Antiphase light and temperature cycles disrupt rhythmic plant growth : the Arabidopsis jetlag

R.M.E.H. Bours

Research output: Thesisinternal PhD, WU



Light and temperature are important determinants of plant growth and development. Plant elongation is stimulated by positively increasing differences between day and night temperature (+DIF, phased cycles). In contrast, a negative temperature difference (-DIF, antiphased cycles) reduces elongation growth. In chapter 1the different responses of plants to light and temperature are described. We focus on how light and temperature are perceived and integrated with physiological and molecular pathways to control plant development and architecture. As both light and temperature converge at the circadian oscillator attention is given to temperature entrainment and temperature compensation of the Arabidopsis circadian clock. Finally we discuss the importance of temperature effects on plant growth for horticulture. -DIF is frequently applied in commercial greenhouses to inhibit unwanted elongation of crops. Despite the economic importance, the response of plants to -DIF was poorly understood. Using Arabidopsis thaliana,our research aimed to understand the mechanisms underlying the -DIF response. The main questions that needed to be answered at the start of this project were:


(1) At what time during the diurnal day is growth affected by -DIF?

(2) What are key genes in the diurnal signalling pathways that result in reduced growth under -DIF?

(3) Is the temporal effect of -DIF on growth linked to the circadian clock, and if so how?


To answer question 1 (when is growth affected by -DIF?) it was important to develop a monitoring system through which growth could be analysed over the full day, including the dark period. This would allow us to determine how growth proceeds over the day and whether there is a specific period of the day at which growth is most affected by the -DIF regime. Elongation in plants is not constant throughout the day, but exhibits a diurnal rhythm. However, the effect of treatments on growth is usually scored as a cumulative effect after many days. Thus the precise relationship between environmental changes and the daily cycles in the growth of the plant remain mostly unnoticed. More detailed analysis can reveal whether the window of growth or the growth rate itself is affected by the environmental conditions. For this purpose, OSCILLATOR, a growth monitoring system, which allows the analysis and parameterisation of diurnal growth of rosette plants was constructed. The demonstration and validation of OSCILLATOR as growth monitoring system is described in chapter 2. The system consists of IR sensitive cameras and allows time-lapse imaging and subsequent analysis of leaf growth and leaf movement of Arabidopsis, tomato and petunia. We use this system to examine how fluctuating diurnal temperature cycles affect leaf movement in different Arabidopsis ecotypes, demonstrating that this approach allows comparison of various genotypes through parameterisation of rhythmic growth. The analysis by OSCILLATOR showed that diurnal growth is accompanied by a cyclic movement of the growing leaves, and parameters (phase and amplitude) of this diurnal leaf movement can be used as a proxy for growth rate. This facilitated the characterisation of the effect of -DIF on growth.

To answer question 2 (what are key genes affected by -DIF) we tested many different mutants impaired in either light signalling, hormone perception, or hormone biosynthesis and studied their response to -DIF in comparison with wild-type plants. Chapter 3 describes how, using this approach, we unravel the light and hormonal signalling processes that mediate the effect of -DIF on leaf movement. Pharmacological treatments combined with the genetic screens identify ethylene signalling as limiting for leaf growth and movement under -DIF. We demonstrate that specifically the activity of the ethylene biosynthesis gene ACC synthase 2activity in the petiole relates to the -DIF leaf phenotype. In addition, the effect of -DIF on ethylene sensitivity and biosynthesis is shown to depend on active PHYB.

To further characterise how light and hormone signalling affect growth under -DIF, we set out to identify factors limiting cell elongation. In chapter 4, local cell elongation in the hypocotyl is linked to local auxin signalling capacity. We demonstrate that ethylene, similar to its role in rosette leaves, becomes limiting in this tissue under -DIF as a result of reduced auxin production. While previously overall auxin was shown to be reduced in Arabidopsis inflorescence tissue developed under -DIF, we now demonstrate that it is mainly the effect of tissue specific auxin signalling that limits growth under -DIF. Moreover, we show that auxin can complement growth inhibition under -DIF in wild-type plants but not in ethylene signalling or biosynthesis mutants, placing the effect of auxin on growth upstream of ethylene. Downstream, ethylene signalling activates the growth promoting transcription factor PIF3, which is known to activate genes controlling cell elongation. In contrast, PIF5 acts upstream, possibly regulating the input of the signalling cascade. Remarkably, PIF4, which is a main regulator of heat induced hypocotyl elongation, is not required for the response to -DIF.

To answer question 3 (does -DIF affect the clock?) we used luciferase reporter plants and developed a unique luminometer set-up with which we could monitor gene promoter activity in mature rosette plants under different diurnal light regimes. This system was used in penultimate chapter 5 where we demonstrate that an altered function of the circadian clock under -DIF is responsible for altered output processes identified in the other chapters. Analysis of expression patterns of core clock genes under diurnal conditions reveals that -DIF reduces the amplitude of most clock genes and differentially shifts the phase of core clock components. The magnitude and direction of these shifts differ for each clock gene, suggesting that -DIF alters the coordination within the circadian clock itself. We subsequently showed that the phase shifts occurring under -DIF relate to a temperature compensation mechanism controlled by GI. GIwas previously identified to be required for temperature compensation in the amplitude of clock controlled genes at low and high temperature. Moreover, GIwas identified to be responsible for the effect of -DIF on the phase of clock genes. Indeed, gi loss-of-function mutants are insensitive to the effects of -DIF on growth. We demonstrate that under –DIF starch biosynthesis during the day, and starch degradation rates at night are altered. Carbohydrate availability during the night is essential for growth and therefore part of the sugars generated during the photoperiod are stored as starch. Throughout the night this starch is degraded in a controlled rate, which is adjusted to the predicted length of the dark period. The starch degradation rate under different photoperiod lengths is therefore tightly controlled by the circadian clock in anticipation of the expected dawn, to prevent running out of carbohydrates at the end of the night. Indeed, under -DIF starch metabolism is disturbed, resulting in an apparent starch shortage at the end of the night. This was monitored by activation of a reporter gene for carbohydrate starvation under -DIF. Furthermore, the phase of leaf movement of starch mutants under control (+DIF) conditions resembles the phase of wild-type plants developing under -DIF, indicating that the carbohydrate status of a plants determines rhythmic leaf movement.

In chapter 6the results obtained in this thesis are discussed and a conceptual model that aims to integrate all findings with recently published literature is proposed. In this model, -DIF affects growth by directly affecting the phase and amplitude of clock genes, which in turn control downstream processes such as starch metabolism and hormone signalling pathways. The auxin and ethylene signalling pathways affected by -DIF show significant crosstalk and interconnect with the circadian clock at several positions, by direct interaction with the PIFs, which are regulated by PHYB, of which transcription is under circadian control. Therefore, special focus is given to the unique position of the photoreceptor PHYB in this model. PHYB is essential for PIF protein stability and in addition is an important component for light entrainment of the clock. Finally we discuss the potential applications of the results described for horticulture and speculate on possible ways to improve the efficiency of DIF like treatments.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Bouwmeester, Harro, Promotor
  • van der Krol, Sander, Co-promotor
Award date2 Jun 2014
Place of PublicationWageningen
Print ISBNs9789461739575
Publication statusPublished - 2014


  • arabidopsis
  • growth
  • light
  • temperature
  • biological rhythms
  • plant development
  • models

Fingerprint Dive into the research topics of 'Antiphase light and temperature cycles disrupt rhythmic plant growth : the Arabidopsis jetlag'. Together they form a unique fingerprint.

  • Projects

    Cite this