Genes 2019, 10, genes-581348; doi: FOR PEER REVIEW www.mdpi.com/journal/genes
Article
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LIP1 regulates the plant circadian clock via the
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oscillator component GIGANTEA
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Anita Hajdu1, †, Kata Terecskei1, †, Péter Gyula2, Éva Ádám1, Anna Nyakó1, Orsolya Dobos1, 3, and
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László Kozma-Bognár 1, 4,*
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1 Institute of Plant Biology, Biological Research Centre, Szeged H-6726, Hungary; hajdu.anita@brc.mta.hu
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(A.H.); kata@gmail.com (K.T.); adam.eva@brc.mta.hu (É.Á.); nyako.anna@gmail.com (A.N.);
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dobos.orsoly@brc.mta.hu (O.D.);e-mail@e-mail.com
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2 Agricultural Biotechnology Institute, Epigenetics Group, National Agricultural Research and Innovation
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Center, Gödöllő H-2100, Hungary; gyula.peter@abc.naik.hu (P.G.)
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3 Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged H-6726,
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Hungary
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4 Department of Genetics, Faculty of Sciences and Informatics, University of Szeged, Szeged H-6726, Hungary;
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* Correspondence: kozma_bognar.laszlo@brc.mta.hu (L.K.-B.)
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† These authors contributed equally to this work
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Received: June 18, 2019; Accepted: September 19, 2019; Published:
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Abstract: Circadian clocks are biochemical timers regulating many physiological and molecular
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processes according to the day/night cycles. The function of the oscillator relies on negative
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transcriptional/translational feedback loops operated by the so-called clock genes and the encoded
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clock proteins. The small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) is a circadian clock-
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associated protein that regulates light input to the clock in the model plant Arabidopsis thaliana. In
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the absence of LIP1, the effect of light on free-running period length is much reduced. We showed
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that LIP1 is also required for suppressing red and blue light-mediated photomorphogenesis, light-
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controlled inhibition of endoreplication and tolerance to salt stress. Here we demonstrate that LIP1
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is present in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB
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1 (TOC1). LIP1 participates in this complex via GUANINE EXCHANGE FACTOR 7. Analysis of
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genetic interactions proved that LIP1 affect the oscillator via modulating GI function. Moreover, we
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showed that GI also connects LIP1 to the regulation of salt stress and endoreplication, but these two
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proteins control photomorphogenesis by separate routes. Collectively, our results suggest that LIP1
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attenuates selected functions of GI, possibly by interfering with binding of GI to downstream
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signalling components.
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Keywords: Arabidopsis; circadian clock; GIGANTEA, small GTPase LIP1.
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1. Introduction
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Circadian clocks are endogenous timekeepers that coordinate internal physiological responses to the
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predicted external environment. In Arabidopsis, 30% of the transcriptome is circadian regulated and
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this includes processes such as metabolism, the induction of flowering, growth, responsiveness to
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hormones and biotic and abiotic stress (1-3). Consequently, having an internal oscillator that closely
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matches external time enhances plant fitness (4). Studies that investigated responsiveness to periodic
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stress cues or metabolism have highlighted the importance of metabolic oscillations in the regulation
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of circadian rhythms (5, 6). It has been proposed that endogenous timekeepers not only predict
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external stress cues, but also provide the basis for temporal segregation between incompatible
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cellular metabolic processes that would otherwise be energetically futile and stressful (7, 8).
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Plant circadian rhythms are generated through a series of transcriptional-translational loops. At the
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center of the oscillator is a repressive feedback loop formed between the morning expressed MYB
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transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED
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HYPOCOTYL (LHY) and the night-phased TIMING OF CAB EXPRESSION 1 (TOC1), also known as
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PSEUDO RESPONSE REGULATOR 1 (PRR1) (9, 10). This core loop is subsequently regulated by a
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series of morning and evening loops. In the morning, sequential expression of PRR9/7/5 starting with
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PRR9 just after dawn results in the repression of CCA1/LHY expression throughout the day and early
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evening (11). At dusk, GIGANTEA (GI) and ZEITLUPPE (ZTL) co-associate to degrade TOC1 (12,
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13), and the evening complex(14), composed of EARLY FLOWERING3, EARLY FLOWERING4 and
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LUX ARRYTHMO (LUX), represses the expression of GI, LUX, PRR9 and PRR7 (15, 16).
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The small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) is a circadian clock- associated protein
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that regulates light input to the clock in the model plant Arabidopsis thaliana. In the absence of LIP1,
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the effect of light on free-running period length is much reduced. We showed that LIP1 is also
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required for suppressing red and blue light-mediated photomorphogenesis, light-controlled
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inhibition of endoreplication and tolerance to salt stress. Here we demonstrate that LIP1 is present
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in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 1 (TOC1).
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LIP1 participates in this complex via GUANINE EXCHANGE FACTOR 7. Analysis of genetic
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interactions proved that LIP1 affect the oscillator via modulating GI function. Moreover, we showed
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that GI also connects LIP1 to the regulation of salt stress and endoreplication, but these two proteins
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control photomorphogenesis by separate routes. Collectively, our results suggest that LIP1
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attenuates selected functions of GI, possibly by interfering with binding of GI to downstream
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signalling components.
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2. Materials and Methods
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2.1. Plant materials and growth conditions.
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The Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) was used as the background
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for all the experimental lines. pifQ (pif1pif3pif4pif5), pifQ CCA1:LUCIFERASE (LUC) and wild-type
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(wt) CCA1:LUC transgenic plants are described in (Leivar et al., 2008; Shor et al. 2017). The PIF-
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overexpression (PIF-ox) lines used were 35spro:PIF1-HA (Zhu et al., 2015), 35spro:PIF3-myc (Park et
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al., 2004), 35spro:PIF4-myc and 35spro:PIF5-myc (Sakuraba et al., 2014). For all experiments, seeds
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were imbibed and cold treated at 4°C for 4 days and sown onto Petri dishes with Murashige and
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Skoog (MS) medium (Duchefa Biochemie, Netherlands) with or without 3% (w/v) sucrose (for
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luciferase assay), or 2% sucrose (w/v) (for leaf movement assays and RT-PCR). Unless otherwise
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stated, plants were grown in 14 hours light: 10 hours dark (14 L:10 D) with 100 μmol m−2 s−1 white
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light supplied by Philips fluorescent lights TLD 18W/840 at 23°C.
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2.2. Bioluminescence assays.
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For the circadian bioluminescence assays, 6-8 seedlings from each of 3-4 independent lines
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carrying the CCA1:LUCIFERASE (CCA1:LUC) reporter (pifQ CCA1:LUC and wt CCA1:LUC) were
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grown for 8 days in 14 L:10 D. After spraying with 2.5mM luciferin (D-Luciferin, Potassium salt, Gold
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Biotechnology, St Louis, MO, USA) in 0.01% Triton X-100 the seedlings were transferred to a growth
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chamber mounted with a Hamamatsu ORCA II ER CCD camera (C4742-98 ERG; Hamamatsu
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Photonics, Hamamatsu City, Japan). Light was provided by red and blue light emitting diodes
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(LEDs), with regulated total fluence rates. Luciferase activity was imaged every two hours for at least
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four days. Images were analyzed with ImagePro software (Media Cybernetics, Inc., Bethesda, MD,
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USA). Data were imported into the Biological Rhythms Analysis Software System (BRASS;
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http://www.amillar.org) and analyzed with the FFT-NLLS (Fourier Transform-NonLinear Least
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Squares) suite of the program, as previously described (Plautz et al., 1997). Rhythms with a period
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between 15 and 35 hours were taken to be within the circadian range.
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2.3. RNA isolation and quantitative RT-PCR.
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Ten to twelve seedlings were harvested per sample and total RNA extracted as previously
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described (Green and Tobin, 1999). RNA samples were treated with DNase I (PerfeCTa DNAse from
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Quanta bio, Beverly, MA, USA) according to the manufacturer’s instructions. From each DNA-free
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RNA sample, 5 μl aliquots were used as a template to produce cDNA, using the qScript cDNA
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SuperMix (Quanta bio). 2.5 μl of template cDNA was used for quantitative RT-PCR reaction with
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SYBR green reagent (KAPA SYBR FAST qPCR kit Master Mix, Kapa Biosystems, MA, USA) according
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to the supplier’s protocol. Three technical repeats were made for each sample. Fluorescence was
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detected using the QuantStudio 12K Flex system (Thermo Fisher Scientific, MA, USA). PROTEIN
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PHOSPHATASE 2A (PP2A, AT1G13320), was used as a control for normalization (Czechowski et al.,
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2005). Quantitation calculations were carried out using the 2–ΔΔCT formula as described (Nozue et
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al., 2007). The primers are shown in Supplementary Table 1.
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2.4. Yeast two-hybrid tests for protein interactions.
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Yeast cells were co-transformed with different cDNA-fragments cloned into pGADT7 and
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pGBKT7 vectors (Clontech) in frame with the GAL4 transcriptional activator domain or with the GAL4
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DNA-binding domain, respectively. Transformed cells were plated on Leu-/Trp- (LW) and Ade-/Leu-
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/Trp- (ALW) CSM agar plates and were grown at 30 oC for 5 days. For -galactosidase enzyme activity
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assay, three independent colonies were picked up from LW or ALW plates and inoculated into LW
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CSM liquid media, and were shaken at 30 oC until density reached OD600 0.8, when the assay (using
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O-nitrophenyl-β-D-galactopyranoside as substrate) was carried out (Yasuhara et al, 2004). For
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documentation of the growth on selective medium, single colonies from LW or ALW plates were
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diluted in 100 l of sterile water and 5 ls of them were dropped on fresh LW and ALW plates. After 5
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days of growth plates were scanned by a flat-bed scanner. Expression of the mutated ZTL proteins in
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yeast were confirmed by Western blotting using anti c-Myc and anti HA antibodies (Sigma).
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3. Results
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3.1.1. Pattern and level of clock gene expression in the lip1-2 mutant
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It has been demonstrated previously that LIP1 affects the circadian clock by mediating light
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signalling to the oscillator (Kevei 2007). Input light signals may affect the level, the activity or
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subcellular localization of oscillator components to set the pace and phase of the circadian clock. In
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order to test the effect of LIP1 on clock gene expression, Col wild type and lip1-2 mutant seedlings
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were entrained to 12 h light:12 h dark (12:12 LD) photocycles for a week and then transferred to
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continuous red light at relatively low fluence rate (5 μmol m-2 s-1), where the short period phenotype
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of lip1 mutants is readily detectable (Kevei 2007)(Terecskei 2013). The accumulation pattern and level
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of selected clock genes was analysed by qPCR assays. The genes were selected to represent the
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different regulatory loops, but also to include the first identified main components (CCA1, TOC1),
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genes with sequential peak times during the day (PRR5, 7, 9) and key elements of the Evening
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Complex (LUX, ELF4) as well. Figure 1 shows that rhythmic accumulation of all tested genes
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displayed shorter periods in the mutant compared with the wild type control. However, mRNA
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levels did not change consistently in the mutant. These data indicate that altered level of clock gene
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expression probably does not underlay the period phenotype of lip1-2, thus the primary and direct
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effect of LIP1 on the oscillator is not the transcriptional regulation of clock genes.
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3.1.2. Identification of proteins interacting with LIP1
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Since the results above suggested that LIP1 may exerts its clock-related function at
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posttranscriptional/protein level, we aimed at identifying proteins thorough which this regulation
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could take place. First we tested the interactions between LIP1 and several clock proteins (CCA1,
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TOC1, GI, ZTL, ELF4, ELF3, LUX, PRR9) using the GAL4-based yeast two-hybrid (Y2H) system
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without any positive results (data not shown). To expand the range of potential partners, in the next
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step we performed a yeast two-hybrid (Y2H) screen employing LIP1 as bait. We isolated 7 clones
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encoding protein fragments that interacted with LIP1 in a reproducible manner. However, only one
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of these retained the ability for interaction when the corresponding full-length protein was co-
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expressed with LIP1 (Figure 2A). The gene is designated as AT5G02010 and encodes for ROP (RHO
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OF PLANTS) GUANINE NUCLEOTIDE EXCHANGE FACTOR 7 (ROPGEF7, GEF7 hereafter in the
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text). GEF7 belongs to the family of ROPGEF proteins consisting of 14 members in Arabidopsis. These
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proteins facilitate the replacement of GDP by GTP bound to the plant-specific Rop GTPases, leading
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to the activation of these signalling factors. The LIP1-GEF7 interaction was verified by a Luciferase
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Complementation Assay (Figure 2B) in E. coli cells, overcoming the problem of transactivation by
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GEF7 in the Y2H system. This result also suggested that no plant-specific posttranslational
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modifications are required for establishment of LIP1-GEF7 interaction. To reveal potential links to
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the oscillator, we tested interactions between GEF7 and clock proteins CCA1, TOC1, GI, ZTL, ELF4,
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ELF3, LUX and PRR9. Significant binding to LIP1 was detected in the case of GI, TOC1 and ZTL
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(Fig.2C-E). Interestingly, it has been demonstrated earlier that ZTL plays a role in the degradation of
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TOC1 (Mas 2003), whereas GI stabilizes ZTL in a light dependent manner (Kim 2007) via direct
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interactions. We verified these interactions in the Y2H system (Fig.2G, H), but also demonstrated
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physical association between TOC1 and GI (Fig.2F) that has not been reported before. These data
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indicate that despite the lack of direct interactions with oscillator components, LIP1 may be present
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in clock protein complexes through GEF7.
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3.1.3. Genetic analysis identifies GI as the clock component targeted by LIP1
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In order to reveal the functional consequences of the indirect interactions described above and
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to test if one of the complex-forming clock proteins represents the entry point of LIP1-derived in the
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oscillator, double mutants were generated by crossing lip1-2 to gi-101, toc1-4, ztl-3 or cca1-1. The
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cca1-1 mutant was used as control, since neither direct nor indirect interaction was detected between
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CCA1 and LIP1. The mutant combinations carried the CCR2:LUC or the CAB2:LUC reporters
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facilitating the analysis of the circadian phenotypes. Plants, including the wild type and the single
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mutant controls were assayed in low intensity red light (Fig.3). Visual inspection of rhythmic traces
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indicated additive period phenotypes for lip1-2 and toc1-4 (Fig.3B), ztl-3 (Fig.3C) and cca1-1 (Fig.3D).
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Estimates of free-running periods verified this observation with quantitative data (Table 1). The
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period of lip1-2 ztl-3 was in between the two parent singles, whereas periods of lip1-2 toc1-4 and lip1-
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2 cca1-1 were significantly shorter compared with the parental lines. In contrast, lip1-2 gi-101
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produced CCR2:LUC rhythms with periods indistinguishable from that of gi-101, but significantly
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longer than that of the lip1-2 single (Fig.3A, Table 1). Moreover, the reduction of amplitude seen in
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gi-101 was also clearly observable in lip1-2 gi-101. These data demonstrate that GI is epistatic to LIP1
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in the regulation of the circadian oscillator.
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3.1.4. Functions of LIP1 in the regulation of endoreplication and salt stress responses are mediated via GI
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In addition to its function in the regulation of the circadian clock, LIP1 was shown to control
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ploidy levels, responses to salt stress and light-dependent hypocotyl elongation (Terecskei 2013).
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Since we showed that LIP1 affects the clock through GI, we aimed at testing if other phenotypes of
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the lip1-2 mutants also depend on GI.
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Previously we demonstrated that the rounded shape of epidermal pavement cells of lip1 mutants is
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due to increased ploidy levels, which is the result of impaired suppression of endoreplication
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(Terecskei 2013). We monitored this phenotype as a proxy for ploidy levels in the different genetic
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backgrounds. Figure 4 illustrates that in agreement with previous results the shape of pavement cells
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in 7-day-old light-grown lip1-2 seedlings (Fig.4B) was dramatically different from that of the WT
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plants (Fig.4A). The gi-101 mutant did not show obvious alterations compared with the WT (Fig.4C).
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Interestingly, the lip1-2 gí-101 double mutant (Fig.4D) phenocopied the gi-101 single indicating the
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functional GI is required for the expression of the ploidy phenotype of lip1-2.
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LIP1 was shown be required for efficient tolerance of high salinity as germination and development
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of lip1 mutants was severely impaired at 100 mM NaCl, which was clearly tolerated by WT plants
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(Figure 5) (Terecskei 2013). The gi-101 mutant did not show any symptoms in these conditions, in
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agreement with previous reports (Kim 2013)(Sakuraba 2017). The lip1-2 gi-101 double mutant
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behaved like gi-101 indicating that GI functions downstream of LIP1 in controlling salt stress
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responses.
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3.1.5. Functions of GI in the regulation of photomorphogenesis and flowering time are not affected by LIP1
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Both LIP1 and GI play a role in light-controlled hypocotyl elongation, although they exert
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opposite effects: compared with WT, lip1 or gi mutants produce shorter or longer hypocotyls,
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respectively, when grown in continuous red or blue light (Martin-Tryon 2007)(Kevei 2007)(Terecskei
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2013). However, none of them are involved in far-red light signal transduction (Huq 2000) (Terecskei
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2013). To test genetic interaction between LIP1 and GI for these phenotypes, hypocotyl lengths of
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seedlings of different genotypes were determined after 4 days of growth in continuous red, blue and
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far-red light and in darkness. In order to reflect the light-controlled component of hypocotyl
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elongation, height of light-grown seedlings was normalized to the height of the corresponding dark-
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grown plants. Figure 6A shows that regarding the inhibition of hypocotyl elongation by red and blue
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light lip1-2 was hypersensitive, whereas gi-101 was hyposensitive, as reported earlier. The lip1-2 gi-
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101 double produced hypocotyl lengths intermediate between the two single mutants and mimicking
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WT. As expected, none of the mutants showed alterations from the WT in far-red light.
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GI is a key player of photoperiodic flowering upregulating FT transcription by CO-dependent (Sawa
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2007) and CO-independent (Sawa 2011) routes. Accordingly, flowering is dramatically delayed in gi
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mutants. Owing to the lack of information on the flowering phenotype of lip1 mutants, plants of the
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different genotypes were grown in short day (8 h light/16 h dark, SD) or long day (16 h light/8 h dark,
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LD) conditions. The time of flowering was determined as the number of rosette leaves at bolting.
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Figure 6B demonstrates that flowering time was not altered in the lip1-2 mutant in neither conditions.
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The gi-101 single flowered later then the WT, whereas the lip1-2 gi-101 double was indistinguishable
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from the gi-101 single.
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These results suggest that GI and LIP1 regulate hypocotyl elongation via largely different signalling
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routes and that the function of GI in flowering time initiation is not modulated by LIP1.
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3.2. Figures, Tables and Schemes
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Figure 1. mRNA accumulation pattern of clock genes in the lip1-2 mutant
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Wild type (Col) and lip1-2 mutant seedlings were grown in 12 h light: 12 h dark photocycles for 7 days and
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transferred to continuous red light (5 µmol m-2 s-1). Samples were harvested at 3-hour intervalls, starting 27 h
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after the transfer. mRNA levels of TOC1 (A), CCA1 (B), GI (C), PRR5 (D), PRR7 (E), PRR9 (F), ELF4 (G),
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and LUX (H) were determined by qPCR and normalised to the corresponding TUB mRNA levels. Average
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values of 3 indpendent replicates are plotted, error bars represent Standard Error values.
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Figure 2. Identification of proteins directly or indirectly interacting with LIP1
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Full-length LIP1, GEF7, GI, TOC1 and ZTL proteins fused to the transcriptional activation domain (AD) or the
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DNA-binding domain (BD) of the GAL4 transcription factor were coexpressed in yeast (PJ69-4A) cells (A, C-
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H). Interactions were tested in either (AD and BD) configurations. For each combination, the first or the second
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indicated protein carried the AD or the BD fusion tag, respectively. AD and BD correspond to controls, where
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these GAL4 derivatives were expressed without foreign fusion partners. β-galactosidase activity, reporting the
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activation of the lacZ marker and therefore the strength of interaction between the two given fusion proteins,
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was determined from liquid-cultured transformant cells. Green bars indicate activation above the background
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levels (grey bars). GEF7 and GI fused to BD were able to activate the markergene without any interacting
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partners (transactivation, red bars). The assays were repeated 3-4 times with essentially the same results. Error
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bars represent Standard Error values of 3 technical repeats of a representative assay. M.-u.: Miller-units.
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The interaction between LIP1 and GEF7 was also tested by luciferase complementation assays (B). LIP1 or
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GEF7 fused to the N- or C-terminal fragment of firefly luciferase (nLUC or cLUC) in either configurations
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were coexpressed in E. coli BL21 Rosetta cells along with the corresponding controls. Luminesce of bacterial
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patches (5 patches for each combinations) was detected by a cooled CCD camera. Error bars represent the
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Standard Error values of 3 independent assays. a.u.: arbitrary units = counts/patch/5 min.
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Figure 3. LIP1 requires GI to affect the circadian clock
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Seedlings of the indicated genotypes carrying CCR2:LUC (A, C, D) or CAB2:LUC (B) reporter genes were
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grown in 12 h light: 12 h dark photoperiods for 7 days and transferred to continuous red light (5 µmol m-2 s-1),
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where luminescence was monitored. For each individual seedlings, values were normalised to the average of
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all counts collected during the course of the assay. The means of normalised data from 24 seedlings for each
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genotypes are plotted. Experiments were repeated 3 or 4 times.
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Table 1. Period estimates demonstrate genetic interaction between LIP1 and GI.
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Luminescence data of plants shown in Figure 3 were analysed by the BRASS3 software package. Free-running
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periods were estimated by FFT-NLLS analysis. p-values were calculated from pairwise t-tests to determine the
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significance of differences from the corresponding double mutant in terms of periods.
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Figure 4. The pavement cell morphology phanotype of lip1-2 mutants is dependent on GI.
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Pavement cell morphology of Col (A), lip1-2 (B), gi-101 (C) and lip1-2 gi-101 plants grown in 12 h light: 12h
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dark photocycles for 7 days. Scale bars: 100 µm.
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Figure 5. The lack of GI function supresses the salt sensitivity phenotype of lip1-2 mutants.
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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
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media with or without 100mM NaCl.
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Figure 6. LIP1 affect photomorphogenic responses independently of GI
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(A) Wild type Col, lip1-2, gi-101 and lip1-2 gi-101 mutant seedlings were grown in continuous red (cR, 20
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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
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measured and normalised to the corresponding dark-grown hypocotyl lengths. 30-40 seedlings were analysed
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for each genotype and fluence rate. Error bars indicate Standard Error, and different letters show significant
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differences at P < 0.05 (Duncan’s test).
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(B) Plants were grown in 16 h light: 8 h dark (LD) or 8 h light: 16 h dark (SD) photocycles. Rosette leaves were
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counted when inflorescences reached 1 cm. 12-15 plants were analysed for each genotypes and conditions.
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Error bars indicate Standard Error, and different letters show significant differences at P < 0.01 (Duncan’s test).
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4. Discussion
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LIP1 is the first small GTPase that has been functionally linked to the circadian clock in plants.
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Lack of LIP1 function results in an accelerated circadian oscillator producing short period rhythms.
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However, the mechanism by which LIP1 affects the oscillator remained unknown. In the present
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work we aimed at revealing an essential piece of this regulation and identify the particular oscillator
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component that is primarily targeted by LIP1.
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We tested the mRNA accumulation of several key clock genes in the loss-of-function allele lip1-
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2 plants in continuous low fluence red light, where the short period phenotype is most pronounced.
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No significant changes in mRNA levels of clock genes were found suggesting that transcriptional
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modulation is not the principal effect of LIP1 on the clock. The pace of the oscillator can also be
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influenced by altering the function or the turnover of one or more clock proteins. This effect may be
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mediated via protein-protein interactions. Although direct interaction between LIP1 and clock
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proteins was not detected, a search for binding partners identified a guanine exchange factor (GEF7),
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which in turn showed physical interaction with GI, TOC1 and ZTL proteins. GEF7 is a member of the
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RopGEF protein family and acts as a functional guanine exchange factor to activate Rop GTPase
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AtRAC1, required for root meristem maintenance (Chen 2011). Interestingly, pairwise interactions
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between GI, TOC1 and ZTL was also found in Y2H. Supposing that these interactions take place in
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vivo, the results suggest the existence of a multiprotein complex to which LIP1 may bind through
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GEF7. It is notable that these factors, including LIP1, operate in the evening. To link LIP1 to the
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oscillator, one can assume that the complex of GI-TOC1-ZTL could modulate the activity of LIP1
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through GEF7 and then LIP1 affect the function of one or more clock proteins. Alternatively, LIP1,
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brought in proximity by GEF7, could influence the activities of GI, TOC1 or ZTL. Analysis of epistasis
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between LIP1 and the three clock components supported the latter option and identified GI as the
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downstream target of LIP1-derived signalling to the oscillator. Moreover, we showed that in addition
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to the circadian phenotype, the ploidy and salt stress phenotypes of lip1 mutants also depend on GI.
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However, the clear additivity of photomorphogenic phenotypes of gi and lip1 mutants demonstrated
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that LIP1 does not act exclusively through the modulation of GI function.
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A simple way to control the function of GI is to regulate the level/turnover or the subcellular
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localisation of the protein. However, these changes to the GI protein should alter all functions of GI,
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including flowering time determination (Mizoguchi 2005)(Günl 2009), which phenotype was not
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observed in lip1 mutants. This indicates that LIP1 does not affect the function of GI in general, but
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probably acts selectively on particular branches of GI signalling. GI has diverse pleiotropic functions,
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which appear to be realized via specific protein-protein interactions (see the Introduction). Based on
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our current results and considering published data we hypothesise that LIP1 affects interaction of GI
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with downstream effector proteins that relay the effect of GI on the clock and salt stress tolerance.
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The fact that the cell shape/ploidy phenotype of lip1-2 is supressed in the lip1-2 gi-101 double mutant,
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strongly indicates that GI plays a role in the regulation of this process as well. This function of GI has
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not been described and the related specific interactors were not identified yet, but our results suggest
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that the corresponding hypothetic interactions are also impacted by LIP1. In contrast, interactions
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representing outputs of GI towards the regulation of photomorphogenesis and flowering should not
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be affected by LIP1.
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GI regulates the circadian oscillator via at least two distinct mechanisms. GI triggers
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destabilization of PIF transcription factors, and thus relieves repression on CCA1 transcription
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(Nohales 2019). On the other hand, GI stabilizes ZTL in the light, which in turn mediate degradation
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of clock proteins, among them TOC1, in the dark (Cha 2017). Since CCA1 mRNA levels were not
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altered in lip1-2 and ztl-3 was not epistatic to lip1-2, we conclude that these two regulatory
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mechanisms of GI are probably not influenced by LIP1. Rather, these data indicate the existence of
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an additional, LIP1-modulated functional link from GI to the clock.
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Plants overexpressing GI (GI-OX) show increased sensitivity to salt stress (Kim 2013) and
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display circadian rhythms with shortened periods (Mizoguchi 2005). These phenotypes of GI-OX
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plants are shared with lip1 mutants (Kevei 2007)(Trecskei 2013). Thus we conclude that LIP1
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attenuates these functions of GI. This very likely holds true of the cell shape phenotype as well, so
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one could predict that epidermal pavement cells of GI-OX plants have rounded shape, similarly to
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those of the lip1 mutants.
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In summary, we demonstrated that LIP1 affects the circadian clock, salt stress responses and
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epidermal cell shaping via the inhibition of selected functions of GI.
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Author Contributions: conceptualization, L.K-B. and A.H.; methodology, A.H., K.T., P.G., É.Á., A.N. and O.D.;
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validation, L.K-B., A.H. and T.K.; formal analysis, P.G., É.Á. and O.D.; writing—original draft preparation, A.H.
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and T.K.; writing—review and editing, L.K-B.; funding acquisition, L.K-B.
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Funding: This research was funded by National Research, Development and Innovation Office, grant numbers
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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
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by GINOP-2.3.2-15-2016-00001.
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Conflicts of Interest: The authors declare no conflict of interest.
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44.© 2019 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
