The mechanisms of NAD-dependent abiotic stress resilience

Crop improvement strategies require improved varieties with increased ability to tolerate environmental stresses. BBSRC's Food Security Strategy Advisory Panel in its advice for a 5-year Wheat Strategy recommended development of new traits for improved sustainability which include resilience to climatic variation and disease. Data from our laboratories and others demonstrate that reduction in the activity of enzymes called poly ADPR polymerases (PARPs) results in plants more resilient to stressful treatments. PARPs are multifunctional enzymes and thus it is not clear how altered activity improves stress tolerance. PARPs are members of a class of enzymes that consume NAD, an important energy containing molecule. Possibly, reducing PARP activity increases the amount of NAD in the plant, resulting in more resilience to stress. We will test this hypothesis by measuring NAD levels in plants with no functional PARPs.
Our preliminary data suggest that not all the benefits conferred by reduced PARP activity are associated with increased NAD levels and therefore we will also test alternative potential mechanisms. We will investigate the regulation of gene expression by PARP activity. We will identify those genes whose expression is altered in PARP loss-of-function plants, also plants in which an enzyme Poly(ADP-ribose) glycohydrolases (PARG1), which counteracts PARP activity, is reduced and in plants with altered Sirtuin (SRT) activity. SRTs are another class of NAD consuming enzymes whose activity is intertwined with the PARPs. We will also identify the regions of DNA bound by the PARPs, PARG1 and SRTs, since this will identify those genes that are likely to be the direct targets for regulation by these enzymes.
We will investigate the effect of loss-of-function of the PARPs, SRTs and PARG1 on the ability of plants to tolerate a wide range of stresses and also the consequence for the functioning of the 24 hour circadian clock. The role of these enzymes in regulating the circadian clock will be investigated because the circadian clock is major regulator of stress signalling in plants and it has been proposed that enzymes with similar function to the PARPs, SRTs and PARG1 have a role in circadian regulation.
Having investigated the effects of PARPs, SRTs and PARGs on gene expression, stress responses and circadian signalling, we will next proceed to addressing the mechanism by which these effects might occur. The activity of the PARPs, SRTs and PARG1 are associated with NAD-dependent modification of chromatin, a complex of proteins bound to the DNA that participates in gene regulation. It is thought that NAD-dependent chemical modifications of chromatin affect DNA folding and thereby regulate gene expression. We will test this hypothesis by studying the activity of the HSP70 gene, which is strongly affected by chemical modifications to the chromatin proteins that bind HSP70. By studying the activity of HSP70 we will be able to determine if loss of function of the PARPs, SRTs and PARG1 has contrasting effects on HSP70 activity, as is predicted by our current models. We also will investigate the effect of loss of each of these enzymes on the chemical content of the plants, the so called metabolome, to identify mechanisms of action and also potential agricultural benefit in altering NAD-consuming enzyme activity.
We have formed an international, multidisciplinary group to address the function and mechanism of action of a major group of enzymes that are thought to contribute to stress tolerance. We combine the academic excellence of laboratories in Cambridge, with world-leading chemical analysis platforms in Golm, Germany and the industrial resource base and potential for translation to real world solutions offered by our industrial partners at Bayer CropScience, Ghent.

We have formed an interdisciplinary team to understand the function of the Poly(ADP-ribose) polymerases (PARPs) in stress tolerance of Arabidopsis. Data from our laboratories and others demonstrate that reduced PARP activity can increase stress tolerance. We aim to identify the mechanism(s) by which PARPs regulate stress tolerance. We also will study the sirtuins (SRTs), another class of NAD consuming enzymes that have close regulatory links with the PARPs and also Poly(ADP-ribose) glycohydrolase 1 (PARG1) as this counters PARP activity.
PARPs add Poly(ADP-ribose) polymers to both DNA and proteins. The SRTs can act as deacetylases and are targets for the PARPs and thus the activity of these two enzymes are interwoven. PARGs remove Poly(ADP-ribose). Such is the complexity of these interactions, only a comprehensive and system wide analysis will reveal the mechanism by which PARPs regulate stress tolerance.
Our approach will be to investigate the consequence of PARPs, SRTs or PARG1 loss-of-function lines and perform molecular, phenotypic and mechanistic analyses. Molecular studies will comprise identification of the regions of DNA bound by the PARPs, SRTs and PARG1 and transcriptomic analysis in loss-of-function lines. Bioinformatic analyses of the data will identify direct targets and biological pathways under the control of the PARPs, SRTs and PARG1.
Phenotypic analysis in loss-of-function lines will determine the role of members of the PARPs, SRT and PARG gene families in stress tolerance and circadian regulation. The goal is to identify gene targets for crop improvement.
Mechanistic insight in to the mode of action of the NAD-consuming enzymes will be provided by analysis of the regulation of the HSP70 promoter, which is marker for chromatin regulation, in loss-of-function lines and analysis metabolic profiling of loss-of-function plants, with particular focus on NAD.
Our studies will provide the foundation dataset of a major regulatory pathway in plants

WHO WILL BENEFIT?
(1) Academic scientists interested in epigenetics, gene regulation, NAD, poly(ADP)ribose, (de)methylases, stress signalling, circadian clocks and metabolism of plants.
(2) Industrial scientists interested in generating crop varieties with enhanced stress tolerance.
(3) Research staff.
(4) The general public.

HOW WILL THEY BENEFIT?
(1) Modification of chromatin and gene regulation is an area of intense research activity. Our proposal provides foundation datasets that will identify the major molecular targets, pathways and mode of action for the NAD-consuming enzymes the Poly(ADP)ribose polymerases (PARPs), sirtuins (SRTs) and the Poly(ADP)ribose glycohydrolases. These enzymes have been implicated as major regulators of chromatin modification but a systematic analysis has not been performed. The PARPs and SRTs are considered to be sensors of cellular energy status, an emerging research are of high impact. Our approach will determine the role of the PARPs and SRTs in sensing and regulating energy status, through metabolic analyses and phenotypic measurement of responses to stress. Our mechanistic findings have the potential for wide significance across many fields, including circadian rhythms, stress signalling and epigenetics. We will ensure maximum impact for academic scientists by publishing our research in a timely manner, dissemination at international meetings and making new biological tools available as soon as practicable.

(2) Industrial scientists will benefit because a central goal of the research is to understand the regulation of plant tolerance of environmental stresses. We work with Bayer Cropscience to identify routes for commercialisation of our findings. To maximise the potential for impact we will perform our studies in the model, Arabidopsis thaliana, since the tools exist only in this organism to make rapid progress.

(3) The PDRAs will gain considerable benefit from being employed on the project. This will include training in state-of-art technologies such as Chip-SEQ and associated bioinformatics. There is a good track record of success for PDRAs in the Webb laboratory; eight former members of the Webb laboratory have obtained faculty positions or equivalent, including five former PDRAs. All BBSRC-funded PDRAs in the Webb laboratory have published their research from the laboratory in Nature or Science The full time technician will receive training in the major techniques associated with the project. The two previous holders of BBSRC-funded technical posts in the Webb laboratory have gained considerable advantage resulting in career progression, one has been accepted as a BBSRC DTP student at the Babraham Institute in Cambridge, the other has started her own business. Both researchers will benefit from interaction with the industrial partner raising their awareness of the industrial context for underpinning research.

(4) The general public will benefit from outreach activities at the Department of Plant Sciences, Cambridge. During Science Week numerous interactive and more formal displays on aspects of plant biology and research are presented and 7,000 visit the Plant Sciences displays on 'Science Saturday' which will include dissemination of findings from this project. It is hoped in the long term that the public will benefit from food security generated from the novel agricultural products that arise from our findings. Whilst recognising that in any field of study the translation rate from laboratory finding to industrial product is low, we make every effort with our industrial partners (Bayer Cropscience) to translate our findings for public benefit. We are currently registering IP on one of our discoveries.