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Mechanisms to generate inter-individual variability from single-cell heterogeneity

Reference	BB/V006088/1
Principal Investigator / Supervisor	Dr James Locke
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Sainsbury Laboratory
Funding type	Research
Value (£)	507,756
Status	Current
Type	Research Grant
Start date	01/10/2021
End date	30/09/2024
Duration	36 months

Abstract

Gene expression can be noisy and variable from cell to cell. Despite much work understanding noise in gene expression in unicellular organisms, it's role in multicellular systems is unclear. We will use Arabidopsis thaliana as a model system to investigate how noisy gene expression in individual cells can generate phenotypic differences at the organism level. We recently performed RNA-seq on individual seedlings grown under identical conditions, revealing that a fraction of genes have highly variable expression between individuals. Genes regulated by the hormone ABA and those involved in abiotic stress responses (which are ABA regulated) were enriched for highly variable genes and we recently showed that the ABA-GA pathway underlies phenotypic variability in germination time. We will now use a multidisciplinary approach, to answer: 1) How do differences in gene expression between individuals map to variability at the single-cell level? For candidate ABA-regulated highly variable genes, we will perform time-lapse microscopy for multiple whole seedlings (using LUC reporters) and at the single-cell level using confocal fluorescent microscopy. This will reveal how cellular-level processes generate between-plant variability. 2) We will model the gene circuit dynamics of the ABA-GA bistable switch controlling the balance between growth and stress response, investigating how dynamics at the single-cell level lead to between-individual variability. We will predict and test how the balance between ABA and GA and therefore growth and stress-response is set differently in genetically identical Arabidopsis plants. 3) Do the gene expression differences generate functional phenotypic variability during stress response? We will track gene expression before, during and after stress to see whether noisy activation of a pathway before stress can aid survival. This work will provide a mechanistic understanding of the role of noise in multicellular systems.

Summary

Genetically identical individuals, from populations of bacteria to human twins, often behave differently, even though they have identical genes. We are interested to know how these differences are generated, and what effects these differences might have. Our model system of choice is a small model plant, Arabidopsis, which produces 1000s of genetically identical seeds each generation. Plants grow in a variable and noisy environment, including fluctuating light, water and temperature. Plants also have a noisy internal environment; recent work from our laboratory has revealed that even in plants that are genetically identical and grown in the same environment, around 9% of genes will be highly variable in their level of activation from plant to plant. We found that stress response genes that help plants survive environmental conditions such as drought, high salinity or extreme temperatures were particularly likely to be variable in their expression levels between plants. This variation in gene activation could be useful in nature for populations of genetically similar plants to hedge bets against unpredictable environmental stresses. This is because variable gene expression would mean that there are always a few plants in the population that are prepared to survive different stresses due to their variable gene activation. There might be a cost to having high expression of stress response genes, which could make variable gene expression more advantageous than all plants having high expression levels of a gene all of the time. As well as having potential benefits in the wild, variability between plants can also be a problem, such as in agriculture where environments are more controlled and farmers require uniform crops that germinate and flower at the same time and respond equally to applications of fertilisers and water. In this proposal, we seek to understand how the differences in gene activity between genetically identical plants grown in the same conditions are generated by cellular-level processes. To do this, we will use two different live imaging techniques to monitor gene activity of candidate 'noisy' pathways over time. We will use luminescent reporters (using a luciferase gene from fireflies) to give us a read out of the expression of our genes of interest at the level of whole seedlings. This technique will allow us to monitor how expression of a particular gene varies over time and across tissues such as leaves, stems and roots. We will also use fluorescent reporters (using green fluorescent protein from jelly fish) to allow higher resolution imaging of individual living cells in the tissues of interest. We will test how the variability in the levels of activity of genes in tissues and individual cells leads to the differences at the level of whole plants. To understand the results of our experiments, we will build mathematical models of the gene activation dynamics that will help us test our understanding and design new experiments. We will focus on a pathway controlled by a stress response hormone, ABA, as we have found that many genes regulated by it appear to be highly variable from plant to plant. Finally, we will determine the effects on plant growth and survival of the noisy gene activation we have characterised. We will track gene activity before and after the addition of stress, and examine whether the level of activation of a pathway before the stress correlates with the survival of a plant after stress. It could be that the random activation of the pathway before stress allows the plants that have higher expression to survive the sudden change in conditions (a bet-hedging strategy). In the future, understanding how plants produce and regulate noise in gene activity will be important for the development of more uniform crops and to understand how populations of wild plants can survive more frequent weather extremes due to climate change.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Plant Science, Systems Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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