The circadian clock contributes to the long-term water use efficiency of Arabidopsis

In plants, water use efficiency is a complex trait derived from numerous physiological and developmental characteristics. Here, we investigated the involvement of circadian regulation in long-term water use efficiency. Circadian rhythms are generated by the circadian oscillator, which provides a cellular measure of the time of day. In plants, the circadian oscillator contributes to the regulation of many aspects of physiology, including stomatal opening, the rate of photosynthesis, carbohydrate metabolism and developmental processes. We investigated in Arabidopsis the impact of the misregulation of genes encoding a large number of components of the circadian oscillator upon whole plant, long-term water use efficiency. From this, we identified a role for the circadian oscillator in water use efficiency. This appears to be due to contributions of the circadian clock to the control of transpiration and biomass accumulation. We also identified that the circadian oscillator specifically within guard cells contributes to long-term water use efficiency. Our experiments indicate that knowledge of circadian regulation will be important for developing future crops that use water more efficiently. One-sentence summary The circadian clock in Arabidopsis makes an important contribution to long-term water use efficiency.


Introduction 39
World population growth is increasing the demand for fresh water for agriculture, with 40 climate change predicted to exacerbate this competition for water resources (Ruggiero et al.,41 under our experimental conditions (Fig. S3C), demonstrating that these promoters were 208 appropriate for constitutive overexpression of circadian oscillator components within guard 209 cells in our experiments. 210 To further verify the guard cell-specific overexpression of CCA1 and TOC1 in the GCS 211 plants, we examined CCA1 and TOC1 transcript accumulation within guard cells. Under 212 constant light conditions, we measured CCA1 transcript accumulation in epidermal peels at 213 dusk (when CCA1 transcript abundance is normally low in the wild type) and TOC1 214 transcript accumulation at dawn (when TOC1 transcript abundance is normally low in the 215 wild type). Guard cell CCA1 overexpressors had greater CCA1 transcript abundance in 216 epidermal peels at dusk than the wild type (GC: t₄ = -2.233, p>0.05; MC: t₄ = -7.409, p = 217 0.002) (Fig. S3D), and guard cell TOC1 overexpressors had greater TOC1 transcript 218 abundance at dawn than the wild type (GT: t₄ = -6.636, p = 0.003; MT: t₄ = -2.736, p = 219 0.050) (Fig. S3D). These data indicate that CCA1 and TOC1 were overexpressed within the 220 guard cells of the guard cell-specific CCA1 or TOC1 overexpressor plants that we 221 generated, respectively. 222 We investigated the effect on WUE of overexpression of CCA1 and TOC1 within guard cells. 223 Two independent GC1::CCA1 lines (GC-1 and GC-2) were significantly more water use 224 efficient than the wild type (GC-1: p < 0.001; GC-2: p = 0.002) (Fig. 4B). GC-1 and GC-2 225 were 8% and 4% more water use efficient than the wild type, respectively (Fig. 4B). In 226 comparison, two independent MYB60::CCA1 did not have greater WUE than the wild type 227 (p > 0.05) (Fig. 4B). This suggests that overexpressing CCA1 in guard cells can increase 228 whole plant long-term WUE in a promoter-specific manner. Overexpression of TOC1 in 229 guard cells with both the GC1 and MYB60 promoters did not alter WUE (p > 0.05) (Fig. 4B). 230 This suggests that decreased WUE in constitutive TOC1-ox plants (Fig. 1A, Fig. 4B) might 231 not be explained by overexpression of TOC1 within the guard cells, and that this decreased 232 WUE might instead be due to TOC1 overexpression in other cell types. Because the 233 stomatal density was unaltered relative to the wild type in the guard cell overexpressors of 234 10 CCA1 and TOC1 (Fig. 4C, D), the WUE phenotypes that we identified from these lines might 235 be caused by alterations in processes within guard cells, such as those regulating stomatal 236 aperture, rather than altered stomatal density. 237

Discussion 238
Pervasive influence of the circadian oscillator upon water use efficiency 239 Our data indicate that the circadian oscillator is important for regulating the long-term WUE 240 of Arabidopsis. Misregulation of several functional subsections of the circadian oscillator 241 altered the WUE of Arabidopsis. Misexpression of morning (PRR9, CCA1), late day (GI, 242 PRR5) and evening (TOC1, ZTL, ELF3) components of the circadian oscillator all perturb 243 WUE under our experimental conditions (Fig. 1A, B). Additionally, altered expression of TEJ 244 and GRP7 alters WUE (Fig. 1A). Therefore, oscillator components that impact WUE are not 245 confined to a specific expression phase or architectural feature (e.g. morning loop) within the 246 multi-loop circadian oscillator. Misexpression of genes encoding some proteins that provide 247 environmental inputs to the circadian oscillator (ELF3, TPS1, ZTL, KIN10; (Covington et al., 248 2001;Kim et al., 2007;Shin et al., 2017;Frank et al., 2018)) also alters WUE (Fig. 1A). 249 Together, this suggests that the entire circadian oscillator can influence WUE, and that 250 alterations in water use that are caused by mutations to the circadian oscillator are not 251 confined to a specific sub-loop of the circadian oscillator or restricted to its input or output 252 pathways. One explanation for these circadian-system wide alterations in WUE relates to the 253 nature of feedback within the circadian oscillator. The complex feedback and 254 interconnectivity of the circadian oscillator means that individual components of the circadian 255 oscillator that directly influence stomatal function or water use are likely to be altered by 256 mutations that are distal to that component. Therefore, if correct circadian timing is required 257 for optimum water use efficiency, multiple components of the circadian oscillator are likely to 258 influence water use efficiency. Alternatively, because mutation of a number of components 259 of the circadian oscillator had no effect upon WUE, it is possible that the oscillator 260 11 components that influence WUE do so through roles in directly regulating outputs of the 261 circadian oscillator such as by regulating genes involved in stomatal function. 262 The sugar signalling proteins TPS1 and KIN10 influence a broad range of phenotypes, in 263 addition to participating in circadian entrainment (Baena-González et al., 2007;Gómez et al., 264 2010;Paul et al., 2010;Delatte et al., 2011;Shin et al., 2017;Frank et al., 2018;Nietzsche 265 et al., 2018;Simon et al., 2018). The tps1-12 TILLING mutant of TPS1 decreases stomatal 266 aperture and increases the ABA sensitivity of guard cells (Gómez et al., 2010), whereas we 267 found that tps1-11 and tps1-12 had lower long-term WUE than the wild type (Fig. 1A). Lower 268 biomass accumulation in tps1-12 ( Fig. 2B) was consistent with slow growth of these alleles 269 (Gómez et al., 2010). Overall, this suggests that the decreased stomatal aperture of tps1-12 270 mutants does not translate into an overall increase in WUE, potentially due to slower growth 271 of the tps1 mutants ( Fig. 2B) (Gómez et al., 2010). The broad range of phenotypes that are 272 altered in tps1-11, tps1-12 and KIN10-ox 6.5 indicates that these genotypes might alter 273 WUE through mechanisms other than circadian regulation. 274

Potential roles for the evening complex in WUE 275
Our finding that ELF3 can influence WUE (Fig. 1A) (Thines and Harmon, 2010;Dixon et al., 2011;Herrero et al., 2012). The low WUE of elf3-1 284 might potentially be caused by altered PRR9 expression, because misregulation of PRR9 285 also affected WUE (Fig. 1A). In a similar fashion, ELF3/ELF4 signalling represses PRR7, 286 and elf3-1 has elevated PRR7 transcript abundance (Herrero et al., 2012). Under light-dark 12 cycles, elf3-1 also has high and constitutive GI expression (Fowler et al., 1999), and elf3-1 288 and gi mutants have opposite WUE phenotypes (Fig. 1). Therefore, the WUE phenotype of 289 elf3-1 (Fig. 1) might be caused by disruption of ELF3 itself, or specific perturbations of 290 PRR7,PRR9 and/or GI expression. 291 Mutating further components of the evening complex (EC) (ELF4 and LUX) did not affect 292 WUE (Fig. 1). This is despite the way that these genes influence circadian oscillator function 293 and plant physiology (Hsu and Harmer, 2014;Huang and Nusinow, 2016), and nocturnal 294 regulation of stomatal aperture impacts WUE (Costa et al., 2015;Coupel-Ledru et al., 2016). 295 One possibility is that the impact of elf3-1 on WUE may be greater than that of elf4 or lux 296 because ELF3 is key to EC scaffolding, with ELF3 operating genetically downstream from 297 ELF4 and LUX (Herrero et al., 2012;Huang and Nusinow, 2016). 298 The EC binds upstream of and regulates a variety of other genes that might also underlie the 299 WUE alterations in elf3-1 mutants (Ezer et al., 2017). This includes regulators of growth, 300 components of the photosynthetic apparatus, and genes associated with phytohormone 301 signalling. This means that potential roles for the EC in WUE might occur through several 302 physiological mechanisms. There also appears to be a negative relationship between 303 temperature and EC promoter binding (Ezer et al., 2017), so it is possible that any influence 304 of the EC upon WUE might be temperature-sensitive. 305 ELF4 appears to play a greater role in circadian regulation in the vascular tissue than 306 stomatal guard cells, with vasculature expression up to ten times higher than other tissues 307 (Endo et al., 2014). Processes within the vasculature can affect WUE; for example, 308 mutations in CELLULOSE SYNTHASE CATALYTIC SUBUNIT7 (ATCESA7) might impact 309 water use through effects of the collapse of the vasculature upon guard cell size (Liang et 310 al., 2010). Because elf3-1 affects WUE differently from elf4-101 and lux-1 (Fig. 1), it appears 311 that ELF3 regulates WUE independently from ELF4 and LUX. 312 Our data suggest that changes in WUE caused by misexpression of circadian clock 314 components might be due to a combination of physiological factors. Many mutants or 315 overexpressors tested alter both biomass accumulation and water loss, often in the same 316 direction ( Fig. 2A, B), so mutations to the circadian oscillator did not alter water use by 317 specifically altering either carbon assimilation or transpiration. This is consistent with 318 previous work demonstrating that both stomatal opening and CO 2 fixation is perturbed in 319 circadian arrhythmic plants under light/dark cycles (Dodd et al., 2005), and with the findings 320 that daily carbohydrate management is dependent upon correct circadian regulation (Graf et 321 al., 2010). We speculate that delayed or advanced stomatal and photosynthetic responses 322 to the day-night cycle might occur in circadian period mutants, because period mutants 323 inaccurately anticipate the onset of dawn ( It has been reported previously that during the light period of light/dark cycles, CCA1-ox has 334 greater stomatal conductance than the wild type and decreased CO 2 assimilation and 335 biomass accumulation (Dodd et al., 2005;Graf et al., 2010). If these alterations in CO 2 336 fixation and transpiration persist throughout the vegetative growth phase, it might be 337 predicted that CCA1-ox would have lower long-term WUE than the wild type. However, we 338 found here that long-term WUE was unaltered relative to the wild type in CCA1-ox under our 339 experimental conditions (Fig. 1). When the quantitative changes in water loss and biomass 340 accumulation in CCA1-ox are examined, it appears that both biomass accumulation and 341 water loss were decreased relative to the wild type in CCA1-ox (Fig. 2). This means that 342 whilst the ratiometric measure of WUE is unaltered in CCA1-ox, the plants are smaller and 343 use less water overall, potentially due to the smaller leaf area. This difference between 344 short-term gas exchange characteristics of CCA1-ox (Dodd et al., 2005) and its long-term 345 WUE (Fig. 1) shows that there can be differences between short-term measures of gas 346 exchange compared with WUE measured over long periods of growth. It also underlines the 347 importance of the type of experiments performed here for understanding how specific 348 molecular mechanisms can alter WUE through the plant lifetime. 349

Contribution of circadian regulation in guard cells to water use efficiency 350
Next, we investigated whether the circadian oscillator within guard cells contributes to long-351 term WUE. To investigate this, we overexpressed two circadian clock genes in guard cells 352 using two different guard cell-specific promoters. Comparable approaches have been 353 adopted to investigate roles of specific cell types in the functioning of the circadian system 354 and their relationships with physiology and development (Endo et al., 2014;Shimizu et al., 355 2015;Hassidim et al., 2017). Under our experimental conditions, we did not identify 356 consistent alterations in the long-term WUE of seedlings overexpressing CCA1 or TOC1 in 357 stomatal guard cells (Fig. 4B). This suggests that decreased long-term WUE of TOC1-ox 358 plants ( Fig. 1) arises from altered circadian regulation in cell types other than guard cells. 359 Whilst two lines harbouring a GC1::CCA1 construct had greater WUE than the wild type, 360 WUE was unaltered in comparable lines harbouring MYB60::CCA1 (Fig. 4B). The differing 361 WUE phenotype of GC1::CCA1 and MYB60::CCA1 might be explained by differences in 362 promoter strength, because the GC1 promoter appears to have somewhat greater activity 363 than the MYB60 promoter (Fig. S3D, E). Although both promoters are guard cell-specific in 364 our hands (Fig. S3), we cannot exclude the possibility of ectopic promoter activity. 365 Interestingly, GC1::CCA1 is reported to have greater drought sensitivity of long-term 366 biomass accumulation than the wild type (Hassidim et al., 2017), whereas we found that 367 GC1::CCA1 had greater WUE than the wild type (Fig. 4B). This might reflect the integration 368 of circadian regulation into ABA signalling (Legnaioli et al., 2009;Robertson et al., 2009) with circadian regulation in other tissues also contributing to overall WUE. It would be 376 informative in future to perform reverse genetic screening of the dehydration tolerance or 377 long-term drought tolerance of sets of circadian clock mutants. However, because well-378 watered WUE is not a drought tolerance trait (Blum, 2009), it possible that different circadian 379 clock alleles might confer dehydration or drought tolerance compared with those alleles that 380 alter WUE (Fig. 1). 381

Conclusions 382
We show that circadian regulation contributes to whole plant long-term WUE under cycles of 383 day and night. This control occurs partly through the influence of components of the 384 circadian oscillator upon rosette architecture. Mutation or overexpression of CCA1, TOC1, 385 ELF3, GI, GRP7, PRR5, PRR9, TEJ and ZTL altered WUE under our experimental 386 conditions. The roles of these genes in WUE may be independent or overlapping, and their 387 WUE phenotypes might be due to direct effects of these genes, or indirect effects on 388 transcript and/or protein abundance of other circadian clock gene(s guard-cell misregulation of circadian oscillator genes suggest that the circadian oscillator 397 within guard cells and other cell types influences WUE. Finally, our findings suggest that 398 circadian regulation potentially alters a single trait (WUE) by affecting many aspects of 399 physiology, along with leaf area. Overall, our study demonstrates that the circadian oscillator 400 is important for the water use efficiency of Arabidopsis plants during their entire vegetative 401 growth period. In future, it will be informative to distinguish the contribution to overall WUE of 402 circadian regulation within additional cell types, such as the mesophyll, vascular tissue, and 403 root cell types. It will also be important to identify specific mechanisms underlying the WUE 404 phenotypes, and determine the extent to which these findings scale to crop species.  Table S1, and all have been described previously. For all experiments, at least two 419 completely independent experimental repeats were performed per genotype and per 420 treatment, with multiple replicate plants within each of the experimental repeats. 421

Measurement of water use efficiency 444
The WUE assay was adapted from Wituszynska et al. (2013) (Wituszyńska et al., 2013. 445 Plants were grown for 6 weeks in modified 50 ml Falcon tubes, under an 8 h photoperiod at 446 70% humidity, 20 °C, and photon flux density of 100 µmol m -2 s -1 of overhead lighting 447 supplied by cool white fluorescent tubes (Reftech, Netherlands). The Falcon tube systems 448 consisted of a 50 ml Falcon tube filled with 37.5 ml of a 1:1 ratio of compost: perlite and 35 449 ml of Milli-Q water (Merck), with the remaining volume filled with a 1:1 ratio of compost: Milli-450 Q water (Fig. S4). Each Falcon tube lid had a 2 mm diameter hole drilled in its centre to 451 allow plant growth. The lid was spray-painted black (Hycote) because we found that the 452 orange colour of the Falcon tube lid caused leaf curling (Fig. S4). The system was wrapped 453 in aluminium foil to exclude light (Fig. S4). 10-15 seeds were sown through the Falcon tube 454 lid using a pipette. Following stratification, Falcon tube systems were placed under growth 455 conditions using a randomised experimental design. 7 days after germination, seedlings 456 were thinned to one per Falcon tube system, and initial Falcon tube weight was recorded. 457 The seedling-thinning step was sensitive to seedling damage for genotypes with 458 substantially altered morphologies (e.g. tps1 mutants), reducing the number of replicates 459 available for some genotypes. After 6 weeks of growth, rosette leaf surface area was 460 measured by photography (D50; Nikon) and Fiji software, rosette dry weight was measured 461 (4 d at 60°C), and final Falcon tube weight was recorded. All experiments were stopped 462 before flowering occurred, with the 8 h photoperiod being used to delay flowering as much 463 as possible. Plants were not obviously stressed during the experiment (e.g. leaves did not 464 become purple due to strong anthocyanin accumulation, and plants did not wilt or become 465 contaminated with mildew) (Fig. S4). Negative controls (Falcon tube systems without plants) 466 were used to assess soil water evaporation over 18 experimental repeats, with an overall 467 mean weight loss of 0.513 g ± 0.004 g over 6 weeks for plant-free Falcon tubes. 468 Plant WUE was calculated as follows: 469 Where d is the rosette dry weight at the end of the experiment (mg), t i and t f are the falcon 470 tube weight at the start and end of the experiment, respectively (g), and e is the amount of 471 water evaporation directly from the compost (g). WUE is derived as mg biomass per ml -1 472 water lost. These calculations assumed that 1 g of weight change was equivalent to a 473 change of 1 ml of water. For each of the 2 experimental experimental repeats (Fig. 1, Fig.  474 S1), 15 plants were screened per genotype. Due to variation between the WUE of each 475 background (Fig. S2), the WUE of each circadian oscillator genotype was normalized to its 476 respective background and expressed as a percentage of that background. Statistical 477 comparisons with the wild types were conducted before this normalization. 478

Measurement of stomatal density 479
Plants were grown for 7-8 weeks on compost mix. Dental paste (Coltene) was applied to the 480 abaxial surface of fully expanded leaves. Transparent nail varnish (Rimmel) was applied to 481 these leaf moulds once they had set, and then peeled away from the mould using clear 482 adhesive tape (Scotch Crystal). Stomatal and pavement cells were counted within an 483 800 µm x 800 µm square at the centre of each leaf half, using an epifluorescence 484 microscope (HAL100; Zeiss) and Volocity (Perkin Elmer) and Fiji software. For each 485 experimental repeat, two leaves were sampled per plant and eight plants sampled per 486 genotype. Stomatal index was calculated as follows: 487 Where SI is the stomatal index, s the number of stomata in the field of view (800 µm x 488 800 µm), and p the number of pavement cells in the field of view. 489