https://orcid.org/0000-0002-2713-5293
Abstract
Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4–PAR1/HFR1 and CIB1–PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.
Introduction
The regulation of plant organ elongation is important for proper growth and development and for acclimatization to changes in the environment. For example, far-red light (characteristic of shady conditions) induces the elongation of shoots, including hypocotyls, petioles, and stems (Franklin, 2008; Wang et al., 2020). This phenomenon, called the shade avoidance response, helps plants escape from the shade cast by other plants and obtain sufficient light for photosynthesis. Mild high temperature (~28 °C) also induces the elongation of shoots, including hypocotyls and petioles (Gray et al., 1998; Koini et al., 2009; Kumar and Wigge, 2010), but blue light inhibits their elongation (Briggs and Huala, 1999; Stamm and Kumar, 2010).
Various regulators play important roles in organ elongation, including the PHYTOCHROME-INTERACTING FACTOR (PIF) basic helix-loop-helix (bHLH) transcription factors (Castillon et al., 2007; Leivar and Quail, 2011; Jeong and Choi, 2013; Leivar and Monte, 2014). PIF4 and PIF5 positively regulate shade-induced shoot elongation, as pif4 and pif5 mutants show reduced hypocotyl elongation and decreased shade-responsive gene expression (Lorrain et al., 2008). The pif4 mutants also show reduced temperature-induced hypocotyl elongation (Koini et al., 2009). Therefore, PIF4 likely acts as a hub that integrates shade and high temperature signals.
Phytochrome (phy) pigments and multiple phytohormones regulate the transcriptional activation activity of PIF4. PIF4 protein accumulates in the dark; when plants are exposed to light, nucleus-localized phy in the PFR state (the conformational state resulting from exposure to far-red light) interacts with PIF4, resulting in the rapid degradation of PIF4 (Lorrain et al., 2008; Leivar and Quail, 2011; Jeong and Choi, 2013; Leivar and Monte, 2014). Moreover, phyB activity is regulated by warm temperatures (Jung et al., 2016; Legris et al., 2016), indicating that PIF4 degradation is controlled by temperature signals via phyB. Gibberellins (GAs) regulate PIF4 activity. The DELLA proteins REPRESSOR OF GA1-3 (RGA) and GA insensitive (GAI), which negatively regulate GA responses, interact with PIF4 and inhibit its DNA binding function, thus reducing its transcriptional activation activity (de Lucas et al., 2008). GA accumulation destabilizes DELLAs, releases PIF proteins from RGA, and allows PIF to activate the transcription of its target genes to induce organ elongation (de Lucas et al., 2008).
PHY RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) inhibit the DNA binding activity of PIF4. HFR1 and PAR1 are non-DNA binding atypical HLH proteins that inhibit the shade avoidance response, including shade-responsive gene expression and hypocotyl elongation (Galstyan et al., 2011). HFR1 and PAR1 form heterodimers with PIF4 to inhibit its G-box binding activity (Fairchild et al., 2000; Hornitschek et al., 2009; Hao et al., 2012). PAR1 participates in regulating the expression of several shade-responsive genes, such as PHYTOCHROME INTERACTING FACTOR 3-LIKE1 (PIL1) and HFR1, which are direct targets of PIF4 (Hornitschek et al., 2009; Hao et al., 2012).
We previously showed that an HLH/bHLH system composed of two atypical HLH factors, ILI1 BINDING bHLH 1 (IBH1) and PACLOBUTRAZOL RESISTANCE 1 (PRE1), and a group of bHLH transcriptional activators, ACTIVATOR OF CELL ELONGATIONs (ACEs) and CRYPTOCHROME INTERACTING BASIC HELIX-LOOP-HELIX 5 (CIB5), regulate cell elongation in response to GA and brassinosteroid (BR) signaling (Ikeda et al., 2012). In this system, ACE1 and CIB5 positively regulate cell elongation by directly activating the expression of genes including EXPANSIN8 (EXP8;Ikeda et al., 2012). IBH1 negatively regulates cell elongation by forming heterodimers with ACEs, thus suppressing their G-box-binding activity. PRE1 positively regulates cell elongation by forming heterodimers with IBH1, thus interfering with the ability of IBH1 to inhibit ACEs (Ikeda et al., 2012). We named this regulatory system the tri-antagonistic HLH/bHLH system because the balance of ACEs, IBH1, and PRE1 regulates cell elongation. The expression of PRE1 and IBH1 is regulated by GA and brassinosteroid signaling, respectively (Lee et al., 2006; Zhang et al., 2009).
Here we show that in Arabidopsis thaliana, CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1), a novel bHLH transcriptional activator related to ACE1 and CIB5, positively controls light- and temperature-regulated cell elongation. CIB1 expression is regulated by an antagonistic HLH/bHLH system composed of PIF4, HFR1, and PAR1. PAR1 also interacts with CIB1 to interfere with its DNA binding activity. Our findings demonstrate that two antagonistic HLH/bHLH systems regulate cell elongation in response to light and temperature in plants.
Materials and methods
Growth, plant transformation, hypocotyl and cotyledon measurements, and phytohormone treatments
Arabidopsis thaliana Col-0 plants were used in all experiments. Plants used for transient expression assays and transformation were grown in soil at 22 °C under 10 h/14 h or 16 h/8 h light/dark photoperiods, respectively. Arabidopsis transformation was performed using the floral dip method (Clough and Bent, 1998). The copy number of transgene insertions in the genome for all transgenic lines (except the experiment shown in Fig. 6A, B) was estimated based on the segregation ratio of antibiotic resistance. Only putative homozygous progeny (T3 and T4 generations) of single insertion lines were used for the experiments. Seedlings used for RNA isolation were grown on agar plates or in soil at 22 °C under a 16 h/8 h light/dark photoperiod.
To measure the length of hypocotyls and cotyledons, seven day-old seedlings were grown on agar plates containing Gamborg’s B5 medium at 22 °C under continuous light. For treatments with and without far-red (FR) light, five day-old seedlings were grown under white light (W; photon flux density of 60 µmol m-2 s-1) or exposed to FR light source plus white light. The red/FR ratio under white light and W + FR conditions was 1.57 and 0.015, respectively (Supplementary Fig. S1). Observations were performed using an Axioskop2 Plus system (Carl Zeiss Inc., Germany) and a WG-4 camera (Ricoh Imaging Company Ltd, Japan). For treatments with and without naphthaleneacetic acid (NAA) or brassinolide (BL), seven day-old seedlings grown under white light were transferred to liquid Murashige and Skoog (MS) medium containing 0.2 mg l-1 NAA or 0.1 µM BL.
Generation of constructs
The coding regions and the 5’ upstream regions of the genes used in this study were amplified from a cDNA library or from Arabidopsis genomic DNA using the appropriate primers (Supplementary Table S1). The constructs used for overexpression and Chimeric REpressor genes Silencing Technology (CRES-T) for each gene were based on modified vectors derived from pGreenII0029 (Hellens et al., 2000) and p35SSRDXG (Mitsuda et al., 2006). Methods used to prepare the cDNA library and bait construct for the yeast two-hybrid assay have been described previously (Mitsuda et al., 2010). The effector and reporter plasmids used for the transient expression assays have been described previously (Hiratsu et al., 2002). Effector plasmids fused with the Gal4 DNA-binding domain (Gal4DB) were constructed by fusing the yeast Gal4DB coding region to the coding sequence of each gene, in-frame, under the control of the Cauliflower mosaic virus 35S promoter (–800 to +8; CaMV35S). The reporter gene 35S-Gal4-TATA-LUC-NOS was described previously (Hiratsu et al., 2002). Synthetic sense and antisense DNAs encoding the CIB-binding element (CIBE) (Supplementary Table S1; Liu et al., 2008) were annealed and introduced into the p190LUC vector (Fujimoto et al., 2000; Mitsuda et al., 2010) with the appropriate restriction enzyme, and used as the CIBE×4 constructs. The 2130 bp 5’ upstream region of CIB1, which extends from the translation initiation site of CIB1, was used to prepare the ProCIB1:LUC reporter construct. Constructs used for protein production were generated using the pMAL-c2 (New England Biolabs Japan inc., Japan) vector. The cDNA fragments were amplified with the relevant primer set (Supplementary Table S1), digested with the appropriate restriction enzymes, such as SalI and HindIII, or EcoRI and SalI, and cloned into pMAL-c2.
Transient expression assays
Transient expression assays were performed using expanded Arabidopsis leaves from two month-old plants, as described previously (Hiratsu et al., 2004). Following particle bombardment of a mixture of reporter, effector and reference constructs suitable for each experiment, the leaves were incubated at 22 °C in continuous darkness.
Yeast two-hybrid assay
The yeast two-hybrid assays were performed as described previously (Mitsuda et al., 2010). To generate the bait and prey constructs, cDNAs of CIB1, PAR1, and HFR1 with stop codons were amplified using primer pairs with the attB1/B2 site (Supplementary Table S1) and cloned into the pDONR207 vector (Life Technologies Inc., USA) by BP cloning. The cloned cDNAs were transferred to pDEST_BTM116 or pDEST_VP16S1 using LR Clonase (Thermo Fisher Scientific Inc., USA).
Observation of fluorescent signals
Bimolecular fluorescence complementation (BiFC) assays were performed as described previously (Bracha-Drori et al., 2004; Walter et al., 2004). For the BiFC assays, the NSC and C2 vectors were constructed by inserting the coding sequences for amino acids 1–154 and 155–239 of EYFP, respectively, into the Aor51HI site of the pUGW2 vector (Nakagawa et al., 2007). The CIB1, PAR1, and PRE1 coding sequences without the stop codon were amplified using primer pairs with an attB1/B2 site (Supplementary Table S1) and cloned into the pDONR207 vector by BP cloning. The cloned coding sequences were transferred into the NSC or C2 vectors using LR Clonase. The 35S-driven mRFP-fused VAM3, whose product localizes to the vacuolar membrane (Uemura et al., 2010), was used as a control. YFP and GFP fluorescence was visualized after bombardment. All light and fluorescence microscopy was performed using an All-in-one Fluorescence Microscope (KEYENCE Inc., Japan).
Isolation of RNA and gene expression analysis
Total RNA was isolated from at least ten individual whole plants using an RNeasy Plant Mini Kit (Qiagen Inc., Germany). Following treatment with DNase I, cDNAs were prepared using a PrimeScript RT Reagent Kit (Perfect Real Time; Takara-bio Inc., Japan). Quantitative RT–PCR was performed using the SYBR green method on an ABI 7300 real-time PCR system (Life Technologies Inc., USA) with the appropriate primers (Supplementary Table S1). To quantify the relative expression of each gene in each sample, a standard curve was prepared by plotting the cycle threshold (Ct) value for a series of four dilutions of the standard sample, in which all cDNA samples were mixed. The amount of transcript in each sample with respect to the standard was calculated using the standard curve and normalized against the amount of UBIQUITIN1 (UBQ1) control transcripts in each sample. At least three replicates were included in each experiment. Results are presented as the mean ±SD. All statistical tests were performed by two-sided Welch’s t-test.
Electrophoretic mobility shift assays
PIF4 and CIB1 proteins were synthesized and purified using a pMAL system (New England Biolabs Japan inc., Japan). IRDye-labeled synthetic oligonucleotides (GWTf; LI-COR Inc., USA) were annealed with GWTr oligonucleotides (Supplementary Table S1) and used as the DNA probes. A DNA–protein binding reaction was performed using an Odyssey infrared EMSA Kit (LI-COR). The reaction contained 0.5 ng DNA, 10 mM Tris, 50 mM KCl, 3.5 mM DTT, 0.25% Tween-20, 1 µg Poly dI-dC and 5% glycerol, and the indicated amount of unlabeled competitor or protein. The reactions were incubated at 24 °C for 20 min in the dark and resolved by electrophoresis on a 5% native polyacrylamide gel containing 2.5% glycerol and Tris-borate-EDTA buffer. The signals were detected with an Odyssey infrared imaging system (LI-COR).
Accession numbers
Accession numbers of the genes in this study are as follows: CIB1 (At4G34530), PIF4 (At2g43010), PAR1 (At2g42870), HFR1 (At1g02340), SAUR15 (At4g38850), HAT4 (At4g16780), XTR7 (At4g14130), IAA19 (At3g15540), and UBQ1 (At3g52590).
Results
CIB1 positively regulates cell elongation
To identify novel transcription factors that regulate cell elongation in plants, we screened a set of transgenic Arabidopsis plants expressing chimeric repressors (CRES-T lines; Hiratsu et al., 2003). The CRES-T constructs encode a transcription factor fused with the plant-specific repression domain SRDX derived from SUPERMAN (SUPERMAN REPRESSION DOMAIN X; SRDX: LDLDLELRLGFA; Hiratsu et al., 2003). We surveyed almost 20 000 transgenic lines that expressed the chimeric repressor of 1700 independent Arabidopsis transcription factors and identified a CRES-T line for CIB1 (Pro35S:CIB1-SRDX) that exhibited severe dwarfism, round leaves, and small flowers in which all floral organs (petals, sepals, stamens, and carpels) were short (Fig. 1).
Morphological analyses of Pro35S:CIB1-SRDX transgenic Arabidopsis plants. (A) Morphological analysis of one month-old Pro35S:CIB1-SRDX (CIB1sx) plants. (B) Morphological analysis of one month-old wild-type (WT) plants. (C) Flowering stage of a Pro35S:CIB1-SRDX plant. (D and E) Leaves (D) and flowers (E) of Pro35S:CIB1-SRDX (left) and wild-type plants (right). Scale bars, 1 mm (E) and 1 cm (A, B, C, D).
CIB1 was previously isolated as an interactor of the blue-light receptors CRYPTOCHROME2 (CRY2; Liu et al., 2008) and CRY1 (Y. Liu et al., 2013). To further analyse the role of CIB1 in cell elongation, we generated transgenic Arabidopsis plants ectopically expressing CIB1 (Pro35S:CIB1). The hypocotyls of seven day-old Pro35S:CIB1 seedlings were slightly, but significantly longer than those of wild-type seedlings (Fig. 2A-E). The cotyledons of Pro35S:CIB1 seedlings were slender and longer than those of the wild type; these phenotypes were opposite to those of Pro35S:CIB1-SRDX plants (Fig. 1). These results indicate that CIB1 positively regulates organ elongation.
Morphological analyses of Pro35S:CIB1 transgenic Arabidopsis plants. (A) Seven day-old wild-type (WT) seedlings grown under 60 µmol m-2s-1 light. (B) Seven day-old Pro35S:CIB1 (CIB1ox) line 2 seedlings grown under 60 µmol m-2s-1 light. (C) Seven day-old WT (left) and Pro35S:CIB1 line 2 (right) seedlings grown under 60 µmol m-2s-1 light. (D, E) Hypocotyl (D) and cotyledon (E) lengths of seven day-old seedlings of two independent Pro35S:CIB1 lines (2, 16 CIB1ox; black bars) and two independent lots of wild-type plants (lot1, lot2; gray bars) under 60 µmol m-2s-1 light. (F) Hypocotyl lengths of seven day-old seedlings of two independent Pro35S:CIB1 lines (2,4 CIB1ox; black bars) and two independent lots of wild-type plants (lot1, lot2; gray bars) grown under 80 µmol m-2s-1 light. (G) Seven day-old wild-type and Pro35S:CIB1 line 2 (CIB1ox) seedlings grown under 80 µmol m-2s-1 light. Asterisks indicate significant data (P<0.001, Student's t-test) between wild-type lot 1 and other lines. Error bars indicate SD (n>47). Scale bars=1 mm (A, B, C and G).
CIB1 regulates light- and temperature-induced cell elongation
Cell elongation in plants is altered in response to various environmental conditions, including light and high temperature. At a photon flux density of 80 µmol m-2 s-1, the hypocotyl length of wild-type seedlings decreased by 40% compared with those grown at 60 µmol m-2 s-1 (Fig. 2C, D, F, G). The hypocotyls of Pro35S:CIB1 seedlings line 2 and line 4 were longer than those of the wild type at both 80 µmol m-2 s-1 and 60 µmol m-2 s-1; this effect was more pronounced at 80 µmol m-2 s-1, at least for line 2 (Fig. 2C, D, F, G). These results suggest that Pro35S:CIB1 plants are less sensitive to the effects of light on cell elongation. Because CIB1 overexpression strongly affected seed yield, we could not obtain enough seeds from line #4 or #16 for both experiments.
To examine the CIB1 loss-of-function phenotype more closely, we generated transgenic plants expressing CIB1-SRDX under the control of the CIB1 promoter (ProCIB1:CIB1-SRDX). The hypocotyl length of ProCIB1:CIB1-SRDX seedlings grown at a photon flux of 80 µmol m-2 s-1 was not significantly different from that of the wild type (the P values for lines 6 and 8 are 0.33 and 0.03, respectively, Student's t-test) (Fig. 3A, B; Supplementary Fig. S2). When seedlings were treated with FR light in addition to white light (Supplementary Fig. S1), both ProCIB1:CIB1-SRDX and wild-type seedlings had elongated hypocotyls, but ProCIB1:CIB1-SRDX hypocotyls were significantly shorter than wild-type hypocotyls (the P-values on lines 6 and 8 are 2.18E-10 and 3.70E-15, respectively, Student's t-test) Fig. 3A, B). These results indicate that ProCIB1:CIB1-SRDX plants had reduced sensitivity to FR light.
Morphological analyses of CIB1 loss-of-function plants. (A) Five day-old ProCIB1:CIB1-SRDX (pCIB1:CIB1sx) line 6 and wild-type (WT) seedlings grown under white light (left) and white + FR light (right) at 22 °C. Scale bars=1 mm. (B) Hypocotyl lengths of five day-old seedlings of two independent ProCIB1:CIB1-SRDX lines (6, 8 pCIB1:CIB1sx; green bars) and wild-type plants (WT; gray bars). For the white + FR light treatment, three day-old seedlings grown on agar plates under white light were transferred to white + FR light and incubated for 2 d. Asterisks indicate significant data (P<0.01, Student's t-test) between the WT and other lines. Error bars indicate SD (n>84). (C) Morphological analysis of ProCIB1:CIB1-SRDX (pCIB1:CIB1sx) plants under continuous dark (DD) treatment. Left, seven day-old seedlings of two independent lines (6, 8) of ProCIB1:CIB1-SRDX (pCIB1:CIB1sx; two seedlings on the right) and wild-type (left) seedlings under DD. Scale bar=1 mm. Right, hypocotyl lengths of seven day-old seedlings of two independent ProCIB1:CIB1-SRDX (6, 8 pCIB1:CIB1sx; green bars) lines and wild-type plants (WT; gray bar) under DD. Asterisks indicate P<0.001 between the WT and other lines. Error bars indicate SD (n>35). (D) Left, morphological analysis of ProCIB1:CIB1-SRDX line 6 (pCIB1:CIB1sx) plants under high temperature (28 °C) conditions. Four day-old seedlings grown on agar plates at 22 °C were transferred to 28 °C and incubated for 3 d. Scale bar=1 mm. Right, hypocotyl lengths of seven day-old seedlings of two independent ProCIB1:CIB1-SRDX (6, 8 pCIB1:CIB1sx; green bars) lines and wild-type (WT; gray bar) plants grown under high temperature (28 °C) conditions. Asterisks indicate significant data (P<0.01, Student's t-test) between the WT and other lines. Error bars indicate SD (n>75). (E) Schematic representation of the point mutations obtained by genome editing of CIB1. (F) Hypocotyl lengths of five day-old seedlings of three independent cib1 lines (pink bars) and two independent lots of wild-type plants (WT; gray bars). For the white + FR light treatment, three day-old seedlings grown on agar plates under white light were transferred to white + FR light and incubated for 2 d. Asterisks indicate significant data (P<0.02, Student's t-test) between the wild type and other lines. Error bars indicate SD (n>103).
Next, we examined the phenotype of ProCIB1:CIB1-SRDX seedlings in the dark. These seedlings had shorter hypocotyls than wild-type seedlings when grown under continuous dark conditions (Fig. 3C). In addition, we observed defects in apical hook formation in ProCIB1:CIB1-SRDX seedlings under continuous dark conditions (Fig. 3C, left panel). These results suggest that CIB1 is involved in light-regulated hypocotyl elongation and skotomorphogenesis.
We analysed whether CIB1 is involved in regulating hypocotyl elongation at high temperatures. Seven day-old ProCIB1:CIB1-SRDX seedlings (four day-old seedlings grown on agar plates at 22 °C were transferred to 28 °C and incubated for 3 d) had shorter hypocotyls than wild-type seedlings (Fig. 3D). By contrast, the hypocotyl lengths of ProCIB1:CIB1-SRDX seedlings grown at 22 °C under white light were similar to those of the wild type (Fig. 3A, B). These data suggest that ProCIB1:CIB1-SRDX plants were less sensitive to high temperature than the wild type. Morphological analysis of ProCIB1:CIB1-SRDX and Pro35S:CIB1 plants indicated that CIB1 positively regulates heat- and light-induced hypocotyl elongation.
To confirm the function of CIB1, we generated three CIB1 knock-out lines using CRISPR/Cas9 technique (Fig. 3E; Tsutsui and Higashiyama, 2017) and analysed the hypocotyl lengths of these cib1 lines under white light, white + FR light, and continuous dark conditions. The hypocotyl lengths of cib1 seedlings grown in the dark and white light were not significantly different (the P-values under dark condition on cib1-3 is 0.03 and under white light consition on cib1-1, cib1-2 and cib1-3 are 0.73, 0.01 and 0.68, respectively, Student's t-test) from those of the wild type (Fig. 3F, Supplementary Fig. S3). When seedlings were treated with FR light, however, the hypocotyls of cib1 seedlings were slightly but significantly shorter than those of the wild type (Fig. 3F). These results support the idea that CIB1 is involved in cell elongation under FR light.
CIB1 expression is positively regulated by PIF4
Next, we analysed the expression of CIB1. Quantitative RT–PCR analysis revealed that CIB1 expression was induced by FR light, auxin (naphthaleneacetic acid; NAA), and high temperature (28 °C), but not by brassinolide (BL) treatment (Fig. 4A–C; Supplementary Figs S4A, B; S5A, B). PAR1, SAUR15 (SMALL AUXIN UP RNAS 15), and SAUR68 were used as positive controls when we examined the responses to FR light and auxin treatment. PIFs regulate cell elongation via various signaling pathways, including pathways mediating the responses to FR light and high temperature (Castillon et al., 2007; Jeong and Choi, 2013; Leivar and Monte, 2014). Publicly available microarray data (Leivar et al., 2009) show that CIB1 expression was lower in the pif1 pif3 pif4 pif5 quadruple mutant (pifq) than in the wild type under continuous dark treatment (Shin et al., 2009). Using qRT–PCR, we analysed the expression of CIB1 under white light, FR light, and continuous dark conditions. CIB1 expression was significantly lower in the pif3 pif4 pif5 triple mutants and the pifq quadruple mutant than in the wild type under continuous dark treatment (the P values on the pif3 pif4 pif5 triple mutants and pifq quadruple mutant are 0.007 and 0.002, respectively, Student's t-test) (Fig. 4D; Supplementary Fig. S5C).
Expression analysis of CIB1. (A) Expression of CIB1, PAR1, and SAUR15 analysed by RT–qPCR in wild-type plants under FR treatment for 0, 1, and 3 h. The expression of UBIQUITIN1 was used to normalize the results; for each gene, the expression in wild-type plants without FR treatment was set to 1. (B, C) Expression of CIB1 analysed by RT–qPCR in wild-type plants treated with 0.2 mg l-1 NAA for 0, 0.5, 1, 3 and 6 h (B) and under high temperature conditions of 28 °C. (C). The expression of UBIQUITIN1 was used to normalize the results; the expression at 0 h (22 °C) was set to 1. Asterisks indicate P<0.01 compared with each non-NAA-treated control. (D) CIB1 expression analysed by RT–qPCR in wild-type and pif plants under continuous dark treatment (DD). The expression of UBIQUITIN1 was used to normalize the results; the expression in the wild type was set to 1. Asterisks indicate significant data (P<0.01, Student's t-test) compared with the wild type. (E) CIB1 expression analysed by RT–qPCR in wild-type plants and Pro35S:PIF4 (PIF4ox) plants with the long hypocotyl phenotype. The expression of UBIQUITIN1 was used to normalize the results; the expression in wild-type lot 1 was set to 1.
We generated transgenic plants ectopically expressing PIF4 (Pro35S:PIF4). The hypocotyls of Pro35S:PIF4 seedlings were elongated (Supplementary Fig. S6A), and CIB1 expression was up-regulated in these plants depending on the expression of PIF4 (Fig. 4E; Supplementary Fig. S6B). These results indicate that PIF4 positively regulates CIB1 expression.
PIF4 directly activates CIB1 expression, and HFR1 and PAR1 antagonistically suppress CIB1 expression
Transgenic Arabidopsis expressing the PIF4 chimeric repressor (Pro35S:PIF4-SRDX) exhibited a dwarf phenotype and had rounded leaves, as did Pro35S:CIB1-SRDX plants (Supplementary Fig. S6C), indicating that PIF4 acts as a transcriptional activator of organ elongation. We performed transient expression assays using a reporter construct in which 2130 bp of the 5′ region upstream of the translation initiation site of CIB1 was placed upstream of the LUC reporter gene (ProCIB1:LUC; Fig. 5A). The ProCIB1:LUC reporter gene was up-regulated several-fold when co-expressed with the Pro35S:PIF4 effector construct (Fig. 5B). These results indicate that PIF4 positively regulates the CIB1 promoter.
Regulation of CIB1 expression by PIF4, PAR1, and HFR1. (A) Schematic representation of the constructs used for transient expression analysis using the ProCIB1:LUC reporter gene. The ProCIB1:LUC reporter gene contained 2130 bp of the 5’ region upstream of the translation initiation site of CIB1 fused upstream of LUC (black box), and the effector constructs for PIF4, HFR1, and PAR1 driven by the CaMV 35S promoter are shown. Ω, translation enhancer sequence derived from Tobacco mosaic virus. (B) Relative luciferase activities after co-bombardment of Arabidopsis leaves with the PIF4 effector construct and the ProCIB1:LUC reporter construct. The relative activity in the vector control (top bar) was set to 1. Error bars indicate SD (n=6). (C) EMSA of the binding of PIF4 to the CIB1 promoter. Above, gene structure of CIB1. The open box represents the coding region of CIB1, the black line represents the 5′ upstream region of CIB1, and the black boxes represent G-box motifs. The regions used as probes and competitors are indicated by a short line (CIB1 promoter probe, CIB1pro). Below, EMSA using the CIB1pro fragment as a probe; PIF4 was fused with MBP (PIF4-MBP) as the test protein and the CIB1 promoter fragment as competitors. The CIB1promoter probe was incubated with PIF4-MBP (0.5 µg). Unlabeled CIB1pro (lanes 2–4) and mutated CIB1 promoter fragment (mut; lanes 5 and 6) were used as competitor DNAs. The arrow indicates the specific PIF4-MBP–DNA complex. (D) Competitive transient expression assays using the ProCIB1:LUC reporter. The relative luciferase activities after co-bombarding Arabidopsis leaves with PIF4, HFR1, and PAR1 effectors and the ProCIB1:LUC reporter. The relative activity in the vector control (top bar) was set to 1. Error bars indicate SD (n=6). Asterisks indicate P<0.05 between the PIF4 effector and others. Numbers indicate the ratio of each effector construct (1=0.3 µg for one experiment). (E) PAR1 inhibits the binding of PIF4 to the CIB1 promoter probe. EMSA using the PIF4-MBP fusion protein, PAR1, and MBP proteins, which were produced in Escherichia coli. MBP was used as a control protein. The CIB1 promoter probe (all lanes) was incubated with PIF4-MBP (lanes 1–5), PAR1 (lanes 3–6), and MBP (lane 1). Numbers indicate the ratio of protein (1=0.5 µg for one lane). The arrow indicates the specific PIF4-MBP–DNA complex.
The 5’ upstream region of CIB1 contains two G-box sequences (upper panel of Fig. 5C), which are known binding sites of PIF4 (Huq and Quail, 2002). In an electrophoretic mobility shift assay (EMSA) using the fragment at position –424 to –394 of the 5′ upstream region of CIB1 (CIB1pro), the specific shifted band of the CIB1pro DNA and PIF4 complex disappeared in a dose-dependent manner when unlabeled CIB1pro competitor was added to the assay (at lanes 3 and 4 of the lower panel of Fig. 5C). Such inhibition was not observed when a mutated competitor (Supplementary Table S1) was used (Fig. 5C). These results indicate that PIF4 binds specifically to the CIB1 promoter, likely to the G-box region.
HFR1 and PAR1 negatively regulate cell elongation (Fairchild et al., 2000; Roig-Villanova et al., 2007; Galstyan et al., 2011) by interacting with PIF4 and interfering with its DNA binding activity (Hornitschek et al., 2009; Hao et al., 2012). To analyse whether PAR1 and HFR1 inhibit the PIF4-mediated expression of CIB1, we performed competition experiments via transient expression of the ProCIB1:LUC reporter gene (Fig. 5A) in Arabidopsis leaves. The expression of the ProCIB1:LUC reporter, which was activated by PIF4, was suppressed when either three-fold dose of Pro35S:HFR1 or Pro35S:PAR1 was co-expressed in Arabidopsis leaves (Fig. 5D), indicating that PAR1 and HFR1 inhibit the ability of PIF4 to activate gene expression from the CIB1 promoter.
In competitive EMSAs, the binding of PIF4 to the CIB1pro probe was disrupted in the presence of PAR1 in a dose-dependent manner (Fig. 5E). Because PAR1 did not bind to the CIB1pro fragment (lane 6 of Fig. 5E), it is likely that PAR1 interferes with the binding of PIF4 to the CIB1 promoter, possibly by forming a heterodimer with PIF4, as described in a previous report (Hao et al., 2012). We also generated transgenic plants ectopically expressing PAR1 and HFR1 (Pro35S:PAR1 and Pro35S:HFR1). qRT–PCR assays revealed that CIB1 was significantly down-regulated in Pro35S:PAR1 and Pro35S:HFR1 seedlings compared with wild-type seedlings (Supplementary Fig. S7), indicating that PAR1 and HFR1 negatively regulate the expression of CIB1.
CIB1 promotes cell elongation downstream of PIF4
To confirm our hypothesis that CIB1 regulates cell elongation downstream of the PIF4 signaling cascade, we generated transgenic plants expressing both Pro35S:CIB1-SRDX and Pro35S:PIF4 (PIF4ox CIB1sx). Approximately 24% of the Pro35S:PIF4 (PIF4ox) seedlings had very long hypocotyls (Fig. 6B; Supplementary Fig. S6A), but the hypocotyls of most PIF4ox CIB1sx seedlings were similar to those of the wild type, as only 5.6% of these seedlings had long hypocotyls (Fig. 6A, B). These results indicate that CIB1-SRDX suppresses the effects of ectopically expressed PIF4.
Analysis of the role of CIB1 in the PIF4 signaling pathway. (A, B) Morphological analysis of plants expressing P35S:PIF4 and/or P35S:CIB1-SRDX transgenic plants. (A) Phenotypes of seven day-old Pro35S:PIF4/Pro35S:CIB1-SRDX seedlings, which express PIF4 as well as the chimeric CIB1 repressor. Scale bar=1 mm. (B) Hypocotyl phenotypes of P35S:PIF4 and P35S:CIB1-SRDX transgenic plants. (C, D) Morphological analysis of plants expressing Pro35S:CIB1 in the pifq mutant background (pifqCIB1ox). (C) Phenotypes of WT, pifq, and pifqCIB1ox seedlings (22, 11) under continuous dark treatment. Scale bar=1 mm. (D) Hypocotyl lengths of seven day-old seedlings of three independent lines (10, 22 11) of pifqCIB1ox, or pifq, and wild type (WT) under continuous dark treatment. Asterisks indicate significant data (P<0.05 in Student's t-test) between the wild type and pifq. Error bars indicate SD (n>25).
We also expressed Pro35S:CIB1 in the pifq mutants. The hypocotyl lengths of Pro35S:CIB1 pifq seedlings (in which the transgene was homozygous and present as a single insertion) were similar to those of the wild type, but longer than those of the pifq mutant under continuous dark treatment (Fig. 6C, D). Therefore, Pro35S:CIB1 appears to suppress the short hypocotyl phenotype of the pifq mutants under dark treatment. Apical hook formation was observed in wild-type seedlings but not in Pro35S:CIB1 pifq seedlings, and the cotyledon-opening phenotype of the pifq mutants was suppressed by the expression of Pro35S:CIB1 (CIB1ox) in dark-treated seedlings (Fig. 6C; Table 1). These results suggest that CIB1 partially suppresses the skotomorphogenesis defect of the pifq mutants under continuous dark treatment, and that CIB1 regulates hypocotyl elongation and opening of cotyledons by acting downstream of PIF4.
Hook formation in pifq and P35S:CIB1 plants
| Line no. | Hook formation (%) | No hooks (%) | Cotyledon opening (%) | n | |
|---|---|---|---|---|---|
| pifq | 0% | 3.4% | 96.6% | 116 | |
| CIB1oxpifq | #40 | 0% | 44.4% | 55.6% | 72 |
| #22 | 0% | 50.0% | 50.0% | 50 | |
| #11 | 0% | 50.9% | 49.1% | 57 | |
| Wild type | 100% | 0% | 0% | 69 |
| Line no. | Hook formation (%) | No hooks (%) | Cotyledon opening (%) | n | |
|---|---|---|---|---|---|
| pifq | 0% | 3.4% | 96.6% | 116 | |
| CIB1oxpifq | #40 | 0% | 44.4% | 55.6% | 72 |
| #22 | 0% | 50.0% | 50.0% | 50 | |
| #11 | 0% | 50.9% | 49.1% | 57 | |
| Wild type | 100% | 0% | 0% | 69 |
Hook formation in pifq and P35S:CIB1 plants
| Line no. | Hook formation (%) | No hooks (%) | Cotyledon opening (%) | n | |
|---|---|---|---|---|---|
| pifq | 0% | 3.4% | 96.6% | 116 | |
| CIB1oxpifq | #40 | 0% | 44.4% | 55.6% | 72 |
| #22 | 0% | 50.0% | 50.0% | 50 | |
| #11 | 0% | 50.9% | 49.1% | 57 | |
| Wild type | 100% | 0% | 0% | 69 |
| Line no. | Hook formation (%) | No hooks (%) | Cotyledon opening (%) | n | |
|---|---|---|---|---|---|
| pifq | 0% | 3.4% | 96.6% | 116 | |
| CIB1oxpifq | #40 | 0% | 44.4% | 55.6% | 72 |
| #22 | 0% | 50.0% | 50.0% | 50 | |
| #11 | 0% | 50.9% | 49.1% | 57 | |
| Wild type | 100% | 0% | 0% | 69 |
To examine whether CIB1 indeed regulates the expression of genes downstream of PIF4, we analysed the expression of the shade-inducible genes PAR1, HFR1, homeodomain-leucine zipper protein 4 (HAT4), XYLOGLUCAN ENDOTRANSGLYCOSYLASE 7 (XTR7), and INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19); these genes are down-regulated in pifq mutants in the dark (Lorrain et al., 2008; Hornitschek et al., 2009, 2012; Hao et al., 2012). In Pro35S:CIB1 plants, PAR1, HFR1, and HAT4 expression increased, but XTR7 and IAA19 expression did not change compared with the wild type (Supplementary Fig. S8), indicating that CIB1 regulates only a subset of genes downstream of PIF4.
Even though all five genes contain an E/G box (CACGTG; the typical binding sequence of bHLH) in their 3000 bp upstream regions, publicly available DNA affinity purification (DAP)-seq data showed that only the upstream region of PAR1 is directly bound by bHLH031. Among the bHLHs with DAP-seq data, bHLH031 is the most closely related to CIB1 (Supplementary Fig. S9; O’Malley et al., 2016). These data suggest that CIB1 could directly regulate PAR1 expression.
CIB1 regulates cell elongation in this HLH/bHLH system
It has previously been reported that CIB1 functions as a transcriptional activator (Liu et al., 2008), which is consistent with our observation that Pro35S:CIB1-SRDX and Pro35S:CIB1 plants exhibited opposite phenotypes (Figs 1, 2). In addition, our transient expression assay using the CIBEx4:LUC reporter construct with four tandem repeats of the CIB-binding element (CIBE; Supplementary Table S1; Liu et al., 2008; Fig. 7A) showed that expression of the CIBEx4:LUC reporter gene was up-regulated several fold when co-expressed with the Pro35S:CIB1 (CIB1) effector (Fig. 7A). These results support our hypothesis that CIB1 acts as a transcriptional activator.
Analysis of the antagonistic inhibition of CIB1 by PAR1. (A) Transient expression assays using the CIBEx4:LUC reporter gene. Above, schematic representation of the constructs used in transient expression analysis. The CIBEx4:LUC reporter gene, which contains four copies of the CIB-binding element (4×CIBE; Supplementary Table S1; Liu et al., 2008) fused upstream of the LUC gene (LUC; shown as a black box), and effector constructs for CIB1, HFR1, and PAR1 driven by the CaMV 35S promoter are shown. Ω, translation enhancer sequence derived from Tobacco mosaic virus. Below, relative luciferase activities after co-bombarding Arabidopsis leaves with CIB1, HFR1, and PAR1 effector constructs and the CIBEx4:LUC reporter. The relative activity in the vector control (top bar) is denoted as 1. Error bars indicate SD (n=6). Asterisks indicate significant data (P<0.05, Student's t-test) between the CIB1 effector and other constructs. Numbers indicate the amount of effector construct (1=0.3 µg for one experiment). (B) Interaction between CIB1 and the non-DNA-binding HLH factors HFR1 and PAR1 in a yeast two-hybrid assay on –leucine –histidine medium containing 5 mM 3AT using CIB1 as prey. (C) BiFC assay to detect the interaction between CIB1 and PAR1 in onion epidermal cells. Percentage of cells with bright green fluorescence in the nucleus among cells with red fluorescence is shown (n≥90). (D) PAR1 inhibits the binding of CIB1 to the CIBE probe. Electrophoretic mobility shift assays (EMSAs) were performed using CIB1, PAR1, and MBP proteins produced in E. coli. MBP was used as a control protein. The CIBE probe (all lanes) was incubated with CIB1 (lanes 1–6), PAR1 (lanes 4–7), or MBP (lane 2). Unlabeled CIBE probe (CIBE; lane 1) was used as the cold competitor DNA. Numbers indicate the ratio of protein (1=0.6 µg for one lane). The arrow indicates the specific CIB1–DNA complex.
To reveal the mechanisms by which CIB1 regulates cell elongation, we searched for proteins that interact with CIB1. Yeast two-hybrid analysis revealed that PAR1 interacted with CIB1, but HFR1 did not (Fig. 7B). In addition, we confirmed the interaction of PAR1 with CIB1 in vivo by BiFC assays (Fig. 7C). Competitive transient expression analysis revealed that the activation of the CIBE×4:LUC reporter gene by the Pro35S:CIB1 effector was suppressed to basal amounts when co-expressed with the Pro35S:PAR1 effector (Fig. 7A). By contrast, the Pro35S:HFR1 effector did not suppress activation of the CIBE×4:LUC reporter by CIB1 (Fig. 7A). This is likely because HFR1 did not interact with CIB1, which is consistent with the results of the yeast two-hybrid assay. These results indicate that the non-DNA-binding HLH protein PAR1 inhibits the transcriptional activation activity of the CIB1 effector.
Like Pro35S:CIB1-SRDX plants, Pro35S:PAR1 plants had a dwarf stature and round, dark-green leaves (Fig. 1A; Supplementary Fig. S10; Roig-Villanova et al., 2007). Pro35S:HFR1 plants had a similar phenotype, although the dwarfism and dark-green color were less pronounced (Supplementary Fig. S10). The ability of PAR1 to interfere with DNA binding by CIB1 was confirmed by EMSA (Fig. 7D). The shifted bands of the CIB1–CIBE complex disappeared in a dose-dependent manner when PAR1 protein was added to the assay (Fig. 7D). Because PAR1 did not bind to the CIBE fragment, it is likely that PAR1 interferes with the ability of CIB1 to bind to the CIBE fragment by forming a heterodimer with CIB1, not by competing with CIB1 (Fig. 7D). These results indicate that PAR1, a non-DNA-binding HLH, inhibits CIB1 activity by forming PAR1–CIB1 HLH/bHLH heterodimers.
Discussion
We previously demonstrated that tri-antagonistic HLH/bHLH systems composed of typical bHLH transcriptional activators (ACEs and CIB5) and two groups of non-DNA-binding atypical HLH proteins (IBH1 and PRE1) regulate cell elongation in response to GA and BR (Bai et al., 2012; Ikeda et al., 2012, 2013). In the current study, we demonstrated that cell elongation mediated by light and high temperature signals is regulated by a two-step antagonistic HLH/bHLH system composed of two typical bHLH factors, PIF4 and CIB1, and two non-DNA-binding atypical HLH proteins, PAR1 and HFR1. In this system (summarized in Fig. 8), PIF4 directly activates the expression of CIB1, and PAR1 and HFR1 inhibit the activation of PIF4 (Fig. 5). Meanwhile, CIB1 positively regulates cell elongation, and PAR1 interacts with CIB1 to inhibit its activation activity (Figs 1, 2, 3, 7).
Summary of the multi-layer antagonistic HLH/bHLH cascade that regulates cell elongation in response to light and high temperatures in Arabidopsis. PIF4 (open circle), PAR1 (black semi-circle), and HFR1 (gray semi-circle) function in the first step of the antagonistic HLH/bHLH system, which regulates CIB1 expression under FR light and high temperature signaling. PIF4 can bind to the CIB1 promoter and activate its transcription. PAR1 and HFR1 interfere with the formation of the PIF4 DNA-binding complex by forming heterodimers with PIF4. FR light, BR, and GA regulate PIF4 activity, and FR light regulates PAR1 expression. CIB1 (gray circle) and PAR1 (black semi-circle) act in the second step of the antagonistic HLH/bHLH system, which regulates cell elongation in Arabidopsis. CIB1 proteins form dimers, which can bind to cis-elements in the promoters of genes required for cell elongation and activate their transcription. PAR1 interferes with the formation of the CIB1 DNA-binding complex by forming heterodimers with CIB1. PRE1 interacts with PAR1 and inhibits the formation of PAR1–CIB1 and PAR1–PIF4 heterodimers.
PIF4 regulates CIB1 via an antagonistic HLH/bHLH system
PIF4 functions as a key factor in light- and high temperature-regulated cell elongation (Castillon et al., 2007; Lorrain et al., 2008; Koini et al., 2009; Leivar and Quail, 2011; Jeong and Choi, 2013; Leivar and Monte, 2014). PIF4 directly regulates the expression of shade response factors, such as HFR1 and PIL1, and auxin biosynthesis and signaling factors under shade and high temperature conditions (Hornitschek et al., 2009, 2012; Franklin et al., 2011). In addition, PIF4 interacts with BRASSINAZOLE RESISTANT 1 (BZR1), an important factor for BR signaling, to activate PRE gene expression (Hyun and Lee, 2006; Lee et al., 2006; Oh et al., 2012). CIB1 was previously identified as a high-confidence PIF4 target gene based on ChIP-seq experiments with an anti-PIF4 antibody (Oh et al., 2012). Our transient expression assay and EMSAs clearly showed that PIF4 binds to the promoter of CIB1 and activates its expression (Fig. 5), indicating that PIF4 directly regulates the expression of CIB1.
DELLAs, phyB, and the atypical HLH proteins HFR1 and PAR1 inhibit the DNA binding activity of PIF proteins (Fairchild et al., 2000; de Lucas et al., 2008; Feng et al., 2008; Hornitschek et al., 2009; Hao et al., 2012; Park et al., 2012). On the other hand, the non-DNA-binding atypical HLH protein PRE1 interacts with PAR1 and interferes with its inhibition of PIF4 function (Fig. 7C; Hao et al., 2012). To regulate CIB1 expression, HFR1 and PAR1 interact with PIF4 and interfere with its binding to the CIB1 promoter (Fig. 5). These findings indicate that the expression of CIB1 is regulated by an antagonistic HLH/bHLH system composed of PIF4, HFR1/PAR1, and PRE1 (Fig. 8).
CIB1 activity is regulated by an antagonistic HLH/bHLH system
CIB1 promotes floral initiation by interacting with CRY2 in a blue-light-specific manner (Liu et al., 2008) and functions in immunity and leaf senescence (Meng et al., 2013; Malinovsky et al., 2014). CIB5, which functions redundantly with CIB1, also interacts with CRY2 to promote floral initiation (Liu et al., 2008). CIB5 and ACE1 function redundantly as positive regulators of cell elongation (Ikeda et al., 2012). Here, we demonstrated that CIB1 is a positive regulator of light- and high temperature-mediated cell elongation. The ectopic expression of CIB1 enhanced hypocotyl elongation, even at 80 µmol m-2 s-1 (Fig. 2), and the suppression of CIB1 expression inhibited organ elongation under FR light, continuous dark, and high temperature conditions (Figs 1, 3). CIB1 and genes with similar activity also positively regulate skotomorphogenesis. Transgenic plants with suppressed CIB1 activity showed defects in apical hook formation in the dark (Fig. 3C). These results point to the involvement of CIB1 in dark-mediated morphogenesis in addition to blue light responses.
We previously demonstrated that CIB5 and ACE1 directly activate the expression of genes related to cell elongation (Ikeda et al., 2012). Moreover, CIB1 acts as a transcriptional activator in tobacco leaves (Meng et al., 2013). Our EMSAs and transient expression assays revealed that CIB1 positively regulated the expression of a reporter gene in Arabidopsis leaves by binding to CIBE in the promoter of LUC reporter construct driving its expression (Fig. 7). CIB5 and ACE1 regulate downstream genes through a tri-antagonistic HLH/bHLH system (Ikeda et al., 2012). The non-DNA-binding HLH protein IBH1 inhibits the DNA binding activities of ACE1 and CIB5 (Ikeda et al., 2012). We found that non-DNA binding protein PAR1 interacts with CIB1 and interferes with its DNA-binding activity (Fig. 7). These results indicate that CIB1 and PAR1 constitute an antagonistic HLH/bHLH system that regulates the expression of its downstream genes (Fig. 8).
Two-step antagonistic HLH/bHLH systems regulate cell elongation
Our complementation analyses demonstrated that two antagonistic HLH/bHLH systems regulate cell elongation in the same regulatory cascade. We showed that the ectopic expression of CIB1 suppressed the short hypocotyl and skotomorphogenesis phenotypes of pif quadruple mutants under continuous dark treatment (Fig. 6; Table 1). In addition, the chimeric CIB1 repressor suppressed the long hypocotyl phenotype of Pro35S:PIF4 plants (Fig. 6). These data indicate that an antagonistic HLH/bHLH system composed of CIB1 and PAR1 regulates light- and high temperature-mediated cell elongation downstream of the PIF4–HFR1/PAR1 antagonistic HLH/bHLH cascade.
CIB1 might act as an enhancer to maintain signals from PIFs. PIF proteins have very short half-lives of only 5–20 min under various light treatments (Leivar and Quail, 2011; Jeong and Choi, 2013; Leivar and Monte, 2014). Following their interaction with phyA and/or phyB, PIFs are rapidly phosphorylated, ubiquitinated, and degraded via the ubiquitin-proteasome system (Park et al., 2004; Al-Sady et al., 2006; Shen et al., 2007, 2008; Lorrain et al., 2008). The BR signaling kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) is involved in the phosphorylation of PIF4 (Bernardo-Garcia et al., 2014), indicating that BR signaling also regulates the degradation of PIFs. This suggests that it is difficult to maintain PIF4-mediated cell elongation because the abundance and activity of PIF4 protein can easily decrease. By contrast, the half-life of CIB1 appears to be longer than those of PIFs. CIB1 protein is degraded by the 26S proteasome system under darkness, red light, or FR light (H. Liu et al., 2013). However, a small amount of CIB1 protein was detected in P35S:Myc-CIB1 plants when they were transferred from white light to darkness or red light for 16 h (H. Liu et al., 2013). In the current study, ectopically expressed CIB1 induced cell elongation in the pifq mutants under dark treatment (Fig. 6). These results suggest that a small amount of CIB1 protein might induce cell elongation independent of PIFs, and that cell elongation signaling might be maintained by the expression of CIB1 proteins after PIF proteins have degraded.
The abundance and activity of each protein involved in the two-step antagonistic HLH/bHLH cascade, including the transcriptional activators PIF4 and CIB1, and the atypical HLH factors PAR1 and HFR1, might be important for the regulation of cell elongation. PIF4 and CIB1 bind to the promoter of a target gene and activate its transcription. PAR1 and HFR1 interfere with the formation of the PIF4 DNA-binding complex by forming heterodimers with PIF4. PAR1 also interferes with the formation of the CIB1 DNA-binding complex by forming heterodimers with CIB1. Multiple phytohormones, environmental conditions, and the atypical HLH protein PRE1 might regulate the abundance and activity of each protein (Fig. 8). PRE1, an atypical HLH protein, interacts with PAR1 (Fig. 7C) and interferes with its suppression of PIF4 function (Hao et al., 2012). GA enhances the DNA binding activity of PIF4 by destabilizing DELLAs, which in turn inhibit DNA binding by interacting with PIF4 (de Lucas et al., 2008). Red light induces the degradation of PIF4 and CIB1 (Park et al., 2004, 2012; Al-Sady et al., 2006; Shen et al., 2007, 2008; Lorrain et al., 2008; Leivar and Quail, 2011; Jeong and Choi, 2013; H. Liu et al., 2013; Leivar and Monte, 2014), while BR inhibits the degradation of PIF4 (Bernardo-Garcia et al., 2014). These observations suggest that phytohormones and light conditions control the balance of the two-step antagonistic HLH/bHLH cascade. This system might play essential roles in the interactions of various environmental factors and the control of cell elongation.
Supplementary data
The following supplementary data are available at JXB online.
Table S1. Oligonucleotides used in this study.
Fig. S1. Light spectra of W and W + FR conditions.
Fig. S2. Rosettes of 15 day-old ProCIB1:CIB1SRDX and wild-type plants.
Fig. S3. Hypocotyl lengths of the seedlings of cib1 knock-out lines under continuous dark conditions.
Fig. S4. Effect of BL treatment on CIB1 expression.
Fig. S5. Expression analysis of CIB1.
Fig. S6. Analysis of PIF4-overexpressing plants and PIF4 CRES-T plants.
Fig. S7. CIB1 expression in Pro35S:PAR1 and Pro35S:HFR1 plants.
Fig. S8. Gene expression analysis of Pro35S:CIB1 plants.
Fig. S9. Phylogenetic tree of Arabidopsis bHLH genes closely related to CIB1.
Fig. S10. Morphological analyses of plants overexpressing CIB1-interacting bHLHs.
Acknowledgements
The authors thank E. Ito and Dr T. Ueda in the University of Tokyo for the NSC and C2 vectors and also thank Dr T. Uemura in the University of Tokyo for the 35S-driven mRFP-fused VAM3 plasmid. The authors thank Ms Yoko Ooi, Ms Fumie Tobe, and Ms Yuko Takiguchi for their skilled technical assistance and Ms Sumiko Takahashi, Ms Yoshimi Sugimoto, Ms Tomomi Kimura, and Ms Haruko Nishiyama for cultivating the plants. This study was supported by Japan Society for the Promotion of Science (JSPS) Grant Numbers JP26840102, 20K05953 and by the Naito Foundation.
Author contributions
MI and NM conceived the research and designed the experiments; NM provided the materials, MI, TI and MS performed the experiments; MI and NM analysed the data; MI and MO-T acquired the financial support; MI and NM wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Data availability
The data supporting the findings of this study are available from the corresponding author (Miho Ikeda) upon request.
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