lip1 regulates the plant circadian clock via the

Genes 2019, 10, genes-581348; doi: FOR PEER REVIEW www.mdpi.com/journal/genes

Article 1

LIP1 regulates the plant circadian clock via the 2

oscillator component GIGANTEA 3

Anita Hajdu1, †, Kata Terecskei1, †, Péter Gyula2, Éva Ádám1, Anna Nyakó1, Orsolya Dobos1, 3, and 4 László Kozma-Bognár 1, 4,* 5

1 Institute of Plant Biology, Biological Research Centre, Szeged H-6726, Hungary; [email protected] 6 (A.H.); [email protected] (K.T.); [email protected] (É.Á.); [email protected] (A.N.); 7 [email protected] (O.D.);[email protected] 8

2 Agricultural Biotechnology Institute, Epigenetics Group, National Agricultural Research and Innovation 9 Center, Gödöllő H-2100, Hungary; [email protected] (P.G.) 10

3 Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged H-6726, 11 Hungary 12

4 Department of Genetics, Faculty of Sciences and Informatics, University of Szeged, Szeged H-6726, Hungary; 13 * Correspondence: [email protected] (L.K.-B.) 14 † These authors contributed equally to this work 15 Received: June 18, 2019; Accepted: September 19, 2019; Published: 16

Abstract: Circadian clocks are biochemical timers regulating many physiological and molecular 17 processes according to the day/night cycles. The function of the oscillator relies on negative 18 transcriptional/translational feedback loops operated by the so-called clock genes and the encoded 19 clock proteins. The small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) is a circadian clock- 20 associated protein that regulates light input to the clock in the model plant Arabidopsis thaliana. In 21 the absence of LIP1, the effect of light on free-running period length is much reduced. We showed 22 that LIP1 is also required for suppressing red and blue light-mediated photomorphogenesis, light-23 controlled inhibition of endoreplication and tolerance to salt stress. Here we demonstrate that LIP1 24 is present in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 25 1 (TOC1). LIP1 participates in this complex via GUANINE EXCHANGE FACTOR 7. Analysis of 26 genetic interactions proved that LIP1 affect the oscillator via modulating GI function. Moreover, we 27 showed that GI also connects LIP1 to the regulation of salt stress and endoreplication, but these two 28 proteins control photomorphogenesis by separate routes. Collectively, our results suggest that LIP1 29 attenuates selected functions of GI, possibly by interfering with binding of GI to downstream 30 signalling components. 31

Keywords: Arabidopsis; circadian clock; GIGANTEA, small GTPase LIP1. 32 33

1. Introduction 34

Circadian clocks are endogenous timekeepers that coordinate internal physiological responses to the 35 predicted external environment. In Arabidopsis, 30% of the transcriptome is circadian regulated and 36 this includes processes such as metabolism, the induction of flowering, growth, responsiveness to 37 hormones and biotic and abiotic stress (1-3). Consequently, having an internal oscillator that closely 38 matches external time enhances plant fitness (4). Studies that investigated responsiveness to periodic 39 stress cues or metabolism have highlighted the importance of metabolic oscillations in the regulation 40 of circadian rhythms (5, 6). It has been proposed that endogenous timekeepers not only predict 41 external stress cues, but also provide the basis for temporal segregation between incompatible 42 cellular metabolic processes that would otherwise be energetically futile and stressful (7, 8). 43

Genes 2019, 10, genes-581348; FOR PEER REVIEW 2 of 13

Plant circadian rhythms are generated through a series of transcriptional-translational loops. At the 44 center of the oscillator is a repressive feedback loop formed between the morning expressed MYB 45 transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED 46 HYPOCOTYL (LHY) and the night-phased TIMING OF CAB EXPRESSION 1 (TOC1), also known as 47 PSEUDO RESPONSE REGULATOR 1 (PRR1) (9, 10). This core loop is subsequently regulated by a 48 series of morning and evening loops. In the morning, sequential expression of PRR9/7/5 starting with 49 PRR9 just after dawn results in the repression of CCA1/LHY expression throughout the day and early 50 evening (11). At dusk, GIGANTEA (GI) and ZEITLUPPE (ZTL) co-associate to degrade TOC1 (12, 51 13), and the evening complex(14), composed of EARLY FLOWERING3, EARLY FLOWERING4 and 52 LUX ARRYTHMO (LUX), represses the expression of GI, LUX, PRR9 and PRR7 (15, 16). 53

The small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) is a circadian clock- associated protein 54 that regulates light input to the clock in the model plant Arabidopsis thaliana. In the absence of LIP1, 55 the effect of light on free-running period length is much reduced. We showed that LIP1 is also 56 required for suppressing red and blue light-mediated photomorphogenesis, light-controlled 57 inhibition of endoreplication and tolerance to salt stress. Here we demonstrate that LIP1 is present 58 in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 1 (TOC1). 59 LIP1 participates in this complex via GUANINE EXCHANGE FACTOR 7. Analysis of genetic 60 interactions proved that LIP1 affect the oscillator via modulating GI function. Moreover, we showed 61 that GI also connects LIP1 to the regulation of salt stress and endoreplication, but these two proteins 62 control photomorphogenesis by separate routes. Collectively, our results suggest that LIP1 63 attenuates selected functions of GI, possibly by interfering with binding of GI to downstream 64 signalling components. 65

2. Materials and Methods 66 2.1. Plant materials and growth conditions. 67 The Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) was used as the background 68

for all the experimental lines. pifQ (pif1pif3pif4pif5), pifQ CCA1:LUCIFERASE (LUC) and wild-type 69 (wt) CCA1:LUC transgenic plants are described in (Leivar et al., 2008; Shor et al. 2017). The PIF- 70 overexpression (PIF-ox) lines used were 35spro:PIF1-HA (Zhu et al., 2015), 35spro:PIF3-myc (Park et 71 al., 2004), 35spro:PIF4-myc and 35spro:PIF5-myc (Sakuraba et al., 2014). For all experiments, seeds 72 were imbibed and cold treated at 4°C for 4 days and sown onto Petri dishes with Murashige and 73 Skoog (MS) medium (Duchefa Biochemie, Netherlands) with or without 3% (w/v) sucrose (for 74 luciferase assay), or 2% sucrose (w/v) (for leaf movement assays and RT-PCR). Unless otherwise 75 stated, plants were grown in 14 hours light: 10 hours dark (14 L:10 D) with 100 μmol m−2 s−1 white 76 light supplied by Philips fluorescent lights TLD 18W/840 at 23°C. 77

2.2. Bioluminescence assays. 78 For the circadian bioluminescence assays, 6-8 seedlings from each of 3-4 independent lines 79

carrying the CCA1:LUCIFERASE (CCA1:LUC) reporter (pifQ CCA1:LUC and wt CCA1:LUC) were 80 grown for 8 days in 14 L:10 D. After spraying with 2.5mM luciferin (D-Luciferin, Potassium salt, Gold 81 Biotechnology, St Louis, MO, USA) in 0.01% Triton X-100 the seedlings were transferred to a growth 82 chamber mounted with a Hamamatsu ORCA II ER CCD camera (C4742-98 ERG; Hamamatsu 83 Photonics, Hamamatsu City, Japan). Light was provided by red and blue light emitting diodes 84 (LEDs), with regulated total fluence rates. Luciferase activity was imaged every two hours for at least 85 four days. Images were analyzed with ImagePro software (Media Cybernetics, Inc., Bethesda, MD, 86 USA). Data were imported into the Biological Rhythms Analysis Software System (BRASS; 87 http://www.amillar.org) and analyzed with the FFT-NLLS (Fourier Transform-NonLinear Least 88 Squares) suite of the program, as previously described (Plautz et al., 1997). Rhythms with a period 89 between 15 and 35 hours were taken to be within the circadian range. 90

2.3. RNA isolation and quantitative RT-PCR. 91 Ten to twelve seedlings were harvested per sample and total RNA extracted as previously 92

described (Green and Tobin, 1999). RNA samples were treated with DNase I (PerfeCTa DNAse from 93


Quanta bio, Beverly, MA, USA) according to the manufacturer’s instructions. From each DNA-free 94 RNA sample, 5 μl aliquots were used as a template to produce cDNA, using the qScript cDNA 95 SuperMix (Quanta bio). 2.5 μl of template cDNA was used for quantitative RT-PCR reaction with 96 SYBR green reagent (KAPA SYBR FAST qPCR kit Master Mix, Kapa Biosystems, MA, USA) according 97 to the supplier’s protocol. Three technical repeats were made for each sample. Fluorescence was 98 detected using the QuantStudio 12K Flex system (Thermo Fisher Scientific, MA, USA). PROTEIN 99 PHOSPHATASE 2A (PP2A, AT1G13320), was used as a control for normalization (Czechowski et al., 100 2005). Quantitation calculations were carried out using the 2–ΔΔCT formula as described (Nozue et 101 al., 2007). The primers are shown in Supplementary Table 1. 102

2.4. Yeast two-hybrid tests for protein interactions. 103 Yeast cells were co-transformed with different cDNA-fragments cloned into pGADT7 and 104

pGBKT7 vectors (Clontech) in frame with the GAL4 transcriptional activator domain or with the GAL4 105 DNA-binding domain, respectively. Transformed cells were plated on Leu-/Trp- (LW) and Ade-/Leu-106 /Trp- (ALW) CSM agar plates and were grown at 30 oC for 5 days. For -galactosidase enzyme activity 107 assay, three independent colonies were picked up from LW or ALW plates and inoculated into LW 108 CSM liquid media, and were shaken at 30 oC until density reached OD600 0.8, when the assay (using 109 O-nitrophenyl-β-D-galactopyranoside as substrate) was carried out (Yasuhara et al, 2004). For 110 documentation of the growth on selective medium, single colonies from LW or ALW plates were 111 diluted in 100 l of sterile water and 5 ls of them were dropped on fresh LW and ALW plates. After 5 112 days of growth plates were scanned by a flat-bed scanner. Expression of the mutated ZTL proteins in 113 yeast were confirmed by Western blotting using anti c-Myc and anti HA antibodies (Sigma). 114

3. Results 115

3.1.1. Pattern and level of clock gene expression in the lip1-2 mutant 116

It has been demonstrated previously that LIP1 affects the circadian clock by mediating light 117 signalling to the oscillator (Kevei 2007). Input light signals may affect the level, the activity or 118 subcellular localization of oscillator components to set the pace and phase of the circadian clock. In 119 order to test the effect of LIP1 on clock gene expression, Col wild type and lip1-2 mutant seedlings 120 were entrained to 12 h light:12 h dark (12:12 LD) photocycles for a week and then transferred to 121 continuous red light at relatively low fluence rate (5 μmol m-2 s-1), where the short period phenotype 122 of lip1 mutants is readily detectable (Kevei 2007)(Terecskei 2013). The accumulation pattern and level 123 of selected clock genes was analysed by qPCR assays. The genes were selected to represent the 124 different regulatory loops, but also to include the first identified main components (CCA1, TOC1), 125 genes with sequential peak times during the day (PRR5, 7, 9) and key elements of the Evening 126 Complex (LUX, ELF4) as well. Figure 1 shows that rhythmic accumulation of all tested genes 127 displayed shorter periods in the mutant compared with the wild type control. However, mRNA 128 levels did not change consistently in the mutant. These data indicate that altered level of clock gene 129 expression probably does not underlay the period phenotype of lip1-2, thus the primary and direct 130 effect of LIP1 on the oscillator is not the transcriptional regulation of clock genes. 131

3.1.2. Identification of proteins interacting with LIP1 132

Since the results above suggested that LIP1 may exerts its clock-related function at 133 posttranscriptional/protein level, we aimed at identifying proteins thorough which this regulation 134 could take place. First we tested the interactions between LIP1 and several clock proteins (CCA1, 135 TOC1, GI, ZTL, ELF4, ELF3, LUX, PRR9) using the GAL4-based yeast two-hybrid (Y2H) system 136 without any positive results (data not shown). To expand the range of potential partners, in the next 137 step we performed a yeast two-hybrid (Y2H) screen employing LIP1 as bait. We isolated 7 clones 138 encoding protein fragments that interacted with LIP1 in a reproducible manner. However, only one 139 of these retained the ability for interaction when the corresponding full-length protein was co-140 expressed with LIP1 (Figure 2A). The gene is designated as AT5G02010 and encodes for ROP (RHO 141


OF PLANTS) GUANINE NUCLEOTIDE EXCHANGE FACTOR 7 (ROPGEF7, GEF7 hereafter in the 142 text). GEF7 belongs to the family of ROPGEF proteins consisting of 14 members in Arabidopsis. These 143 proteins facilitate the replacement of GDP by GTP bound to the plant-specific Rop GTPases, leading 144 to the activation of these signalling factors. The LIP1-GEF7 interaction was verified by a Luciferase 145 Complementation Assay (Figure 2B) in E. coli cells, overcoming the problem of transactivation by 146 GEF7 in the Y2H system. This result also suggested that no plant-specific posttranslational 147 modifications are required for establishment of LIP1-GEF7 interaction. To reveal potential links to 148 the oscillator, we tested interactions between GEF7 and clock proteins CCA1, TOC1, GI, ZTL, ELF4, 149 ELF3, LUX and PRR9. Significant binding to LIP1 was detected in the case of GI, TOC1 and ZTL 150 (Fig.2C-E). Interestingly, it has been demonstrated earlier that ZTL plays a role in the degradation of 151 TOC1 (Mas 2003), whereas GI stabilizes ZTL in a light dependent manner (Kim 2007) via direct 152 interactions. We verified these interactions in the Y2H system (Fig.2G, H), but also demonstrated 153 physical association between TOC1 and GI (Fig.2F) that has not been reported before. These data 154 indicate that despite the lack of direct interactions with oscillator components, LIP1 may be present 155 in clock protein complexes through GEF7. 156

157

3.1.3. Genetic analysis identifies GI as the clock component targeted by LIP1 158

In order to reveal the functional consequences of the indirect interactions described above and 159 to test if one of the complex-forming clock proteins represents the entry point of LIP1-derived in the 160 oscillator, double mutants were generated by crossing lip1-2 to gi-101, toc1-4, ztl-3 or cca1-1. The 161 cca1-1 mutant was used as control, since neither direct nor indirect interaction was detected between 162 CCA1 and LIP1. The mutant combinations carried the CCR2:LUC or the CAB2:LUC reporters 163 facilitating the analysis of the circadian phenotypes. Plants, including the wild type and the single 164 mutant controls were assayed in low intensity red light (Fig.3). Visual inspection of rhythmic traces 165 indicated additive period phenotypes for lip1-2 and toc1-4 (Fig.3B), ztl-3 (Fig.3C) and cca1-1 (Fig.3D). 166 Estimates of free-running periods verified this observation with quantitative data (Table 1). The 167 period of lip1-2 ztl-3 was in between the two parent singles, whereas periods of lip1-2 toc1-4 and lip1-168 2 cca1-1 were significantly shorter compared with the parental lines. In contrast, lip1-2 gi-101 169 produced CCR2:LUC rhythms with periods indistinguishable from that of gi-101, but significantly 170 longer than that of the lip1-2 single (Fig.3A, Table 1). Moreover, the reduction of amplitude seen in 171 gi-101 was also clearly observable in lip1-2 gi-101. These data demonstrate that GI is epistatic to LIP1 172 in the regulation of the circadian oscillator. 173

3.1.4. Functions of LIP1 in the regulation of endoreplication and salt stress responses are mediated via GI 174

In addition to its function in the regulation of the circadian clock, LIP1 was shown to control 175 ploidy levels, responses to salt stress and light-dependent hypocotyl elongation (Terecskei 2013). 176 Since we showed that LIP1 affects the clock through GI, we aimed at testing if other phenotypes of 177 the lip1-2 mutants also depend on GI. 178

Previously we demonstrated that the rounded shape of epidermal pavement cells of lip1 mutants is 179 due to increased ploidy levels, which is the result of impaired suppression of endoreplication 180 (Terecskei 2013). We monitored this phenotype as a proxy for ploidy levels in the different genetic 181 backgrounds. Figure 4 illustrates that in agreement with previous results the shape of pavement cells 182 in 7-day-old light-grown lip1-2 seedlings (Fig.4B) was dramatically different from that of the WT 183 plants (Fig.4A). The gi-101 mutant did not show obvious alterations compared with the WT (Fig.4C). 184 Interestingly, the lip1-2 gí-101 double mutant (Fig.4D) phenocopied the gi-101 single indicating the 185 functional GI is required for the expression of the ploidy phenotype of lip1-2. 186


LIP1 was shown be required for efficient tolerance of high salinity as germination and development 187 of lip1 mutants was severely impaired at 100 mM NaCl, which was clearly tolerated by WT plants 188 (Figure 5) (Terecskei 2013). The gi-101 mutant did not show any symptoms in these conditions, in 189 agreement with previous reports (Kim 2013)(Sakuraba 2017). The lip1-2 gi-101 double mutant 190 behaved like gi-101 indicating that GI functions downstream of LIP1 in controlling salt stress 191 responses. 192

3.1.5. Functions of GI in the regulation of photomorphogenesis and flowering time are not affected by LIP1 193

Both LIP1 and GI play a role in light-controlled hypocotyl elongation, although they exert 194 opposite effects: compared with WT, lip1 or gi mutants produce shorter or longer hypocotyls, 195 respectively, when grown in continuous red or blue light (Martin-Tryon 2007)(Kevei 2007)(Terecskei 196 2013). However, none of them are involved in far-red light signal transduction (Huq 2000) (Terecskei 197 2013). To test genetic interaction between LIP1 and GI for these phenotypes, hypocotyl lengths of 198 seedlings of different genotypes were determined after 4 days of growth in continuous red, blue and 199 far-red light and in darkness. In order to reflect the light-controlled component of hypocotyl 200 elongation, height of light-grown seedlings was normalized to the height of the corresponding dark-201 grown plants. Figure 6A shows that regarding the inhibition of hypocotyl elongation by red and blue 202 light lip1-2 was hypersensitive, whereas gi-101 was hyposensitive, as reported earlier. The lip1-2 gi-203 101 double produced hypocotyl lengths intermediate between the two single mutants and mimicking 204 WT. As expected, none of the mutants showed alterations from the WT in far-red light. 205

GI is a key player of photoperiodic flowering upregulating FT transcription by CO-dependent (Sawa 206 2007) and CO-independent (Sawa 2011) routes. Accordingly, flowering is dramatically delayed in gi 207 mutants. Owing to the lack of information on the flowering phenotype of lip1 mutants, plants of the 208 different genotypes were grown in short day (8 h light/16 h dark, SD) or long day (16 h light/8 h dark, 209 LD) conditions. The time of flowering was determined as the number of rosette leaves at bolting. 210 Figure 6B demonstrates that flowering time was not altered in the lip1-2 mutant in neither conditions. 211 The gi-101 single flowered later then the WT, whereas the lip1-2 gi-101 double was indistinguishable 212 from the gi-101 single. 213

These results suggest that GI and LIP1 regulate hypocotyl elongation via largely different signalling 214 routes and that the function of GI in flowering time initiation is not modulated by LIP1. 215

3.2. Figures, Tables and Schemes 216


217 Figure 1. mRNA accumulation pattern of clock genes in the lip1-2 mutant 218 Wild type (Col) and lip1-2 mutant seedlings were grown in 12 h light: 12 h dark photocycles for 7 days and 219 transferred to continuous red light (5 µmol m-2 s-1). Samples were harvested at 3-hour intervalls, starting 27 h 220 after the transfer. mRNA levels of TOC1 (A), CCA1 (B), GI (C), PRR5 (D), PRR7 (E), PRR9 (F), ELF4 (G), 221 and LUX (H) were determined by qPCR and normalised to the corresponding TUB mRNA levels. Average 222 values of 3 indpendent replicates are plotted, error bars represent Standard Error values. 223


224 Figure 2. Identification of proteins directly or indirectly interacting with LIP1 225 Full-length LIP1, GEF7, GI, TOC1 and ZTL proteins fused to the transcriptional activation domain (AD) or the 226 DNA-binding domain (BD) of the GAL4 transcription factor were coexpressed in yeast (PJ69-4A) cells (A, C-227 H). Interactions were tested in either (AD and BD) configurations. For each combination, the first or the second 228 indicated protein carried the AD or the BD fusion tag, respectively. AD and BD correspond to controls, where 229 these GAL4 derivatives were expressed without foreign fusion partners. β-galactosidase activity, reporting the 230 activation of the lacZ marker and therefore the strength of interaction between the two given fusion proteins, 231 was determined from liquid-cultured transformant cells. Green bars indicate activation above the background 232 levels (grey bars). GEF7 and GI fused to BD were able to activate the markergene without any interacting 233 partners (transactivation, red bars). The assays were repeated 3-4 times with essentially the same results. Error 234 bars represent Standard Error values of 3 technical repeats of a representative assay. M.-u.: Miller-units. 235 The interaction between LIP1 and GEF7 was also tested by luciferase complementation assays (B). LIP1 or 236 GEF7 fused to the N- or C-terminal fragment of firefly luciferase (nLUC or cLUC) in either configurations 237 were coexpressed in E. coli BL21 Rosetta cells along with the corresponding controls. Luminesce of bacterial 238 patches (5 patches for each combinations) was detected by a cooled CCD camera. Error bars represent the 239 Standard Error values of 3 independent assays. a.u.: arbitrary units = counts/patch/5 min. 240


241

242 Figure 3. LIP1 requires GI to affect the circadian clock 243 Seedlings of the indicated genotypes carrying CCR2:LUC (A, C, D) or CAB2:LUC (B) reporter genes were 244 grown in 12 h light: 12 h dark photoperiods for 7 days and transferred to continuous red light (5 µmol m-2 s-1), 245 where luminescence was monitored. For each individual seedlings, values were normalised to the average of 246 all counts collected during the course of the assay. The means of normalised data from 24 seedlings for each 247 genotypes are plotted. Experiments were repeated 3 or 4 times. 248

249

Table 1. Period estimates demonstrate genetic interaction between LIP1 and GI. 250 Luminescence data of plants shown in Figure 3 were analysed by the BRASS3 software package. Free-running 251 periods were estimated by FFT-NLLS analysis. p-values were calculated from pairwise t-tests to determine the 252 significance of differences from the corresponding double mutant in terms of periods. 253


254 Figure 4. The pavement cell morphology phanotype of lip1-2 mutants is dependent on GI. 255 Pavement cell morphology of Col (A), lip1-2 (B), gi-101 (C) and lip1-2 gi-101 plants grown in 12 h light: 12h 256 dark photocycles for 7 days. Scale bars: 100 µm. 257 258

259 Figure 5. The lack of GI function supresses the salt sensitivity phenotype of lip1-2 mutants. 260 Col, lip1-2, gi-101 and lip1-2 gi-101 seedlings were grown in 12 h light: 12 h dark photocycles for 14 days on 261 media with or without 100mM NaCl. 262


263 Figure 6. LIP1 affect photomorphogenic responses independently of GI 264 (A) Wild type Col, lip1-2, gi-101 and lip1-2 gi-101 mutant seedlings were grown in continuous red (cR, 20 265 µmol m-2 s-1), blue (cB, 2 µmol m-2 s-1) or far-red (cFR, 1 µmol m-2 s-1) light for 4 days. Hypocotyl lengths were 266 measured and normalised to the corresponding dark-grown hypocotyl lengths. 30-40 seedlings were analysed 267 for each genotype and fluence rate. Error bars indicate Standard Error, and different letters show significant 268 differences at P < 0.05 (Duncan’s test). 269 (B) Plants were grown in 16 h light: 8 h dark (LD) or 8 h light: 16 h dark (SD) photocycles. Rosette leaves were 270 counted when inflorescences reached 1 cm. 12-15 plants were analysed for each genotypes and conditions. 271 Error bars indicate Standard Error, and different letters show significant differences at P < 0.01 (Duncan’s test). 272 273

4. Discussion 274

LIP1 is the first small GTPase that has been functionally linked to the circadian clock in plants. 275 Lack of LIP1 function results in an accelerated circadian oscillator producing short period rhythms. 276 However, the mechanism by which LIP1 affects the oscillator remained unknown. In the present 277 work we aimed at revealing an essential piece of this regulation and identify the particular oscillator 278 component that is primarily targeted by LIP1. 279

We tested the mRNA accumulation of several key clock genes in the loss-of-function allele lip1-280 2 plants in continuous low fluence red light, where the short period phenotype is most pronounced. 281 No significant changes in mRNA levels of clock genes were found suggesting that transcriptional 282 modulation is not the principal effect of LIP1 on the clock. The pace of the oscillator can also be 283 influenced by altering the function or the turnover of one or more clock proteins. This effect may be 284 mediated via protein-protein interactions. Although direct interaction between LIP1 and clock 285 proteins was not detected, a search for binding partners identified a guanine exchange factor (GEF7), 286 which in turn showed physical interaction with GI, TOC1 and ZTL proteins. GEF7 is a member of the 287 RopGEF protein family and acts as a functional guanine exchange factor to activate Rop GTPase 288 AtRAC1, required for root meristem maintenance (Chen 2011). Interestingly, pairwise interactions 289 between GI, TOC1 and ZTL was also found in Y2H. Supposing that these interactions take place in 290


vivo, the results suggest the existence of a multiprotein complex to which LIP1 may bind through 291 GEF7. It is notable that these factors, including LIP1, operate in the evening. To link LIP1 to the 292 oscillator, one can assume that the complex of GI-TOC1-ZTL could modulate the activity of LIP1 293 through GEF7 and then LIP1 affect the function of one or more clock proteins. Alternatively, LIP1, 294 brought in proximity by GEF7, could influence the activities of GI, TOC1 or ZTL. Analysis of epistasis 295 between LIP1 and the three clock components supported the latter option and identified GI as the 296 downstream target of LIP1-derived signalling to the oscillator. Moreover, we showed that in addition 297 to the circadian phenotype, the ploidy and salt stress phenotypes of lip1 mutants also depend on GI. 298 However, the clear additivity of photomorphogenic phenotypes of gi and lip1 mutants demonstrated 299 that LIP1 does not act exclusively through the modulation of GI function. 300

A simple way to control the function of GI is to regulate the level/turnover or the subcellular 301 localisation of the protein. However, these changes to the GI protein should alter all functions of GI, 302 including flowering time determination (Mizoguchi 2005)(Günl 2009), which phenotype was not 303 observed in lip1 mutants. This indicates that LIP1 does not affect the function of GI in general, but 304 probably acts selectively on particular branches of GI signalling. GI has diverse pleiotropic functions, 305 which appear to be realized via specific protein-protein interactions (see the Introduction). Based on 306 our current results and considering published data we hypothesise that LIP1 affects interaction of GI 307 with downstream effector proteins that relay the effect of GI on the clock and salt stress tolerance. 308 The fact that the cell shape/ploidy phenotype of lip1-2 is supressed in the lip1-2 gi-101 double mutant, 309 strongly indicates that GI plays a role in the regulation of this process as well. This function of GI has 310 not been described and the related specific interactors were not identified yet, but our results suggest 311 that the corresponding hypothetic interactions are also impacted by LIP1. In contrast, interactions 312 representing outputs of GI towards the regulation of photomorphogenesis and flowering should not 313 be affected by LIP1. 314

GI regulates the circadian oscillator via at least two distinct mechanisms. GI triggers 315 destabilization of PIF transcription factors, and thus relieves repression on CCA1 transcription 316 (Nohales 2019). On the other hand, GI stabilizes ZTL in the light, which in turn mediate degradation 317 of clock proteins, among them TOC1, in the dark (Cha 2017). Since CCA1 mRNA levels were not 318 altered in lip1-2 and ztl-3 was not epistatic to lip1-2, we conclude that these two regulatory 319 mechanisms of GI are probably not influenced by LIP1. Rather, these data indicate the existence of 320 an additional, LIP1-modulated functional link from GI to the clock. 321

Plants overexpressing GI (GI-OX) show increased sensitivity to salt stress (Kim 2013) and 322 display circadian rhythms with shortened periods (Mizoguchi 2005). These phenotypes of GI-OX 323 plants are shared with lip1 mutants (Kevei 2007)(Trecskei 2013). Thus we conclude that LIP1 324 attenuates these functions of GI. This very likely holds true of the cell shape phenotype as well, so 325 one could predict that epidermal pavement cells of GI-OX plants have rounded shape, similarly to 326 those of the lip1 mutants. 327

In summary, we demonstrated that LIP1 affects the circadian clock, salt stress responses and 328 epidermal cell shaping via the inhibition of selected functions of GI. 329 Author Contributions: conceptualization, L.K-B. and A.H.; methodology, A.H., K.T., P.G., É.Á., A.N. and O.D.; 330 validation, L.K-B., A.H. and T.K.; formal analysis, P.G., É.Á. and O.D.; writing—original draft preparation, A.H. 331 and T.K.; writing—review and editing, L.K-B.; funding acquisition, L.K-B. 332 Funding: This research was funded by National Research, Development and Innovation Office, grant numbers 333 GINOP-2.3.2-15-2016-00001, GINOP-2.3.2-15-2016-00015 and GINOP-2.3.2-15-2016-00032. The APC was funded 334 by GINOP-2.3.2-15-2016-00001. 335 Conflicts of Interest: The authors declare no conflict of interest. 336

337

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lip1 regulates the plant circadian clock via the

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