa16 knockout mutants display a reduced dynamic stomatal range, and have a lower photosynthetic rate than wildtype plants. However, these differences do not have a significant effect on whole-plant physiology e.g. plant growth.
• Some data suggest that expa4 knockout plants may also display differences in stomatal function, however further work is necessary to describe to what extent.
• The data in this chapter combined with previous work into the link between altered expansin expression and differences in stomatal function suggest that expansins do play a role in stomatal function. It is likely that expansins provide a degree of elasticity to the guard cell wall which, upon an increase in guard cell turgor pressure, increases loosening of the guard cell wall and allowing cell wall stretch, cell expansion and therefore opening the pore.
Exploitation Route Several stomatal function models have indicated that shape change of guard cells during stomatal opening and closing is essential to stomatal function. The work I did in this thesis provides a detailed method of guard cell shape quantification in 3D which provides characterisation of Arabidopsis wildtype guard cells, and also has a great amount of potential for future work. This can then be used as a point of comparison to characterise, for example, known stomatal mutants or stomata after enzyme modification. Understanding guard cell shape change in Arabidopsis lays the groundwork for translation into crop research. Learning more about genetic contributors to guard cell shape change allows us to identify target genes for guard cell wall/geometry optimisation. Developing methods of quantifying guard cell shape in Arabidopsis encourages the movement of these methods into plant species that may be more complex e.g. stomata with subsidiary cells. Stomatal opening and closing is key to the instantaneous water use efficiency of plants. Altered cell wall geometry may lead to variant dynamics of stomatal conductance which may therefore further provide targets for crop improvements.
Sectors Agriculture, Food and Drink,Other