The role of sugar-responsive bZIP transcription factors in the regulation of the circadian oscillator of Arabidopsis

Due to population growth and changing environments there is an urgent need to understand the basic biology that regulates the yield of crop plants. We have recently discovered a new pathway that regulates the ability of plants to tell the time, balance sugar resources correctly and grow. We have discovered that cellular sugars speed up the circadian clock, whereas low sugars makes the circadian clock run more slowly. We have evidence that this change in the speed of the plant circadian clock is important in regulating the balance between consumption and storage of sugars. Our studies have identified a protein that is required for the correct control of circadian speed in response to sugars, this protein is called BASIC LEUCINE ZIPPER63 (bZIP63). We will discover if other bZIP proteins regulate the circadian clock, we will discover the changes in gene expression controlled by the bZIP proteins, we will work out how the bZIP proteins regulate gene expression and therefore the circadian clock. Lastly, we will discover the changes in plant physiology and metabolism that occur as a result of bZIP proteins regulating the circadian clock. These studies will begin to resolve why bZIP63, a regulator of the response of plants to sugar status, and often associated with starvation responses, regulates the speed of the circadian oscillator. We will resolve how this sugar-sensitive signalling pathway regulates the circadian clock to optimise carbon use in the plant. This is a collaboration between Prof Alex Webb (Cambridge, UK) who studies circadian clocks and Prof Michel Vincentz (Campinas, Brazil) an expert in bZIP transcription factors and carbon homeostasis. Our work is performed in the model plant Arabidopsis and findings will be transferred to breeders of UK crops, such as wheat, and Brazilian crops such as sugar cane, through relationships of the researchers with industrial partners, and through ongoing research programmes in the applicants' laboratories.

Webb and Vincentz have discovered that the transcription factor, BASIC LEUCINE ZIPPER63 (bZIP63), which is normally associated with sugar signalling, binds and regulates the expression of PSEUDO RESPONSE REGULATOR7 (PRR7), a component of the plant circadian clock of Arabidopsis. This finding builds on Webb's discovery that sugars regulate the pace of the Arabidopsis circadian oscillator. We have combined forces to investigate how and why bZIP transcription factors regulate the circadian oscillator. We have evidence that this forms part of a pathway by which sugar signalling feeds in to the circadian clock to regulate circadian timing to balance carbon homeostasis. We will investigate the molecular basis of this pathway by using mutant analysis to identify if bZIPs other than bZIP63 also contribute to the regulation of the circadian oscillator by sugars. We will determine the molecular basis of the pathway by comparing transcriptome data with CHIPseq to identify those genes regulated directly by bZIP63 to those that are downstream targets. We will investigate how the regulation of bZIP63 occurs to bring about changes in circadian function through studies of phosphorylation, binding of bZIP63 to DNA and bZIP63-protein binding. Lastly, we will determine the nature of the physiological and metabolic outputs that are controlled by bZIP63-dependent regulation of the circadian oscillator. An understanding of circadian function in plants requires that this new mode of regulation we have discovered is investigated. We believe that our findings have significance for understanding circadian timing, the mechanisms of sugar sensing within plant cells and the optimal control of growth and carbon metabolism. We will share our findings with our industrial partners with the long-term goal of crop improvement.

IMPACT STATEMENT
Understanding plant growth and biomass accumulation is crucial to maintaining improvements in crop productivity, particularly as climate changes. bZIP transcription factors are central regulators of metabolism. We will determine how bZIPs regulate the circadian clock to ensures that biological events occur at the correct time of day and to balance carbons within the plant to optimise growth. Our work will be performed in the model plant Arabidopsis because the goals of the research can only be achieved with access to the genetic resources of this organism. Our approach considers a network that might apply also to the major crops. The data from this study will be fed in to other studies in the Webb lab that are considering the role of the circadian oscillator in wheat. Our project therefore has potential impact for the agrifood/plant breeding sector, as well as providing great opportunities for public engagement.

AGRIFOOD IMPACT
We anticipate that there will be many agricultural stakeholders interested in the work. Circadian rhythms regulate the interactions of the plant with both the abiotic and biotic environment. As climate changes, the security of many food crops is likely to be compromised, and strategies to maintain food supply will require objective scientific evidence about the relationships between plants and their environment, along with an understanding of how genetic pathways control development of relevant plant traits. Commercial ventures interested in maximising agricultural output are obvious partners
in maximising impact of this work as are crop breeders.
We will disseminate data to farmers and breeders, and increase our engagement with stakeholders, through activities at the University and at the National Institute for Agricultural Botany (NIAB). We will target outreach to these stakeholders through AARW's current contacts with Bayer Crop Science and a number of plant breeders, and through the University's "Enterprise Tuesdays", where research can be presented informally to interested industrial and agricultural partners.
Results will be demonstrated at NIAB Innovation Farm Open Days and Symposia.
IMPACT THROUGH PUBLIC ENGAGEMENT
Other users who will be interested in this work include the general public, who we know, through previous engagement activities, are fascinated to learn about the intricacies of plant biology. The recent Novel Prize for Medicine recognising members of the circadian research community has raised the public profile of the study of circadian rhythms in plants. Public engagement about natural systems is nationally and internationally becoming increasingly important, as public concerns, fuelled by constant media coverage, are an important factor in promoting dialogue at all governmental policy levels concerning our treatment and management of environmental and agricultural systems.
We will present our project at public engagement events such as in-house University events tied with National Science Week (annually in March), Cambridge Festival of Plants (annually in May) and the Cambridge Festival of Ideas (annually in October). In addition, we will work with the Horticulture, Education and Interpretation staff at Cambridge University Botanic Garden to a trail through the Garden, leading visitors to a variety of plants with particular developmental, metabolic or life cycle traits associated with circadian rhythms. The Cambridge University Botanic Garden hosts 250,000 casual visitors per year, plus 12,000 schoolchildren on arranged visits, so this display will reach a large and varied audience.
Research findings will be made freely available on this site after their assessment by Cambridge Enterprise for potential IP issues.