A New Conditional Gene Regulation System in Plants

The ability to switch gene expression on and off is a critical factor in an organism's capacity to respond to internal and external cues. The sessile nature of plants means that the need to be able to respond rapidly to ever changing environmental conditions is of particular importance. Until recently, it was thought that the major pathways of gene regulation occurred before transcription. However, it is now known that post-transcriptional processes play a crucial role in regulating gene expression. Among these processes is the control of mRNA stability. Whereas the regulation of mRNA stability by miRNAs has been well explored in plants and continues to provide new insights into plant growth and development, other forms of mRNA stability control are less well understood. Some mRNA transcripts are specifically targeted for degradation based on inherent features that induce a process known as nonsense-mediated mRNA decay (NMD). NMD regulates the stability of a large number of eukaryotic transcripts (of the order of 10% in a range of species), and by moving transcripts into or out of the influence of NMD a cell is able to respond quickly to urgent signals. We have recently discovered that a highly conserved signal (the Conserved Peptide upstream Open Reading Frame - CPuORF) targets specific mRNAs for destruction by NMD. Our evidence suggests that this happens conditionally, in response to a range of signals including abiotic stress, the presence of specific small molecules and in response to changes in translational efficiency. Together with Bayer CropScience, as part of an Industrial partnership Award, we seek support to investigate this novel form of posttranscriptional regulation of gene expression and to determine its potential for crop improvement.

The production of functional proteins from their encoding genes involves a multitude of regulatory checkpoints at each stage of the process. Many of the regulatory steps that produce the appropriate level of gene expression have been characterised in eukaryotic cells, including those affecting both synthesis and decay of the mRNA. One of the key post-transcriptional mechanisms is nonsense-mediated mRNA decay (NMD), which targets specific transcripts for destruction. Conditional modulation of NMD, or conditional mRNA sensitivity to NMD, are means by which cells can respond rapidly to important signals. Specific features of a transcript determine whether it is targeted by NMD. Amongst these are the presence of short peptide-encoding open reading frames, conserved between species, within the 5'-UTR (known as CPuORFs). Examples of CPuORFs controlling expression of the associated major ORF in response to different signals have been identified. Thus, CPuORFs can be regarded as conditional regulators of gene expression with fundamental and commercial applications. Through the identification of direct NMD targets we have linked CPuORF-directed targeting of transcripts to NMD with abiotic stress, the presence of specific small molecules and changes in translational efficiency. We have identified a CPuORF-containing direct NMD target that responds to drought in Arabidopsis and rice and demonstrated that its overexpression confers drought tolerance in Arabidopsis. Here we will investigate the link between CPuORFs, NMD and stress, small molecules and translation efficiency. In collaboration with our commercial partner, we will assess the feasibility of using this knowledge for crop improvement. The nature of CPuORF regulated control of mRNA stability suggests routes to engineer gene expression by GM and non-GM approaches and raises the possibility of designing condition-CPuORF combinations that do not currently exist for the specific control of gene expression.

The focus of this proposal is the role CPuORFs play in gene regulation by NMD and their potential for crop improvement. Healthy plant development relies on the plant being able to respond rapidly to changes in their micro and macro environment. In terms of food security, it is imperative that crop productivity is maintained or increased despite significant environmental challenges. Finding methods to improve a crop's ability to respond to biotic and abiotic stress is an important challenge within plant sciences, and is the focus of research in both the academic and commercial sectors. Elegant approaches to tackle some of these issues have been developed, but there is still the opportunity to develop crops that respond specifically to a particular stress, switching appropriate genes on or off when the plant is challenged. In the current regulatory climate in the EU and elsewhere, the ability to modify plants without the need for the production of transgenics, would also be of significant benefit. We therefore anticipate that impact will be delivered in this project through fundamental research underpinning future discoveries, applied research based on current findings, training, public engagement and international interactions.

Fundamental research: We are seeking to understand a novel form of posttranscriptional gene regulation, which has obvious impact on our understanding of how cells work. The potential of this work, which could be exploited by people wishing to modulate gene expression in e.g. crops, is that we could discover unknown existing CPuORF-condition relationships and, in the future, design novel ones.
Applied research: Our commercial IPA partner is interested in both the potential of RoJ to enhance drought resistance and yield in drought stressed plants, and in the potential to use the RoJ CPuORF for drought-dependent gene expression. If successful, this could lead to improved crop performance and help mitigate the effects of climate change.
Training: Impact in this area will be delivered by training a PDRA, TA and PhD students in preparation for future employment in academic or commercial environments. The involvement of a commercial partner in this project will enhance these aspects of the training. We will also provide laboratory experience to undergraduate students at various stages of their studies, including provision of work experience to graduates.
Public engagement: Impact in this area will be delivered through a range of individual activities in schools, higher education and popular science initiatives and also via specific activities of the university publicity office.
Internationality: Interaction, translation and dissemination to those concerned with crop security in the developing world will be facilitated by our formal, established links in China, India and Africa.