Investigating how plants sense water

Overview

As a result of climatic change, the world is experiencing unprecedented increases in average global temperatures and extreme weather events leading to intermittent periods of drought. In addition, over-irrigation of agricultural land has resulted in increased salination of once productive arable soils. This has resulted in a situation in which farmers around the world face the problem of ensuring sufficient farm yields, both now and in the future. Drought stress tolerance in plants is therefore a critical goal for plant breeders. To achieve this, arable systems must be resilient to environmental fluctuations, and it is essential that we better understand how it is that plants respond to and adapt to stresses such as drought and salinity. Central to both water and salinity stresses is how plants go about regulating and optimising their use of water. One of the most sensitive stages to water stress during a plant life cycle is the transition from seed to seedling: germination and establishment. This project will investigate the ecological variability in molecular mechanisms by which drought-tolerant and drought-susceptible barley varieties respond to water or salinity stress, particularly during germination, but also during establishment, vegetative growth and seed production. These molecular mechanisms will be compared with the similar mechanisms found in drought tolerant tropical cereals such as millet, quinoa, or sorghum.

To investigate the drought mechanisms, you will work on a recently identified molecular sensor for water stress in plants, and the associated intracellular pathway that is critical for how plants respond to water stress. PM19 is an osmosensor that is conserved in protein sequence all the way back to the first land plants (liverworts). Structurally PM19 resembles the yeast osmosensor Sho1, with four transmembrane domains, and the Arabidopsis gene PM19L1 phenotypically complements the yeast sho1 mutant (Fig.1), showing that it can function as an osmosensor. In plants, PM19 is highly expressed during seed development and germination and is upregulated by dormancy and abscisic acid. Importantly, mutations in the PM19 gene results in plants with enhanced salt sensitivity during germination. The hypothesis that will be explored in this project is that the variability in expression levels of PM19 and pathway components, or variability in gene and protein sequence underlies the differences in plant drought and salt tolerance during germination and later growth.

This project would be ideal for candidates seeking to apply molecular biological methods to real world problems in agriculture.

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Image Captions

Figure 1: PM19L1 complements the yeast sho1 osmosensor mutant. Growth of wildtype, sho1 mutant, sho1 transformed with PM19L1 on basal media, 0.5 M sorbitol and 0.5 M NaCl.

Methodology

The key aims of this project will be to: (i) identify variation in genes in the water sensing pathway for barley varieties differing in drought tolerance, (ii) test the robustness of this tolerance in the field and by genetic complementation of stress compromised mutants in plants, and (iii) compare barley stress tolerance mechanisms with those in tropical cereals.

We have phenotyped over 150 European barley landraces (including both two and six- rowed seed head types), and 20 elite cultivars for germination response under osmotic stress, and have identified striking differences in several different lines. Some are very tolerant to osmotic stress; others are very intolerant. In this project you will devise experiments to investigate selected barley varieties and further characterise them in terms of drought tolerance, and also look at the gene expression patterns of the osmosensor gene PM19, and its associated downstream pathway components. You will also look at the functionality of variability in the protein sequence of these lines. Additionally, you will study the expression patterns, and sequences of the highly conserved gene PM19 from tropical cereals such as millet, quinoa and sorghum, with known drought tolerance traits.

Of the 150 barley cultivars that have been phenotypically characterised with respect to drought, the student will select a range of sensitive and resistant cultivars. These will be grown to maturity under control conditions (green-house and hydroponics) and exposed to drought and salinity conditions to monitor the abiotic stress susceptibility of the mature plant. Fresh weight, dry weight, relative water content, reactive oxygen species and stress gene expression will be recorded in these plants. RNA will be prepared from germinating seeds and seedlings under control and stress conditions and Q-PCR will be used to measure PM19 gene expression, and other signalling genes, known to interact with PM19 and involved in the transduction of water stress. In addition, expression of stress-induced genes such as catalase, ascorbate oxidase, and transcription factors such as MYB1 will be studied. This will yield a thorough description of the role and importance of osmosensor systems in drought tolerance. Gene sequencing will be used to characterise barley alleles of pathway components associated with abiotic stress resistance, and molecular variants will be functionally tested by cloning into a binary vector harbouring native promoters and complementation of corresponding Arabidopsis mutants.
The key aims of this project will be to: (i) identify variation in genes in the water sensing pathway for barley varieties differing in drought tolerance, (ii) test the robustness of this tolerance in the field and by genetic complementation of stress compromised mutants in plants, and (iii) compare barley stress tolerance mechanisms with those in tropical cereals.

We have phenotyped over 150 European barley landraces (including both two and six- rowed seed head types), and 20 elite cultivars for germination response under osmotic stress, and have identified striking differences in several different lines. Some are very tolerant to osmotic stress; others are very intolerant. In this project you will devise experiments to investigate selected barley varieties and further characterise them in terms of drought tolerance, and also look at the gene expression patterns of the osmosensor gene PM19, and its associated downstream pathway components. You will also look at the functionality of variability in the protein sequence of these lines. Additionally, you will study the expression patterns, and sequences of the highly conserved gene PM19 from tropical cereals such as millet, quinoa and sorghum, with known drought tolerance traits.

Of the 150 barley cultivars that have been phenotypically characterised with respect to drought, the student will select a range of sensitive and resistant cultivars. These will be grown to maturity under control conditions (green-house and hydroponics) and exposed to drought and salinity conditions to monitor the abiotic stress susceptibility of the mature plant. Fresh weight, dry weight, relative water content, reactive oxygen species and stress gene expression will be recorded in these plants. RNA will be prepared from germinating seeds and seedlings under control and stress conditions and Q-PCR will be used to measure PM19 gene expression, and other signalling genes, known to interact with PM19 and involved in the transduction of water stress. In addition, expression of stress-induced genes such as catalase, ascorbate oxidase, and transcription factors such as MYB1 will be studied. This will yield a thorough description of the role and importance of osmosensor systems in drought tolerance. Gene sequencing will be used to characterise barley alleles of pathway components associated with abiotic stress resistance, and molecular variants will be functionally tested by cloning into a binary vector harbouring native promoters and complementation of corresponding Arabidopsis mutants.

In addition, the student will devise experiments to grow drought-tolerant tropical cereal species such as millet and sorghum under control and stress conditions, to compare the expression patterns of PM19 and associated pathway proteins. You will then investigate potential similarities in osmosensor associated gene expression among these species.
Overlapping this molecular-based approach will be consideration of the geographic background and field performance of each sub-selected barley landrace. This will enable you to explore the possibility that robust abiotic stress resistance comes with an associated trade-off for other factors that impact on plant growth and crop production. To evaluate this, field trials will be carried out over two years in order to measure agronomic performance such as yield and disease resistance of the abiotic stress tolerant and non-tolerant barley varieties.
These investigations will shed light on how plants have adapted to challenging abiotic stress conditions throughout evolution and to very different climates, and will also provide valuable information on plant breeding strategies for producing climate change resilient crops for the future. We know the climate is warming and we need to plan for the coming generations by breeding crops that have a robust resistance to drought stress. This project will be an important part of the overall effort in achieving that aim.

Project Timeline

Year 1

Growth of barley varieties under control / stress conditions, initiate physiological and molecular analysis of stress tolerant/intolerant barley varieties including sequence analysis of pathway components.

Year 2

Continue with analysis of barley varieties. Comparisons of gene expression patterns and sequences of key genes with stress tolerant tropical cereal species. Field trials of stress tolerant barley varieties. Genetic complementation of Arabidopsis mutants with genes of interest discovered by the student.

Year 3

Field trials of stress tolerant varieties, analysis of data from barley field trials (disease scores, yield).
Preparation of the thesis and journal papers

Year 3.5

Submission of thesis aimed at 3.5 years after commencement of project.

Training
& Skills

The student will have access to the full variety of the extensive IAPETUS2-cohort training, including workshops and cohort meetings, and it is anticipated that they will make full use of these opportunities in order to develop broader transferable skills and knowledge. Furthermore, Heriot-Watt University offers a broad pallet of training opportunities relevant to postgraduate students. Course sessions are typically delivered in three hour sessions facilitated by experts, and mapping onto the four domains of the Researchers Development Framework: A, Knowledge and intellectual abilities: B, Personal effectiveness: C, Research governance and organisation: D, Engagement, influence and impact (www.vitae.ac.uk).
In addition to generic training, the student will be trained in a range of scientific methods: molecular biological methods (e.g. Q-PCR, bioinformatics) Plant physiology (e.g. abiotic stress biology), data analysis (including appropriate statistical analysis). All methodologies are well established in the supervisors’ laboratories.

The student will join a strong set of laboratories working on a variety of aspects of plant stress. They will use a range of innovative techniques in molecular biology, plant physiology and environmental studies on drought and stress. The project provides for excellent multidisciplinary training and an exciting opportunity to interact between the fields of molecular biology, ecology of environmental stress and plant physiology.

We anticipate that the PhD student will present their results in at least two appropriate meetings in the UK (for example Society for Experimental Biology) and at least one international symposium (for example International conference for Plant Physiology). The student will also have opportunities to network with project partners at Heriot-Watt and the UK Centre for Ecology & Hydrology and to become a member of the broader scientific community working on plant stress

References & further reading

Alexander, R.D., Pablo Castillejo-Pons, P., Alsaif, O., Stahl, Y., Madeleine Seale M., Morris, P.C. (2020) The conserved plant PM19 protein functions as an osmosensor and regulator of germination. BioRxiv, doi: https://doi.org/10.1101/2020.08.10.244889

Alexander, R.D., Wendelboe-Nelson, C. Morris, P.C. (2019). The barley transcription factor HvMYB1 is a positive regulator of drought tolerance. Plant Physiol Biochem. 142, 246-253.

Abass, M. and Morris, P. C. (2013). The Hordeum vulgare signalling protein MAP kinase 4 is a regulator of biotic and abiotic stress responses. J. Plant Physiol. 170, 1353-1359.

Qiu, J. L., Zhou, L., Yun, B. W., Nielsen, H. B., Fiil, B. K., Petersen, K., Mackinlay, J., Loake, G. J., Mundy, J. and Morris, P. C. (2008). Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant Physiol. 148, 212-222.

Rae, S. J., Macaulay, M., Ramsay, L., Leigh, F., Matthews, D., O’sullivan, D. M., Donini, P., Morris, P. C., Powell, W. and Marshall, D. F. (2007). Molecular barley breeding. Euphytica 158, 295-303.

Wendelboe-Nelson, C. and Morris, P. C. (2012). Proteins linked to drought tolerance revealed by DIGE analysis of drought resistant and susceptible barley varieties. Proteomics 12, 3374-3385.

Further Information

Peter Morris, Institute of Life and Earth Sciences
Heriot-Watt University, Riccarton
Edinburgh, EH14 4AS
Tel +44 (0)131 451 3452
p.c.morris@hw.ac.uk

Ross Alexander, Institute of Life and Earth Sciences
Heriot-Watt University, Riccarton
Edinburgh, EH14 4AS
Tel +44 (0)131 451 3181
r.alexander@hw.ac.uk

Jill Thompson
UK Centre for Ecology & Hydrology
Bush Estate, Penicuik
EH26 0QB
Tel +44 (0)131 445 8518
jiom@ceh.ac.uk

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