BIG regulates dynamic adjustment of circadian period in Arabidopsis thaliana

Circadian clocks drive rhythms with a period near 24 hours, but the molecular basis 31 of the circadian period’s regulation is poorly understood. We previously 32 demonstrated that metabolites affect the free-running period of the circadian 33 oscillator of Arabidopsis thaliana , with endogenous sugars acting as an accelerator 34 and exogenous nicotinamide acting as a brake. Changes in circadian oscillator 35 period are thought to adjust the timing of biological activities through the process of 36 entrainment, in which the circadian oscillator becomes synchronised to rhythmic 37 signals such as light and dark cycles, as well as changes in internal metabolism. To 38 identify molecular components associated with the dynamic adjustment of circadian 39 period, we performed a forward genetic screen. We identified Arabidopsis mutants 40 that were either period insensitive to nicotinamide ( sin ) or period oversensitive to 41 nicotinamide ( son ). We mapped son1 to BIG, a gene of unknown molecular function that was previously shown to play a role in light signalling. We found that son1 has an early entrained phase, suggesting that the dynamic alteration of circadian period contributes to the correct timing of biological events. Our data provide insight into how dynamic period adjustment of circadian oscillators contributes to establishing a correct phase relationship with the environment, and they show that BIG is involved 47 in this process. provides tool to the and the pathways of this essential feature of the circadian The study of how the circadian clock establishes correct phase with the environment essential to understand the role of the circadian timing cycle constitute


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The circadian clock is an endogenous oscillator that in Arabidopsis thaliana consists 51 of nuclear and cytosolic feedback loops. It is often considered that the circadian 52 oscillator runs with a period of 24-hour but the circadian period is plastic, depending 53 on environmental conditions. For example, in diurnal organisms such as Arabidopsis 54 (Arabidopsis thaliana), the circadian clock has a reduced period with increased light 55 intensity (Aschoff 1960). This is commonly referred to as Aschoff's rule and was the 56 foundation for the model of parametric entrainment that describes how the circadian 57 oscillator synchronises with environmental cycles (Aschoff 1960). We have 58 discovered that exogenous application of two common metabolites also regulates 59 circadian period in Arabidopsis. Sucrose reduces circadian period under dim light We performed a rescreen of the M3 to confirm initial mutants and exclude those that 143 were just free-running circadian period mutants. In the M3 screen, wild-type Ws-2 144 plants responded to 20 mM nicotinamide with an increase in circadian period of 145 CAB2:LUC + from 23.9 ± 0.2 h to 26.2 ± 0.3 h and amplitude reduced from 1.1 ± 0.04 146 normalised luminescence counts (n.c.) to 0.6 ± 0.03 n.c. (Figure 1a). Sixty-three  Twenty-five mutants were confirmed for the sin phenotype with either no significant 151 period increase in the presence of 20 mM nicotinamide, or with a reproducibly 152 smaller increase in period than Ws-2 (Supplemental Table S1). Sixteen mutants 153 were confirmed for the son phenotype with significantly greater period in the 154 presence of 20 mM nicotinamide compared to Ws-2 (Supplemental Table S1). 155 Similarly, 25 san mutants were confirmed to have either no significant decrease in 156 amplitude in response to nicotinamide, or significantly smaller amplitude than Ws-2 (ANOVA: F=7.85 df=19 p<0.01). sin1 was hyposensitive to nicotinamide, as there 173 was no variation in circadian period of CAB2:LUC + between 0.1 mM and 20 mM 174 nicotinamide (Supplemental Figure S1b; ANOVA: F=2.15 df=18 p=0.11). The 175 mutants were backcrossed twice to the parental Ws-2 line carrying 35S:AEQ and 176 CAB2:LUC + for mapping. Here we describe our findings of son1, the first mutant that 177 we have mapped from the population, which has the strongest phenotype of all those 178 identified.
179 180 son1 maps to a mutation in a splice acceptor in BIG 181 We mapped the causal mutation for son1 using a mapping population of 25 BC 1 F 2 182 plants clearly displaying the mutant phenotype and sequenced pooled DNA to 50-183 fold coverage. SHOREmap analysis (Schneeberger et al., 2009) 198 This SNP resulted in a G-A transition causing a mutation in the 3' splice acceptor site 199 of exon 12 of At3G02260 (Figure 2d). At3G02260 encodes BIG, a callosin-like 200 protein of 5098 amino acids and unknown molecular function (Gil et al., 2001). The

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M3 line carrying son1 is slightly short period (Figure 1). This short period phenotype 202 in the M3 generation was reproducible but not significant (period diff = 0.53, p>0.05, 203 Supplemental Figure S3). However, the short period phenotype was not present in 204 the M4 generation, or in the BC 1 F 3 (Supplemental Figure S3) or BC 2 F 3 205 (Supplemental Figure S2), indicating that the phenotype was not linked to the son1 206 phenotype after backcrossing to Ws-2 and that the son1 mutation does not cause a To test the effect of the 3:433767 mutation on transcript splicing in the son1 mutant, 210 PCR products were amplified from cDNA using primers spanning exon 11 -12 of 211 BIG in three independent BC 2 F 3 pedigrees. In addition to the 316 bp product  Figure S4). The smaller fragment was 2 218 bp smaller than the Ws-2 product, with a second AG immediately downstream of the 219 first being used as a splice acceptor instead, whilst the larger 458 bp product 220 contained the full sequence of intron 11 -12 suggesting it is retained in son1, due to inefficient splicing. Thus, the G-A 3:433767 causes both the production of an 222 unspliced transcript, and the use of a cryptic splice site in son1, both of which result 223 in frameshifts and are predicted to cause premature stop codons.

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To confirm that the son1 phenotype was due to the G to A transition in BIG, we 226 assessed the response to nicotinamide in mutants in BIG identified from previous 227 mutant screens, dark over-expresser of cab1-1 (doc1-1) (Li et al., 1994) and auxin 228 transport inhibitor response 3 (tir3-101) (Ruegger et al., 1997), using delayed 229 chlorophyll fluorescence (Gould et al., 2009). doc1-1 has an increase in 230 photosynthesis-related gene expression, including CAB genes, in etiolated seedlings 231 in the dark (Li et al., 1994;Gil et al., 2001) caused by a G-A transition resulting in a 232 Cys to Thr amino acid substitution in the first cysteine rich domain (CRD-1, also 233 known as a UBR box). tir3-101 is reported to have impaired polar auxin transport 234 giving rise to a dwarf phenotype (Ruegger et al., 1997;Prusinkiewicz et al., 2009).

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As confirmation, we tested whether son1 is allelic to doc1-1.   Figure S6). These crosses all behaved as wild-250 type, and had circadian period increases that corresponded to the heterozygous 251 BC 2 F 3 on the segregation analysis ( Figure 2c). This demonstrates that doc1-1 is 252 allelic to son1 and that BIG regulates sensitivity of the circadian oscillator to 253 nicotinamide.

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Having established that son1 and doc1-1 are both nicotinamide-oversensitive for 255 circadian period, we tested whether son1 plants exhibit the doc1 phenotype of 256 increased photosynthesis related gene expression in etiolated seedlings in the dark 257 (Li et al., 1994;Gil et al., 2001). CAB2:LUC + expression was higher in etiolated 258 seedlings of son1 than wild type in constant dark (DD) (Supplemental Figure S7 a-c), 259 indicating that son1 also had a dark-over expresser of CAB phenotype consistent 260 with allelism to doc1-1. Similar to doc1-1, higher CAB2 expression in constant dark 261 was not associated with premature de-etiolation (Supplemental Figure S7 d-e).

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To test if BIG could be part of the transcriptional feedback loops of the oscillator we  As son1 is compromised in the ability to regulate changes in circadian period in 330 response to nicotinamide, we tested whether it was also affected in its ability to 331 adjust period correctly to other stimuli. Response to light is the most well 332 characterised dynamic adjustment of the circadian period and is described by Arabidopsis. Wild-type Ws-2 had a typical phase shift of later phase with increasing at 8.1 ± 0.2 h (16:8). By contrast, son1 was an early phase mutant 353 peak at 3.5 ± 0.2 h (8:16), peak at 5.5 ± 0.2 h (12:12) peak at 5.7 ± 0.1 h (16:8).

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These data demonstrate that BIG is required for correct circadian entrainment. Finally, having identified that BIG regulates dynamic adjustment of circadian period 361 and that it is required for correct circadian entrainment, we wanted to investigate 362 whether oscillator period is associated with entrainment and whether the effect of 363 BIG on phase could be involved in this regulation. To do this, we studied whether 364 entrainment photoperiod affects free-running period in Ws-2 and son1 (Figure 5e).

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The results showed that there is a relationship between length of entrainment 366 photoperiod and the length of circadian period in Ws-2 ( Figure 5e). However, this 367 relationship was lost in son1, whose circadian period was not affected by the 368 duration of the photoperiod during entrainment. As a result of this, son1 did not have Using a forward genetic screen, we found that BIG is a regulator of the dynamic 392 adjustment of circadian period and phase. The period of the circadian oscillator is not 393 fixed to 24 hours, but instead is a dynamically plastic phenotype and dependent on 394 environmental conditions. Typically, experimentalists measure circadian period in 395 constant conditions that allow the circadian oscillator to free run. In these constant   year. This is essential to co-ordinate whole organism responses as circadian period  Table S2). Mutants can have a constitutive effect on circadian period 495 at all intensities of light and the mutation therefore has no effect on dynamic plasticity hyposensitivity to light. rve4 rve6 rve8 (Gray et al., 2017) and phyB-1 (Somers et al.,513 1998b) confer hyposensitivity to red light, and prr7-3 to blue light (Farre et al., 2005).

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The phenotype of son1 for the white light fluence response curve is also 515 hyposensitive. However, as shown in Supplemental Table S2, rve4 rve6 rve8 (Gray 516 et al., 2017) and phyB-1 (Somers et al., 1998b)  input, suggesting that BIG is associated with regulation of plastic period of the 526 oscillator by environmental signals, rather than acting as a core oscillator 527 component. There is variability in the reported phenotypes of prr7 mutants, with them 528 being described as long period (Farre et al., 2005) or wild type (Nakamichi et al., 529 2005;Seki et al., 2016). This and the hyposensitivity to light suggest that prr7 530 mutants might also have a defect in plasticity similar to son1 in terms of responses to 531 light. The mechanisms might be different because PRR7 is an oscillator component, 532 whilst there is no evidence for BIG being so.

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Alterations in circadian period are thought to be required for entrainment though 534 there is not yet a consensus on how this is achieved. It is envisaged that changes in 535 circadian period are a result of phase adjustment of the oscillator. For example, a 536 phase advance will reduce the period of the cycle in which the advance occurred by an amount equal to the phase advance (Johnson, 1992). Additionally, changes in the 538 velocity of the oscillator can affect period. Whilst changes in period are associated 539 with entrainment, it is not known if this is due to changes in velocity, phase or both 540 and whether these occur continuously or discontinuously (Daan, 2000). Our 541 discovery of a mutant that is specifically compromised in the ability to dynamically 542 alter circadian period and has altered entrained phase provides a tool to study the 543 mechanism of entrainment and the pathways of this essential feature of the circadian growth on agar or soil was as described previously (Xu et al., 2007). background frequency <16 were discarded. The workflow was automated in a 620 pipeline using bpipe 0.9.8.5. (Supplemental Table S3). Sliding allele frequencies 621 were generated for SNPs based on the R statistic in SHOREmap. performed using the PCR settings and electrophoresis described above. RT-qPCR 663 was performed as previously described (Haydon et al., 2013).