A mobile ELF4 delivers circadian temperature information from shoots to roots

The circadian clock is synchronized by environmental cues, mostly by light and temperature. Elucidating how the plant circadian clock responds to temperature oscillations is crucial to understand plant responsiveness to the environment. Here we found a prevalent temperature-dependent function of the Arabidopsis clock component ELF4 (EARLY FLOWERING 4) in the root clock. Micrografting assays and mathematical analyses show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information but trafficking is essential to control the period of the root clock in a temperature-dependent manner. At low temperatures, ELF4 mobility is favored and results in a slow-paced root clock while high temperatures decrease movement, leading to a fast clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.


Introduction
Nearly all photosensitive organisms have evolved timing mechanisms or circadian clocks able to synchronize metabolism, physiology and development in anticipation to the 24-hour light/dark cycles 1 .
In Arabidopsis thaliana, the molecular clockwork is based on complex regulatory networks of core clock components that generate rhythms in a myriad of biological outputs 2,3 . Appropriate phasing of biological processes relies on clock resetting by light and temperature cues; a mechanism that requires effective changes in the expression and activity of essential clock components 4 . Circadian clocks are also defined by a conserved feature known as temperature compensation 5 . In contrast to the temperature dependency of many physicochemical and biological activities, the circadian clock is able to maintain a constant period over a range of physiological temperatures. By virtue of different transcriptional, posttranscriptional and post-translational mechanisms [6][7][8][9] , the plant circadian system buffers the circadian period length. Therefore, the circadian clock does not run faster at higher than at lower temperatures, sustaining a period close to 24-hours within a physiological range of temperatures. An ample collection of light-related factors [10][11][12][13][14] and clock-associated components 9,15,16 has been shown to directly or indirectly regulate clock temperature compensation in plants.
Among the Arabidopsis clock components, ELF4 (EARLY FLOWERING 4) was initially identified by its role in photoperiod perception and circadian regulation 17 . ELF4 protein assembles into a tripartite complex (Evening Complex, EC) together with ELF3 and LUX ARRHYTHMO or PHYTOCLOCK1 (LUX/PCL1) 18,19 to regulate growth and repress circadian gene expression 20,21 . ELF4 promotes the nuclear localization of ELF3 18 while LUX directly binds to the promoters of the target genes and thus facilitates the recruitment of ELF4 and ELF3 19,22 . Loss-of-function mutants of any of the EC components lead to arrhythmia 17,[23][24][25] . Through multiple interactions with light, clock and photomorphogenesis related factors 26 , the EC is able to coordinate plant responses to environmental cues including temperature 15,[26][27][28][29][30] .
Regarding the circadian structure and organization within the plant, it is broadly accepted that every plant cell harbors a circadian oscillator. However, circadian communication or coupling among cells and tissues varies at different parts of the plant [31][32][33][34][35] . For instance, while cotyledon cells present circadian autonomy 36 , different degrees of cell-to-cell coupling have been reported in leaves 37,38 , in the vasculature with neighbor mesophyll cells 39 , in guard cells 40 , in cells at the root tip 41,42 and within the shoot apex 43 . Long-distance shoot-to-root photosynthetic signaling is also important for clock entrainment in roots 44 while light piping down the root 45 contributes to this entrainment. Micrografting assays and shoot excision 43 suggest the existence of a long-distance mobile circadian signal from shoots to roots. Here we report that ELF4 moves from shoots to control the pace of the root clock in a temperature-dependent manner.

Prevalent function of ELF4 sustaining rhythms in roots
We first approached the investigation of the circadian mobile signal by simultaneously following rhythms in shoots and roots of intact plants 43 . The waveforms of the morning-expressed CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL) promoter activities displayed a long period, slightly reduced amplitude and phase delay in roots (Rt) compared to shoots (Sh) (Fig. 1a-b and Supplementary Fig. 1a). The mRNA rhythmic accumulation assayed by Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-QPCR) followed the same trend  Fig. 1b-c). Therefore, the clock is fully operative in roots but its overall pace is slower and the phase delayed compared to shoots.
The circadian clock is unable to properly run in mutant plants of any of the EC components 17,[23][24][25] . We therefore examined the role of the EC components in the root clock, and in particular, we focused on ELF4. Circadian time course analyses in WT and elf4-1 mutant roots showed the fully suppressed CCA1 and LHY promoter activities and mRNA expression in elf4-1 ( Fig. 1d and Supplementary Fig.   1d-f) in a similar way to that described in shoots 21 (Supplementary Fig. 1g-i). Conversely, overexpression of ELF4 (ELF4-ox) lengthened the period of LHY::LUC (Fig. 1e) indicating that increased ELF4 activity in roots makes the clock to run slow. The expression of PRR9 (PSEUDO-RESPONSE REGULATOR 9), a previously described direct target of the EC in shoots, was clearly up-regulated in elf4-1 mutant roots (Fig. 1f) suggesting that the EC also represses PRR9 in roots. Thus, ELF4 plays an important regulatory function in the root clock: mutation abolishes rhythms while over-expression lengthens the circadian period.

ELF4 moves from shoots to regulate oscillator gene expression in roots
Our previous study showed that a signal from shoots is important for circadian rhythms in roots 43 .
Micrografting assays are a powerful tool to identify the nature of mobile signals. The grafting technique per se does not alter the rhythms in roots, as grafted WT scions into WT roots show similar rhythms as non-grafted WT plants ( Supplementary Fig. 2a-b) 43 . By micrografting different genotypes, we found that grafts of ELF4-ox shoots into elf4-1 rootstocks [ELF4-ox(Sh)/elf4-1(Rt)] ( Supplementary Fig. 2c) were particularly efficient in recovering the rhythms in roots ( Fig. 2a and Supplementary Fig. 2d-e).
The results are noteworthy as CCA1::LUC rhythms are severely disrupted in elf4-1 mutant roots (Fig.   1d). Restoration of the rhythms was reflecting the circadian function exclusively in roots as water instead of luciferin was applied to shoots (ELF4-ox, Sh, H2O) to exclude the possibility of luminescence signals leaking from shoots into roots of adjacent wells. Rhythms in roots were also recovered when ELF4-ox scion was grafted into elf4-2 mutant ( Supplementary Fig. 2f) rootstocks (Fig.   2b). The influence of shoots as a driving rhythmic force of elf4-1 rootstocks was mathematically analyzed with recurrence plots obtained by delay coordinates of the grafting time series (Fig. 2c-d). The waveforms of the driving rhythmic force reconstructed from the driven system and their autocorrelation analyses showed a strong periodicity after grafting ( Supplementary Fig. 3a-d). In analyses with 10000 randomly shuffled surrogates using as null hypothesis of no serial dependence, we obtained a p-value of 0.2341 (black dash line) (Fig. 2e) before grafting and 0.0004 (gray dash line) after grafting (Fig. 2f).
The statistics suggest that after grafting, roots are being forced by a periodic signal from shoots. To exclude the possibility that the observed results were due to the high over-expression of ELF4-ox plants, we grafted WT shoots into elf4-1 roots. Although the recovery of the rhythms was not as robust as with ELF4-ox grafts, a rhythmic pattern was observed in roots ( Fig.   2h). Thus, ELF4 mRNA or protein are able to move from shoots to roots. This notion was reinforced by the results showing the rhythmic recovery of elf4-1 rootstocks grafted with ELF4 Minigen (E4MG) scion ( Supplementary Fig. 2g-i). To investigate whether the mRNA could be the mobile signal, we performed RT-QPCR time course analyses of roots from ELF4-ox (Sh)/elf4-1(Rt) grafts. Our results showed no detectable amplification of ELF4 mRNA at any time point analyzed (Fig. 2i), which suggest that ELF4 mRNA might not move through the graft junctions. To confirm this notion, we injected purified ELF4 protein into elf4-1 mutant ( Supplementary Fig. 4d-f). Injection in shoots was able to restore rhythms in roots (Fig. 2j). Although the percentage of ELF4-injected plants with recovered rhythms was low (about 5-8%), the fact that rhythms were actually restored is supportive of a mobile ELF4 protein from shoots to roots. Rhythmic recovery was not apparent when purified GFP was injected (Fig. 2j). Other mobile proteins such as FT (FLOWERING LOCUS T), and HY5 (LONG HYPOCOTYL 5) share some features with ELF4 protein in terms of low molecular weight and high isoelectric point (Fig. 2k).

Blocking ELF4 movement by shoot excision alters circadian rhythms in roots
We next attempted to unveil the effects of the mobile ELF4 by blocking ELF4 movement through shoot excision. Analyses of the rhythms showed that excised roots sustained robust oscillations (Supplementary Fig. 5a-b) confirming that the root clock is able to run in the absence of shoots.
However, comparison of intact versus excised roots uncovered a shorter period and advanced phase in excised roots ( Supplementary Fig. 5c-d). As accumulation of ELF4 results in long periods in shoots 21 and roots (Fig. 1e), it is plausible that blocking ELF4 movement by shoot excision leads to shorter periods in excised roots. If that is the case, blocking ELF4 movement should also affect ELF4 target gene expression in excised roots. Time course analyses by RT-QPCR revealed that the expression of PRR9 and PRR7 was up-regulated in excised roots compared to intact roots (Supplementary Fig. 5e-f), which suggest that in the absence of ELF4 movement from shoots, repression of these genes is alleviated in roots. The use of ELF4-ox intact roots confirmed that PRR9 and PRR7 are targets of ELF4 as their expression was clearly down-regulated in intact ELF4-ox roots compared to WT intact roots (Supplementary Fig. 5g-h).

Mobile ELF4 does not regulate the photoperiodic-dependent phase in roots
In aerial tissues, the circadian clock controls the photoperiodic regulation of growth and development 46 .
To determine whether ELF4 movement is important to deliver photoperiodic information, we analyzed rhythms under short day (ShD) and long day (LgD) conditions. In roots, PRR9::LUC waveforms function is not essential to sustain rhythms under entraining conditions. Together, the results suggest that blocking ELF4 movement by excision advances the phase of the root clock but the mobile ELF4 does not directly regulate the photoperiodic-dependent phase shift in roots.
ELF4 movement contributes to the temperature-dependent changes in circadian period of the root clock As the EC also coordinates temperature responses, we examined whether a mobile ELF4 can convey temperature information from shoots to roots. To that end, we first examined the effect of different temperatures (28ºC, 18ºC and 12ºC) on circadian rhythms in roots. We found that LHY::LUC circadian period length was significantly shorter at high than at low temperatures ( Fig. 4a-b). Shortening of period length at increasing temperature was also observed for other circadian reporter lines (Supplementary Fig. 7a-d). Thus, under our conditions, the circadian clock in roots is not able to sustain circadian period length within a range of temperatures. Temperature compensation, one of the defining properties of circadian systems, does not function in the root clock.
As ELF4 accumulation lengthens period length, we next examined the possible contribution of ELF4 to the long period phenotype at low temperatures. Changes in period length could be mediated by increased ELF4 activity and/or by the increased protein movement from shoots to roots. To examine these possibilities, we compared the effects of blocking ELF4 movement by excision and by grafting at low and high temperatures. Essentially, if the long period in roots at 12ºC is independent of movement but results from the increased activity of ELF4, blocking movement from shoots by excision or grafting should not have a major effect on period length. However, if ELF4 movement contributes to the period regulation, abolishing ELF4 traffic should lead to an observable and differential effect on period length at different temperatures.
Our results showed that excision shortened the period length in WT roots and this effect was highly significant at 12ºC as compared to the minor effect at 28ºC ( Fig. 4c and Supplementary Fig. 8a-c).
Grafting elf4-1 scion into WT rootstock resulted in a similar pattern to that of excised WT roots at 12ºC ( Fig. 4c and d). Therefore, blocking ELF4 movement by excision or grafting shortens the long period of WT roots at 12ºC. Analyses of other circadian reporter lines and at 18ºC also showed that excision significantly shortened period length compared to intact roots ( Supplementary Fig. 8d-e). The results suggest a prevalent function of ELF4 movement in the control of period length at lower temperatures.
Interestingly, elf4-1 grafting into WT rootstock did not noticeably affect the rhythms of WT roots at 28ºC ( Supplementary Fig. 8f) consistent with the results of WT excised roots at 28ºC ( Supplementary   Fig. 8a-b). The results suggest that ELF4 movement is controlled by temperature. To further verify this notion, we examined rhythmic recovery in grafts of ELF4-ox scion into elf4-1 rootstock at low and high temperatures and found evident rhythms at 12ºC but not at 28ºC (Fig. 4e-f). Therefore, ELF4 movement contributes to the temperature-dependent control of circadian period in roots.
Altogether, we propose a model by which mobile ELF4 (mbE4) from shoots to roots defines a pool of active ELF4 protein that is competent to repress target circadian gene expression in roots. ELF4 trafficking is favored at low temperatures, which results in a slow-paced clock (Fig. 4g) while high temperatures decrease the movement, leading to a fast root clock (Fig. 4h). The temperature-dependent movement of ELF4 allows a shoot-to-root dialogue that controls the pace of the clock and provides a mechanism by which temperature cues from shoots set the circadian period length in roots.

Discussion
The simultaneous examination of rhythms in shoots and roots of single individual plants shows that the promoter activities and mRNA accumulation of clock genes in roots display a longer period and delayed phase compared to shoots. The trend was observed for morning-and evening-expressed key oscillator genes suggesting that the overall circadian system in roots is not as precise as in other parts of the plant (e.g. the shoot apex) 43 . Despite the long period and delayed phase, the rhythms persist in roots for several days under LL, which is reminiscent of a fully functional clock. The lack of precision might provide circadian flexibility for rapid adjustments and improved responses in roots.
The EC directly represses PRR9 and PRR7 expression 18,22,28,47,48 and indirectly promotes the expression of the morning-expressed oscillator genes CCA1 and LHY [47][48][49][50] . Our analyses with elf4-1 mutant and ELF4-ox plants demonstrate that ELF4 function in roots is also important for proper repression of PRR9 and PRR7 and activation of CCA1 and LHY. ELF4 regulatory function in roots is thus similar to that previously described for the EC using whole plants. Over-expression of ELF4 further delays the phase and lengthens the period of the root clock suggesting that ELF4 slows down the circadian period in roots as in shoots 21 . The fact that accumulation of ELF4 lengthens the period agrees with the results showing that blocking movement by shoot excision or grafting shortens the period while the reduced ELF4 movement at high temperature also leads to shorter periods in roots.
Micrografts of ELF4-ox scion into elf4-1 or elf4-2 rootstocks allow a remarkable recovery of rhythms and the accumulation of ELF4-GFP fluorescent signals in the vasculature of elf4-1 mutant rootstock.
These results suggest that ELF4 moves from shoots to roots, which is in agreement with the assays of ELF4 protein injection in shoots and the subsequent rhythmic recovery in roots. The effects are not due to the over-accumulation of ELF4-ox as micrografts of E4MG and WT plants are also able to recover the rhythms of the elf4-1 mutant roots. Furthermore, our experiments adding water to the scion exclude the possibility that rhythms in grafted roots are due to leakage for the adjacent well containing the shoot. The fact that ELF4 protein shows similar properties in terms of length, molecular weight and isoelectric point to other mobile proteins 52-55 also support our conclusions of an ELF4 movement.
Excision blocks ELF4 movement from shoots and consequently, we observe that oscillator gene expression is affected in WT excised roots. The phase shifts observed following excision prompted us to examine whether ELF4 movement contributed to the photoperiodic-dependent phase shift. However, excised roots still sustained the phase delay under LgD suggesting that other factors are responsible for this regulation. Light piping down the root 45 might be important for this synchronization. Regardless the mechanism, it is able to overcome the miss-expression of ELF4 in shoots and roots as ELF4-ox and elf4-1 mutant plants displayed similar rhythms to WT. Clear alteration of circadian expression under LL but not under entraining conditions has been reported for other clock mutants and over-expressing plants 56 .
As photoperiod does not control ELF4 movement, we focused on temperature. The EC activity is downregulated at high temperatures in whole seedlings 28,30 . Shoot excision and micrografting elf4-1 scion into WT rootstock shortened the period indicating that ELF4 movement is important in the control of circadian period length. Period shortening is more significant at low than at high temperatures confirming that ELF4 movement is favored at low temperatures. As ELF4 accumulation results in long period, the increased movement leads to a clock that runs slower at low than at high temperatures.
Therefore, a mobile ELF4 contributes to the lack of temperature compensation in roots. It would be interesting to elucidate whether period sensitivity to temperature might provide an advantage for optimal root responsiveness to temperature variations.

In vivo luminescence assays
In vivo luminescence assays were performed as previously described 43 . Briefly, 7-15 day-old seedlings synchronized under LD cycles at 22°C were transferred to 96-well plates and released into the different conditions as specific for each experiment. Analyses were performed with a LB960 luminometer (Berthold Technologies) using the Microwin software (Mikrotek Laborsysteme). The period, phase and amplitude were estimated using the Fast Fourier Transform-Non-Linear Least Squares (FFT-NLLS) suite 62 using the Biological Rhythms Analysis Software System (BRASS, http://www.amillar.org). For the simultaneous analysis of rhythms of shoots and roots from the same plant, the connection between the two adjacent wells of the 96-well plates was serrated. Seedlings were then horizontally positioned so the shoot was placed in one well and the roots in the contiguous well. For excision analyses, roots were excised from shoots and placed into the 96-well plates for luminescence analyses. Data from samples that appeared damaged or contaminated were excluded from the analysis. For analyses of grafted samples, water instead of luciferin was applied to the wells containing shoots to avoid possible leaking signals from shoots to roots as specified. At least two biological replicates were performed per experiment. Each biological replicate included 6 to 12 seedlings per condition and/or genotype. protease (Sigma-Aldrich). The purified recombinant proteins were concentrated using Amicon centrifugal filters following the manufacturer recommendations (Millipore). Protein yield was estimated by measuring absorbance at 595 nm using a spectrophotometer (UV-2600, SHIMADZU). Proteins were also examined by Coomassie-Brilliant Blue staining of polyacrylamide gels to confirm protein size and integrity. Purified ELF4 was injected into leaves of 15-day old elf4-1 mutant seedlings harboring the LHY::LUC reporter line. Similar concentration of GFP protein was also injected as a negative control.

Protein purification and injection analyses
Rhythms were subsequently examined in a LB960 luminometer (Berthold Technologies) as described above.

Time course analyses of gene expression by RT-qPCR
Seedlings were synchronized under LD cycles in MS3 medium plates for 8-14 days and subsequently transferred to LL. Shoots are roots from intact plants were taken every 4 hour over the circadian cycle.
For excised roots, shoots and roots were carefully separated with a sterile razor blade and the excised roots were deposited on MS3 agar medium plates for 2 or 3 days as specified. RNA was purified using a Maxwell RSC Plant RNA kit following the manufacturer's recommendations (Promega). Singlestranded cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad). qPCR analyses were performed with cDNAs diluted 50-fold with nuclease-free water using iTag Universal SYBR Green Supermix (Bio-Rad) or Brilliant III Ultra-Fast SYBR Green qPCR Master Mix and an goat anti-mouse horse peroxidase conjugated secondary antibody (1:500 dilution) (Thermo-Fisher). At least two biological replicates were performed per experiment and/or condition.

Micrografting assays
Micrografting was performed essentially as previously described 43 . Data from unsuccessful grafted seedlings that failed to properly join together or grafts that were insufficiently clear to be successful were discarded. Approximately 100-150 grafting events were performed for every combination of grafts. The percentage of successfully micrografted plants was about 30-50 % (possibly higher but only the clearly successful grafted plants were taken into account). From the successfully grafted plants, 30-60 % showed different degrees of recovered rhythms. For in vivo luminescence assays, shoots and roots of grafted plants were simultaneously examined using the protocol described above. For ELF4-ox grafts, water instead of luciferin was added to the wells containing shoots to exclude the possibility that recovery of rhythms in roots were due to leaking signals from shoots. Similarly, grafted WT shoots contained no reporter fused to luciferase.

Reconstruction of driving forces by recurrence plots
Common driving forces were estimated following a several-step procedure. First, we took time differences of consecutive measurements and remove trends. Second, we used delay coordinates 74, 75 of 5 dimensional space to obtain a recurrence plot 76 so that 5% points, except for the central diagonal line, have points plotted. Third, we took the union for the recurrence plots to infer the recurrence plot of the common driving force 77 . Fourth, we applied the assumption of continuity and supplied the points at (i,i+1) and (i+1,i) for each i 78 . Fifth, we applied the method described in 77 to convert the recurrence plot of the common driving force into time series. Lastly, we extracted the component corresponding to the largest eigenvalue. The periodicity of the reconstructed common driving force X(t) was evaluated using the autocorrelation function and 10000 random shuffle surrogates 79 . Here, the null hypothesis was that there was no serial dependence. The autocorrelation with time difference k is the correlation coefficient between X(t) and X(t+k). Thus, it is close to 1 if X(t) and X(t+k) are similar while it is close to 0 if they are not related to each other. If there is a 24h periodicity in the driving force, the autocorrelation with 24h time difference should be a value close to 1.        Fig. 3. Mathematical analyses of the micrografting effects on rhythms. Twodimensional plots of CCA1::LUC rhythms in roots before a, and after b, grating ELF4-ox scion into elf4-1 rootstocks. c, Reconstructed waveforms and d, autocorrelation analyses in roots before and after grafting.