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Abstract

The early morning is an important time period in which light and temperature signals entrain the plant circadian clock and trigger changes in a variety of developmental and physiological processes such as elongation growth and stress responsiveness. Through a high-resolution RNA-seq time series experiment using Arabidopsis seedlings, we demonstrate that there are multiple coordinated bursts of gene expression of transcription factors within the first two hours of light exposure that are both light- and temperature-sensitive.  We find that prr5 prr7 prr9 and phyA phyB cry1 cry2 mutants both show an overall delay in the expression of morning genes and an elevated expression of genes that are usually expressed at high temperatures.  In particular, we find that that phytochromes and cryptochromes induce expression of  the photomorphogenesis-promoting bZIP transcription factors HY5 and HYH, as well as multiple BBX transcription factors that are known to interfere with HY5-mediated light responses. It appears that photoreceptors orchestrate multiple transcriptional cascades to tightly control gene expression at the control gene expression at dawn, which suggests that the dawn burst plays an important role in fine-tuning photomorphogenic responses in response to light.  
The abstract should be fewer than 150 words and should not contain subheadings. It should provide a clear, measured, and concise summary of the work. If the biological system (species names or broader taxonomic groups if appropriate) is not mentioned in the title, it must be included in the abstract. (Note: 150 is very short!  The abstract below is already 107!)

Introduction

In the early morning, plants need to adapt their transcriptional programmes to respond to biotic and abiotic stresses that primarily occur in the daytime. Many researchers have observed bursts of gene expression shortly after dawn in drought-response genes \cite{Grundy_2015}, temperature response genes such as heat shock factor 70 (HSP70) \cite{Dickinson2018}, anthocyanin biosynthesis genes \cite{Seaton2018} and phytohormone genes \cite{Michael2008}.  In the latter case, \citealt{Michael2008} observed that dawn expressed phytohormone genes had a G-box motif in their promoters, and indeed there is a large set of genes with G-box promoter motifs that are expressed within one hour of dawn including many that are involved in response to metals \cite{Ezer2017a}.  Moreover, Arabidopsis is less susceptible to certain fungal pathogens in the morning, linked to jasmonic acid signalling \cite{Ingle2015}.  
For these responses to occur at the appropriate time of day, it is important to have well-calibrated diurnal gene expression cycles.  Dawn (and dusk) are critical periods for entrainment of the circadian clock, in response to changes in light  \cite{Kinmonth_Schultz_2013,Covington_2001,Edwards_2010,Seo_2014}, temperature \cite{Michael_2003,Gould_2006,McClung_2010,Salome_2010,Mizuno_2014} and even humidity \cite{Mwimba_2018}.   The evidence that these transition periods are important for entrainment comes from experiments that show that light or temperature pulses in different times of day cause phase shifts in the circadian clock \cite{Covington_2001,Michael_2003}.   However, dawn and dusk transitions are not exclusively responsible for entrainment-- photosynthesis and sugar production can also gate the circadian clock, producing a second 'metabolic dawn' \cite{Haydon_2013}.  
There are several proposed mechanisms for circadian entrainment by light and temperature.  Red- and blue-light sensors -- including phytochrome A (phyA), phyB and cryptochrome 1 (cry1) -- are involved in entrainment \cite{Hall_2002,Salomé2002,Devlin2000,Somers1998}. In addition, members of the ZEITLUPE (ZTL) family, including ZTL, FLAVIN-BINDING KELCH F-BOX 1 (FKF1) and LOV‐KELCH PROTEIN 2 (LKP2) all have clock-associated functions \cite{Somers2000,Nelson2000,Schultz2001,Somers2004,Kim2007,Baudry2010}.    While phyB has also been implicated as a temperature sensor \cite{Legris2016,Jung2016} and interacts with the Evening Complex (EC) that is part of the evening loop of the circadian clock \cite{Ezer2017,Huang2016}, it is unclear whether phyB plays a role in temperature entrainment of the circadian clock.  However, there is evidence that HEAT SHOCK PROTEIN 90 (HSP90) may be responsible for temperature entrainment \cite{Davis2018}.  
NEW PARAGRAPH, NOT REFERENCED YET:  Intriguingly, many of the genes that are involved in entraining the circadian clock are also key genes in photoperiodism and photomorphogenesis, including the phytochromes/cryptochromes and ZTL family members \cite{Jackson2009,Kami2010,Franklin2005}. phyA in particular has been reported as a key sensor of dawn and photoperiod \cite{Seaton2018}.  It makes sense that there should be a mechanistic link between photoperiod-dependent processes and circadian entrainment, since both depend on day length.   However, it is unclear how entrainment genes (such as phytochromes and cryptochromes) relay information to photomorphogenesis pathways and the circadian clock.  
Although there have been a number of studies that demonstrate that there is a burst of gene expression after light stimulus in the morning, the dynamics of this burst have not been fully characterised because time points were not sampled frequently enough.  Through a high resolution RNA-seq time series, we find five distinct transcriptional waves within the first two hours of the morning.  We characterise how each wave of expression responds to temperature elevation and light signals during the night and subjective day, and how these waves are affected by a light signalling mutant (phyA phyB cry1 cry2), a circadian clock mutant (prr5 prr7 prr9), and a temperature response mutant (hsfa1QK).  Furthermore, we infer a gene regulatory network and validate edges using DNA binding data.  We find that HY5 and BBX31 are among the TFs that are predicted to regulate multiple expression waves.  Phytochromes and cryptochromes are required for a burst of expression of HY5 and BBX family proteins that fine-tune hypocotyl elongation and photomorphogenesis, suggesting that the dawn bursts may play a role in time-of-day dependent growth response to light stimulus.  This work provides unprecedented detail as to how light, temperature, and circadian genes are regulated at dawn, as well as providing evidence of a mechanistic link between morning entrainment and photomorphogenesis.

Results

There is a burst of gene expression of DNA binding proteins at dawn

While previous research found groups of genes that had peak expression at dawn, we wondered whether the dawn burst was a more widespread phenomenon and whether regulatory proteins also exhibited a peak in expression (Table S1).  
Overall, 39% of DNA binding proteins have peak gene expression immediately before or an hour after dawn (Figure S1).  We find that there is a significant enrichment for ABA and ethylene-linked DNA binding proteins that have maximal expression in this time period compared to other DNA binding proteins (58% and 55%, p<0.005 in both cases using a Fisher Exact test with Bonferroni correction), which is consistent with the observations in Michael et al, 2008 (Figure 1Ai,ii).  There is also an enrichment for DNA-binding proteins that are associated with GO terms related to light (53%, p<0.02) and stress (55%, p<0.002)-- see Figure 1Aiii,iv.  However, we found no significant enrichment for auxin-associated DNA binding proteins or circadian clock genes (Figure S1).  
Consistent with the role of phytochromes in regulating the dawn peak \cite{Michael2008}, we observe that DNA binding factors that have peak expression immediately before dawn have increased expression in a  phyABCDE quintuple mutant both before and after dawn (Figure 1B).  In contrast, genes that have peak expression in the hour after dawn do not have perturbed expression in phyABCDE.  This suggests that phytochromes may play a more important role in inhibiting nighttime genes than activating morning genes.  We observe the same trends in elf3-1, which is consistent with evidence that phytochromes interact with ELF3 \cite{Ezer2017} (Figure S2).