Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology

Abstract Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

Two experts in the field have reviewed your manuscript, and I have read it as well.The editor and reviewers were enthusiastic about your approach linking mathematical models of circadian pathways with molecular genetic tests of hypotheses.While your manuscript is not currently acceptable for publication in GENETICS, we would welcome a substantially revised manuscript.Both reviewers have comments and concerns to be addressed in a revised manuscript.You can read their reviews at the end of this email.
The motivation for the current study stems from model predictions on clock regulatory dynamics and plant growth and development.The literature in the field is quite advanced, with a number of existing models and model predictions.Moreover, there are many empirical studies of mutants including studies of epistatic interactions among key elements.This study focuses specifically on ELF3 regulation of PRR genes.One limitation of the study is that it lacks clear motivation or justification for use of the C2016 model (amongst other possibilities), there is little discussion of its limitations, and some relevant citations are missing.Reviewer 2 argues that a potentially more realistic model (U2019.3or U2020.3)would be more appropriate for generating hypotheses and for comparison to the empirical work presented.The reviewer argues that these models would be more powerful compared to the predictions of the "compact model".The manuscript would be strengthened if these issues can be addressed.Both reviewers had questions about the statistical results and their interpretation.It would be helpful to include more detail about the statistical tests performed, including adding p-values in text and figures when appropriate.Be careful that the interpretation and conclusions are consistent with the evidence.Finally, the reviewers suggest a number of additional experiments that could improve the experiment, including new gene expression studies or manipulation of TOC1.While the editor agrees that these experiments would strengthen the paper, adding new empirical work to the existing study is not a requirement for consideration of a resubmission.
Upon resubmission, please include: 1.A clean version of your manuscript; 2. A marked version of your manuscript in which you highlight significant revisions carried out in response to the major points raised by the editor/reviewers (track changes is acceptable if preferred); 3. A detailed response to the editor's/reviewers' feedback and to the concerns listed above.Please reference line numbers in this response to aid the editor and reviewers.
Your paper will likely be sent back out for review.
Additionally, please ensure that your resubmission is formatted for GENETICS https://academic.oup.com/genetics/pages/general-instructionsFollow this link to submit the revised manuscript: Link Not Available Sincerely, Tom Juenger Series Editor, Plant Genetics & Genomics Approved by: David Greenstein Senior Editor GENETICS --------------------------------------------------------------------Reviewer #1 (Comments for the Authors (Required)): In their manuscript entitled "Complex Epistatic Interactions between ELF3, PRR9, and PRR7 Regulate the Circadian Clock and Plant Physiology", Yuan et al. investigate the modeling and genetic interactions among three clock genes, ELF3, PRR9, and PRR7, in Arabidopsis to test the regulatory dynamics in the circadian clock.The authors simulated the impact of the prr9/prr7 mutation on the elf3 circadian phenotype.They also investigated epistatic interactions among three clock genes using single, double, and triple mutants across different growth and development processes.This is a well-designed manuscript with a clear hypothesis.And the entire manuscript is globally easy to follow.The results of the study are not entirely unique.The mathematical model for these core circadian clock genes has previously been explored, as referenced by the authors (De Caluwé et al., 2016).Furthermore, the genetic interactions among these three genes in similar phenotypes have also been reported (though not included in the reference; please refer to the below sources).However, the current study aims to separate the roles of the PRR9 and PRR7 genes within the model and elucidate the complex epistatic interactions involving all three genes across diverse phenotypic outcomes.My comments on the manuscript are listed below.It is a bit challenging to review this manuscript since there are no line numbers in the merged file, but I will do my best to make it clear.
1) The ELF3, PRR9, and PRR7 are well-studied genes.The manuscript should include a comparative analysis, discussion, and appropriate referencing from prior literature.A few examples that are closely related to this manuscript, but are not found in the reference, are provided below: PSEUDO-RESPONSE REGULATORS, PRR9, PRR7, and PRR5, Together Play Essential Roles Close to the Circadian Clock of Arabidopsis thaliana.Plant and Cell Physiology, 2005 (https://doi.org/10.1093/pcp/pci086)In this paper, the authors used single, double, and triple PRR mutants to investigate circadian-associated phenotypes, including photoperiodic flowering time and the length of hypocotyls.Ambient temperature response establishes ELF3 as a required component of the core Arabidopsis circadian clock.PNAS, 2010 (https://doi.org/10.1073/pnas.0911006107)In this paper, the authors investigated the expression change of PRR9/7 in the elf3 mutant background in response to ambient temperature.
2) There are a lot of pairwise comparisons of a series of phenotypes between mutants and the WT in the results (Figures 3, 4,  and 5).For example, on Page 8, the author mentioned that "the elf3 mutant had a long hypocotyl phenotype under these conditions compared to WT Col-0 (Figure 3A)"; on Page 9, the author mentioned that "Root development was strongly impaired in the elf3, prr9, and prr7 single mutants at 20{degree sign}C compared to WT, with each respective single mutant having a similar response to each other (Figure 4A).There was no change in the primary root length of prr9/prr7 double and elf3/prr double combinations compared to the respective single mutants.However, root development was further impaired in the elf3/prr9/prr7 triple mutant compared to the single and double mutants (Figure 4A)."...However, the author did not provide the statistical results for those comparisons.Please include p-values throughout Figures 3, 4, and 5 for those comparisons.Additionally, please clarify the statistical methods used in those comparisons in the Methods section (Page 17).RStudio and/or R are not the methods for the statistical analysis.
3) As the authors mentioned on page 9, those circadian genes, such as ELF3 and PRR9/7, may regulate root development at ambient temperatures.I am curious about the expression levels of ELF3, PRR9, and PRR7 genes across different tissues.Incorporating supplementary data of tissue-specific expression profiles from online databases like the Bio Analytic Resource/eFP could provide valuable information in this regard.4) On page 10, the authors examined the number of rosette leaves and days to flower in WT plants and mutants.I recommend that the authors examine the expression levels of florigen and downstream flowering time genes, including FT, TSF, and SOC1, in those lines.This may help readers in comprehending the link between upstream clock genes and reproductive phenotypes.5) I'm not an expert in modeling, yet I have an open question to discuss.As the authors mentioned the phenotype of root development in those mutants, did they include tissue as a factor in their modeling?Or, in other words, do the authors think the pattern of circadian rhythm in above-ground (seedlings or leaves) is the same as that in below-ground (roots)? 6) Page 9, "PRR9, but not ELF3, regulates regulate root development under warm temperatures."Typing issue?Reviewer #2 (Comments for the Authors (Required)): Comments to the authors: In this manuscript by Yuan L et al., the authors use a compact mathematical model of the clock (De Caluwe et al., 2016) to shed light on the functional significance of the ELF3 regulation of PRR9 and PRR7.They particularly focused on how this regulation affects circadian clock dynamics and plant development.To tackle this question they first model the effects of a prr9/prr7 mutation on an elf3 mutant background.The model they employed suggested that this would rescue the circadian arrhythmicity of elf3.They then generated an Arabidopsis triple mutant to validate the findings of the model and further tested their circadian and development phenotypes.Their data shows that elf3 is needed for hypocotyl growth under both, ambient and warm temperatures, while prr9 is critical for root thermomorphogenesis.Furthermore, the authors conclude that the prr9 or prr7 mutations could rescue the photoperiod insensitive early flowering of elf3.

Major concern:
The authors used the C2016 compact model of the Arabidopsis clock and then split the variables of prr7 and prr9.They performed simulations to model the effects of a prr9/prr7 mutation on an elf3 mutant background and found that this would rescue the circadian arrhythmicity of elf3.However, they do not observe this experimentally.The concern here is the model of choice.For example, running a simulation with either one of the two most realistic models that work with absolute units: U2019.3 and U2020.3 (Urquiza and Millar 2020) results in arrhythmicity (see attached images).In particular, in 2019.3 which has a better network architecture than U2020.3, the levels of TOC1 mRNA are around 2x higher than WT.Moreover, the predicted levels of CCA1/LHY are lower.This could result in two effects: 1) on one hand reduction of PIF activity mediated by TOC1, as it has been shown that TOC1 can suppress PIF3; 2) the reduction in the levels of CCA1/LHY can result in acceleration of flowering.Also, TOC1 has been implicated in root development (https://doi.org/10.1038/ncomms8641)Using a mathematical model in absolute units would provide more powerful predictive power/insights than the compact model of C2016.C2016 lacks this capacity as the scale is compressed to the interval [0,1] and therefore lacks the ability to propose the effect of changing RNA levels.The U2019.3 model suggests that the effect on hypocotyl length reduction in the triple mutant could potentially be attributed to high levels of TOC1 in combination with low levels of CCA1/LHY.This hypothesis would then need to be further verified by the authors.I would suggest abandoning the C2016 model and switching to the U2019.3.Then, verifying the predictions of high levels of TOC1, and introducing a TOC1 mutation (by CRISPR, for example) into the triple prr9/prr7/elf3 mutant to remove the suppression of hypocotyl and observe a delay in flowering.
Other concerns: Results section: In the results section you make the following statement: "Both the prr9 and prr7 mutations were able to partially rescue the elf3 early flowering phenotype under SD and, as with LD, the prr7 mutation had a stronger effect on rescuing the elf3 phenotype than prr9 (Figure 5)."However, Fig. 5A (LD -rosette leaves): the double elf3/prr7 and elf3/prr9 mutants are statistically the same as the single elf3 mutant.Fig. 5B (LD -days to flowering): the double elf3/prr9 mutants are statistically the same as the single elf3 mutant.Here, only the double elf3/prr7 is similar to wild type.Fig. 5C (SD -rosette leaves): the double elf3/prr9 mutants are statistically the same as the single elf3 mutant.The double elf3/prr7 is different to both elf3 and wild type.The triple mutant does not fully rescue the phenotype.Fig. 5D (SD -days to flowering): the double elf3/prr7 and elf3/prr9 mutants partially rescue the flowering phenotype of the elf3 mutant.Please revise your statement accordingly to reflect the data more accurately.Or let the readers know that you specifically refer to the 'days-to-flowering' data.
Methods section: What is the light intensity of constant red and blue light you used for the TopCount experiments?Please add this to the 'Luciferase circadian experiments' subsection.