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Plants balance water availability with gas exchange and photosynthesis by controlling stomatal aperture. This control is regulated in part by the circadian clock, but it remains unclear how signalling pathways of daily rhythms are integrated into stress responses. The serine/threonine protein kinase OPEN STOMATA 1 (OST1) contributes to the regulation of stomatal closure via activation of S-type anion channels. OST1 also mediates gene regulation in response to ABA/drought stress. We show that ZEITLUPE (ZTL), a blue light photoreceptor and clock component, also regulates ABA-induced stomatal closure in Arabidopsis thaliana, establishing a link between clock and ABA-signalling pathways. ZTL sustains expression of OST1 and ABA-signalling genes. Stomatal closure in response to ABA is reduced in ztl mutants, which maintain wider stomatal apertures and show higher rates of gas exchange and water loss than wild-type plants. Detached rosette leaf assays revealed a stronger water loss phenotype in ztl-3, ost1-3 double mutants, indicating that ZTL and OST1 contributed synergistically to the control of stomatal aperture. Experimental studies of Populus sp., revealed that ZTL regulated the circadian clock and stomata, indicating ZTL function was similar in these trees and Arabidopsis. PSEUDO-RESPONSE REGULATOR 5 (PRR5), a known target of ZTL, affects ABA-induced responses, including stomatal regulation. Like ZTL, PRR5 interacted physically with OST1 and contributed to the integration of ABA responses with circadian clock signalling. This suggests a novel mechanism whereby the PRR proteins—which are expressed from dawn to dusk—interact with OST1 to mediate ABA-dependent plant responses to reduce water loss in time of stress.

Tree growth is central to terrestrial ecology and the forestry industry. The overproduction by biotechnological means of hormones such as gibberellins (GAs) has been used as a powerful toolto greatly increase tree yield and wood properties. However, for trees in temperate and boreal regions, overexpressing GAs prevents the ability to induce vegetative dormancy, and results in reduced yield and tree loss over time. Since Populus trees are using an internal 24-h (circadian) clock to synchronize their metabolism and growth with local, predictable changes in the external environment, we focused on circadian control of GA metabolism, to showcase the principle of seasonal growth adaptation. To obtain both maintained growth benefits and a seasonally timed growth, we set out to modulate levels of bioactive GAs by using the endogenous Populus tremula× P. tremuloides CycD3 promoter. We show that both high yield and biotechnical seasonal growth adaptation is obtained with this promoter, which is coordinated by the clock protein LATE ELONGATED HYPOCOTYL 2 (PttLHY2). This work paves the way for future precision breeding of trees with local adaptation and increased yield. 

Due to their sessile nature, plants must precisely time and coordinate their physiological processes with daily and seasonal changes in the environment. In this PhD thesis, I explored the interaction between an internal timekeeper, or circadian clock, and hormonal regulation, and how it may control plant growth and adaptation in the model species Arabidopsis thaliana (Arabidopsis) and hybrid aspen (Populus tremula x P. tremuloides). 

Our research showed how the circadian clock component ZEITLUPE (ZTL) regulates abscisic acid (ABA)-mediated stomatal closure, indicating its central role in environmental adaptation.  

We investigated the effects of manipulating gibberellin (GA) metabolism in hybrid aspen by the strategic expression of the Arabidopsis GIBBERELLIN20-OXIDASE1 gene, using a clock-controlled promoter. This approach struck a delicate balance between enhanced growth and seasonal adaptation and showed potential for increased tree performance through biotechnological means.

Our studies on the circadian clock components LATE ELONGATED HYPOCOTYL (LHY) and EARLY BIRD (EBI) in hybrid aspen suggested their involvement in regulating GA metabolism and overall plant growth.

Taken together, these findings improve our understanding of how plants regulate growth and respond to environmental stresses and help to provide solutions for enhancing plant resilience and productivity.

This study investigates the role of the circadian clock in the regulation of gibberellin (GA) metabolism and growth in hybrid aspen (Populus tremula x P. tremuloides (Ptt)). We revealed a conserved function of the clock homolog PttEARLY BIRD1 (PttEBI1), but also implicated its rolein controlling tree growth. GA metabolite profiling and transcriptomic analysis in hybrid aspenlines with modulated expression of PttEBI1 or the core clock homologs PttLATE ELONGATED HYPOCOTYLs (PttLHYs) revealed significant changes in GA metabolism. These alterations werelinked to the differential expression of PttGA2ox8, a gene encoding an enzyme with both GA2-oxidase and GA20-oxidase activities. Our results indicate that modifications to circadian clockcomponents can significantly influence both GA metabolism and tree growth, providing potential strategies for improving tree biomass production.