Dissection of HY5/HYH expression in Arabidopsis reveals a root-autonomous HY5-mediated photomorphogenic pathway

ELONGATED HYPOCOTYL 5 (HY5), a member of the bZIP gene family, is a positive regulator of the light signaling pathway in Arabidopsis thaliana. Whereas the hy5 mutant exhibits an elongated hypocotyl when grown in the light, the hy5 homolog (hyh) mutant does not. Although the functions of HY5 and HYH in light-mediated seedling development have been revealed, the tissue-specific expression patterns of HY5 and HYH and their interconnected regulation are largely unknown. Here, we report that HY5 regulates HYH expression in roots and contributes to root growth under different light conditions. We generated HY5 and HYH transcriptional and translational fusion reporter lines to investigate their expression patterns. HY5 was constitutively expressed in all root tissues, while HYH was predominantly expressed in root xylem cells. Root growth after a dark-to-light transition was perturbed in the hy5 and hy5hyh mutant lines, but not in the hyh mutant line, indicating that HY5 plays a major role in light-regulated root growth. Light-induced HY5/HYH expression occurred autonomously in roots. HYH expression in roots was decreased in the hy5 mutant, suggesting that HY5 regulates HYH expression. Collectively, these results indicate that an organ-specific HY5-mediated pathway controls root photomorphogenic development independently of light signaling in the shoot.

In nature, the root systems of most terrestrial plants are underground and in darkness; however, roots contain photoreceptors that may detect ambient light through the soil [17,18], and can also respond to direct illumination when grown in transparent Petri dishes [19][20][21]. The root cap is reported to be essential for the photophobic root behavior in maize, which implies that a local light response exists in roots [22]. The SCAR/WAVE complex, which facilitates nucleation of new short F-actin branches from existing filaments, mediates light-induced root elongation independently from the shoot in Arabidopsis [23]. In contrast to the well-studied light responses of aboveground tissues, the mechanism by which light regulates root development is not well understood. Previous work has suggested that COP1-mediated auxin transport is involved in adapting root growth to the ambient light conditions [24], while the light-dependent signaling of HY5, the best-characterized target of COP1-mediated proteolysis, plays an important role in PIN2 intracellular distribution in roots [25]. Recently, it was reported that aboveground light is directly transmitted through the stem and activates phytochrome B in the roots, triggering HY5-mediated light responses [26]. Moreover, HY5 mediates the promotion of root growth and nitrate uptake in response to light, acting as a shoot-to-root mobile signal [27]. However, the mechanism by which HY5 regulates root photomorphogenesis is largely unknown. Root growth in response to light is therefore mostly influenced by shoot-derived signals; however, we speculate that a root-autonomous light response also exists.
There is growing evidence to suggest that HY5 and HYH expression is upregulated by light in the seedling [7,9,28]. However, their expression in roots has yet to be fully elucidated. A genome-wide study using chromatin immunoprecipitation with DNA chip hybridization (ChIP-chip) revealed that the HYH promoter has putative binding sites for HY5 [10]. A number of HY5 putative target genes identified in the ChIP-chip study were confirmed using a yeast one-hybrid (Y1H) assay and/or chromatin immunoprecipitation, including COP1 [29], FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) [12], and the nitrate uptake gene NITRATE TRANSPORTER 1.1 (NRT1.1/CHL1) [30]. Elucidating the regulatory relationship between HY5 and HYH is a key step in deciphering the response of roots to light.
Here, we investigated the expression patterns of HY5 and HYH, and found that HY5 directly regulates HYH expression in roots. We confirmed that HY5, and to a lesser extent HYH, contributes to root growth under dark/light conditions.

Plant materials and growth conditions
All Arabidopsis mutants used in this study were in the Columbia (Col-0) background. The hy5-2 (SALK_056405), hy5-51 (SALK_096651), and hyh (WISCDSLOX253D10) mutants were obtained from the Nottingham Arabidopsis Stock Centre. The primers used for genotyping are listed in S1 Table. Seedlings were germinated on half-strength Murashige and Skoog (MS) agar plates and then incubated in a near-vertical position at 22˚C under continuous white light (120 μmol m −2 s −1 ) or darkness (packed with foil) in a plant growth chamber for 7 d or as indicated.

Plasmid construction and plant transformation
To generate HY5 and HYH promoter fusion constructs, promoter regions upstream of the start codons of HY5 and HYH were fused to a GUS (β-Glucuronidase) or ERGFP (Endoplasmic Reticulum-Green Fluorescent Protein) reporter gene and nopaline synthase terminator in the pGreenII-0229 plasmid (www.pgreen.ac.uk). To create protein fusion constructs of HY5::HY5: GFP and HYH::HYH:GFP, genomic fragments of HY5 and HYH, including the respective 700-bp and 3000-bp promoter region (up to the next upstream gene), were fused in frame to the N terminus of the GFP reporter gene engineered in pGreenII-0229. The resulting constructs were then transformed into WT and/or hy5 plants using the floral dip method [31]. The primers used for cloning are listed in S1 Table. Phenotypic and expression analyses GUS staining was performed as previously described [24]. Briefly, samples of 7-day-old seedlings were incubated in assay buffer [50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 0.1% Triton X-100, 20% methanol, and 0.5 mg/mL X-Gluc A] at 37˚C until sufficient staining was observed. GUS activity was analyzed with a Nikon 80i microscope using Nomarski interference contrast optics. To prepare cross sections, roots were embedded in 2% agarose, fixed in 4% paraformaldehyde, dehydrated in an ethanol series, embedded in Technovit 7100 (Heraeus Kulzer, EBSciences), and sectioned at 15 μm. For analyses of hypocotyl and root phenotypes, 4-day-old etiolated seedlings were transferred to light for additional days (one additional day for hypocotyl and eight additional days for root). Root tip positions were marked with a pen each day. Seedlings were imaged with a Canon 40D camera and root length was measured using ImageJ (http://rsbweb.nih.gov/ ij/). Quantitative analyses of the root apical meristem (RAM) were performed as previously described [24]. Statistical analyses (pairwise two-tailed Student's t-test) were performed using Microsoft Excel. Confocal imaging was performed using a Leica TCS SP2 microscope. Propidium iodide (Sigma-Aldrich; 10 μg/mL dissolved in water) was used to counterstain root cells. Epifluorescence images were taken using a Leica M205FA stereomicroscope. The roots of the seedlings were submerged in water on the microscope slide before the coverslip was placed over the root. For all light/dark transition experiments, seedlings were grown in continuous light or darkness for 5 d. Seedlings were then transferred to the opposite condition for 24 h. Samples were then imaged under a confocal microscope or stereomicroscope. For the decapitation experiment, 5-day-old light-and dark-grown seedlings were decapitated by a double edge razor blade before the light/dark transition. Each experiment was repeated at least three times.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
ViiA™ 7 Real-Time PCR System (Life Technologies). EF1α was used as reference gene for normalization. The primer sequences used for qRT-PCR analysis are listed in S1 Table. Yeast one-hybrid assays (Y1H) For yeast one-hybrid assays, plasmids harboring AD (activation domain) fusions were cotransformed with the LacZ reporter gene driven by four fragments (frag 1-frag 4) of the HYH promoter into the yeast strain EGY48 [12,32]. Transformants were grown on proper dropout plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for blue color development. Yeast transformation was conducted as described in the Yeast Protocols Handbook (Clontech). The primers used for cloning are listed in S1 Table. Electrophoretic mobility shift assay (EMSA) The full-length coding region of HY5 was first cloned in frame into the SalI-EcoRI sites of the pSPUTK in vitro transition vector (Stratagene). The HY5 protein was synthesized using a TNT SP6 Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions in a total volume of 10 μL [33]. 3' FAM oligonucleotides were synthesized and labeled by the Shanghai Sangon Company (S1 Table). To generate double-stranded oligos, an equal amount of the complementary single-stranded oligos was mixed, heated to 95˚C for 2 min, and annealed by gradually cooling down to 25˚C. To perform the EMSA, HY5 was pre-incubated with the Binding buffer [1 μg poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 2.5% glycerol, and 5 mM MgCl 2 ] at room temperature for 20 min. For the competition assay, non-labeled probe was incubated with protein and Binding buffer at room temperature for 20 min. Then, 1 μL of 3' FAM-labeled probe (10 μM) was added and samples were incubated at room temperature for 20 min. The samples were then subjected to electrophoresis on 6% PAGE gels running with 0.5× Tris-borate-EDTA buffer at 4˚C in darkness for 1 h. The fluorescence signaling was captured using a FLA-5100 fluorescent image analyzer (Fuji).

Accession numbers
Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: HY5, AT5G11260; HYH, AT3G17609.

Expression pattern of HY5 and HYH in response to light
To investigate the expression patterns of the light signaling master regulator HY5 and its homolog HYH, we constructed their promoter reporter lines, HY5::GUS and HYH::GUS, respectively. These lines revealed that the two genes exhibited a similar expression pattern in shoots grown in the light (Fig 1A and S1 Fig). In dark-grown etiolated seedlings, both lines showed GUS activity in the cotyledons; however, HY5 was expressed in the upper hypocotyl, whereas HYH was not ( Fig 1A). Immunoblot analysis of total protein extracts from intact seedlings previously revealed that HY5 and HYH were induced after light treatment [7,9]. We confirmed this finding using the HY5::GUS line, in which HY5 was expressed in the entire hypocotyl following a darkto-light transition. By contrast, HYH was not induced in the hypocotyl (Fig 1A). The increase in hypocotyl elongation of etiolated seedlings after light treatment was enhanced in the hy5-2 and hy5-2 hyh mutant lines, but not in the hyh single mutant (Fig 1B and S1 Data). These results confirm the previous findings that HY5, but not HYH, is a major repressor of hypocotyl elongation during shoot photomorphogenesis.
HY5 and HYH were shown to be differentially expressed in the roots (Fig 1C). In plants grown in the light, HY5 was constitutively expressed in all root tissues, while HYH was predominantly expressed in the root vascular tissues and columella cells (Fig 1C). A root cross section of the HYH::GUS line showed GUS activity in the xylem cells ( Fig 1C). The expression of HY5 and HYH in roots suggests that the HY5-mediated light signaling pathway may also function in root development. We measured the root length of wild-type (WT), hy5-2, hyh, and hy5-2 hyh lines under dark conditions. For the nine days measured, there were no significant differences in the hy5-2, hyh, and hy5-2 hyh lines compared with the WT in darkness ( Figure A in S2 Fig and S2 Data). These observations indicate that HY5 and HYH do not function in early root development under dark conditions. Given that HY5 and HYH were induced by light, we also examined the root length and cortex cell number of WT, hy5-2, hyh, and hy5-2 hyh plants following a dark-to-light transition. After transferring four-day-old etiolated seedlings to the light, the hy5-2 and hy5-2 hyh lines, but not the hyh mutant, exhibited shorter roots and smaller root apical meristems than the WT (Fig 1D and 1E and S1 Data). These results indicate that HY5, but not HYH, promotes root elongation after a transition from dark to light conditions. In addition, the hy5-2 hyh double mutant showed a more severe root phenotype than hy5-2 after eight days of growth in light (Fig 1D and 1E and S1 Data). When grown in the light for 9 to 10 d, the hy5-2 and hy5-2 hyh mutants had shorter roots than the WT and hyh lines, with the shortest roots observed in the double mutant ( Figure B in S2 Fig and S2 Data). These results indicate that HYH plays a role in late root development in response to light. The observation that HY5 has different functions in shoot and root elongation suggest that these systems respond differently to light. Although these homologs exhibit different expression patterns, the root phenotypes suggest that there is functional redundancy between them.

Light induces HY5 and HYH transcripts and proteins in roots
Under the typical agar-plate culture system, the entire Arabidopsis seedling, including the root, is exposed to light; however, the light response and corresponding gene expression patterns of roots have not been extensively examined in previous photomorphogenic studies [5,[19][20][21]. In the present study, we generated the HY5 and HYH transcriptional/translational GFP reporter lines (HY5::ERGFP and HY5::HY5-GFP, HYH::ERGFP, and HYH::HYH-GFP) to investigate their expression in roots in response to light. In addition, the GFP fusions driven by the cauliflower mosaic virus 35S promoter were generated and subsequently introduced into WT or hy5-2 mutant backgrounds and confirmed to function properly using a hypocotyl elongation assay ( S3 Fig and S3 Data). Both HY5 and HYH transcripts and proteins accumulated in roots under the light and dark-to-light transition conditions, but were suppressed in darkness and under the light-to-dark transition conditions (Fig 2). A qRT-PCR analysis determined that the transcriptional levels of HY5 and HYH were 4.9 ± 0.5 (mean ± SD) and 4.2 ± 0.3 times greater in the light than in darkness, respectively (S4 Fig and S4 Data). These results indicate that both HY5 and HYH levels are altered in response to light in the root.

Root-autonomous regulation of HY5 and HYH expression in response to light
Changes in HY5 and HYH expression in roots following exposure to light might be regulated by a signal from the shoot or, alternatively, might be root autonomous. To test these hypotheses, we examined the expression patterns of HY5 and HYH in roots after removing the shoots. In decapitated HY5::HY5-GFP and HYH::HYH-GFP lines, the expression levels of both genes increased and decreased when plants were subjected to the dark-to-light and light-to-dark transition conditions, respectively (Fig 3). These responses were similar in both decapitated plants and intact seedlings, indicating that HY5 and HYH expression in the roots respond to light in a root-autonomous manner.

HYH expression in roots is regulated by HY5
To further study the functions of HY5/HYH in root development in response to light, we investigated the interconnected regulation of HY5 and HYH. Given that HY5 and HYH expression were repressed under dark conditions, we first examined the expression of these genes in the reciprocal mutants using qRT-PCR and reporter lines grown in the light. HY5 transcript levels were similar in both the shoots and roots of the hyh mutant compared with those observed in the WT ( Figure A in S5 Fig and S5 Data). Examination of the HY5::HY5-GFP line indicated that there was no difference in the level of HY5 protein in the roots of the WT and the hyh mutant ( Figure B in S5 Fig), suggesting that HY5 is not regulated by HYH. In a previous study, hy5-2 was demonstrated to be a knockout mutant [34]. However, to exclude any possible T-DNA multi-insertion effects in this line, the experiments were repeated using another T-DNA insertion line, hy5-51 (SALK_096651), which has also been repeatedly used in published research [35]. The hy5-51 mutant is also a knockout line, and exhibits the same phenotype as hy5-2 ( Figure A to C in S6 Fig). The HYH transcript level was repressed in the root but not the shoot of both hy5 mutants ( Figure D and E in S6 Fig and S6 Data). To confirm that HYH is repressed in the hy5-2 mutant, we examined the HYH promoter reporter and protein reporter lines in the hy5-2 background and compared these with the WT. The promoter activity of HYH, represented by HYH::GUS, exhibited no differences in the shoots of the WT and the hy5-2 mutant (Fig 4A and 4B). Similarly, the levels of HYH protein in the cotyledons and hypocotyls were unchanged in the WT and hy5-2 mutant seedlings (Fig 4C to 4F). By contrast, HYH transcript and protein levels in the roots were decreased in the hy5-2 mutant compared with the WT (Fig 4G to 4J). These results indicate the existence of a root-specific HY5-mediated pathway that regulates HYH expression. A previous genome-wide study showed that HYH is one of the targets of the HY5 transcription factor [10], suggesting that HYH is regulated by HY5. To confirm this, we performed a yeast one-hybrid (Y1H) assay. HY5 has been shown to bind to ACGT-containing elements (ACEs) and promote the activation of light-induced genes [10,12,36]. Several putative HY5 binding sites were selected from the HYH promoter region for the Y1H assay (Fig 5A). The expression of the LacZ reporter gene was determined by the production of a blue colored   colony on the X-gal plates. We found that HY5 bound to fragment 3 (frag 3) of the HYH promoter region, which contains three ACEs (Fig 5B). Although frag 1 and frag 4 also contain putative HY5 binding sites, they exhibited no binding activity in our Y1H assay. However, frag 2 showed strong trans-activation activity in yeast cells, because the negative control also appeared blue. To further confirm the binding activity at frag 3, three probes containing one single ACE each were designed and used to perform an electrophoretic mobility shift assay (EMSA; Fig 5C). The EMSA results indicated that HY5 could bind to the frag 3-2 and frag 3-3 regions of fragment 3. Collectively, our results indicate that HY5 binds directly to the HYH promoter at the frag 3 region.

Discussion
The roles of HY5 in plant photomorphogenesis have been extensively studied, with a major focus on the shoot system [10,14,37]. Furthermore, large-scale omics data support the notion that HY5 is a key signal integration point during dark-to-light transitions [10,36,38]. It was recently demonstrated that phyB-activated HY5 mediates cellular responses to light in the root, and is important for primary root growth and root gravitropism [26,39]. HY5 has been reported to be a shoot-to-root mobile signal that mediates the light-responsive coupling of shoot growth and carbon assimilation with root growth and nitrogen uptake in Arabidopsis [27]. These findings suggest that HY5 expression in the root affects both its growth and the expression of other genes in this tissue through two distinct pathways [26,27]. Nevertheless, our knowledge of the molecular mechanisms underlying root photomorphogenesis is fragmented. The accelerated root growth of hy5 was reported to be associated with auxin signaling [13]; however, although HY5 is known to integrate the light and hormone signaling pathways [40], the details of this crosstalk are unclear.
In this study, HY5 and HYH expression patterns were examined at the tissue level and under various light/dark growth conditions using GUS and GFP reporter lines (Fig 1A and 1B,  Fig 2 and S1 Fig). Generally, both HY5 and HYH showed similar expression patterns in the shoots in both darkness and in the light (Fig 1A and S1 Fig). Unlike HY5, HYH was not expressed in hypocotyls following the dark-to-light transition (Fig 1A), suggesting that HY5, but not HYH, contributes to hypocotyl elongation. Interestingly, HY5 and HYH had distinct expression patterns in the root; HY5 was expressed in all root tissues, while HYH was predominantly expressed in the root vascular tissues and the root cap ( Fig 1C). A phenotypic analysis of root length in the WT and hy5-2, hyh, and hy5-2 hyh mutants indicated that growth of the primary root of the hy5-2 mutant, but not the hyh mutant, was suppressed during the dark-tolight transition, with the most severe short-root phenotype observed in the hy5-2 hyh double mutant (Fig 1D and 1E and S2 Fig). These results indicate that HY5 and HYH function in root development during de-etiolation.
Previous immunoblot analyses of total protein extracts from intact seedlings revealed that the expression of both HY5 and HYH is induced by light [7,9]. In the present study, we used reporter lines to provide direct tissue-level evidence that the expression of both HY5 and HYH in the roots is induced by light (Fig 2). Additionally, decapitation experiments suggested that the root response of HY5 and HYH to light occurs in a root-autonomous manner (Fig 3). In combination with the root phenotype of the hy5-2 and hy5-2 hyh mutants, our results indicate that root growth is regulated by the HY5/HYH-mediated light signaling pathway.
Roots may perceive light signals directly by photoreceptors [17,18]. Extensive studies showed that interactions between cryptochrome and phototropin play a key role in root phototropism and architecture [18,[41][42][43]. Recent studies have shown that HY5 positively regulates the activity of its own promoter in a tissue-specific manner [44], and that HY5 and HYH bind to the T/G-box in the HY5 promoter and act redundantly to induce HY5 expression upon UV-B exposure [45]. An immunoblot analysis indicated that HYH protein levels are decreased in the hy5 mutant and that HYH and HY5 might heterodimerize with each other in vivo [7]. To further elucidate the interconnected regulation of HY5 and HYH, we examined the reciprocal expression of HY5 and HYH in their mutants using qRT-PCR and reporter lines grown in the light (Fig 4 and S3 Fig). Our findings indicated that HYH expression in roots is regulated by HY5. These results, in addition to previous immunoblot data [7], further demonstrate that the suppression of HYH in the hy5 mutant is achieved in a root-specific manner. Similar to the regulation of HYH, the chalcone synthase gene (CHS1) was previously shown to be strongly induced by light in a HY5-dependent pathway, and the induction of CHS1::GUS was also specific to the root [8].
The results of Y1H and EMSA indicated that HY5 directly binds to a particular section (frag 3) of the HYH promoter that contains putative HY5 binding sites (ACEs) (Fig 5). Consistently, although HY5 and HYH have additive effects in hypocotyl elongation [7], the hy5 and hy5hyh mutants exhibited opposite lateral root architecture phenotypes [13]. Our findings suggest different pathways might be involved in the light-induced expression of HYH in the roots and shoots. We speculate that a possible unidentified factor might regulate HY5 and HYH during shoot photomorphogenesis. Further research is needed to fully elucidate these mechanisms.
Light is an important environmental factor influencing root growth. Long-distance signal transduction has long since been thought to be an important mechanism for the light response in roots [21], and auxin is the master signaling molecule in this pathway [24,25]. Furthermore, root apices generate massive amounts of reactive oxygen species (ROS) after only a few seconds of illumination [19,46]. Since roots grow continuously when placed in transparent Petri dishes, it was concluded that ROS, as a stress introduced by light, stimulates root growth [19]. How roots integrate shoot-derived and local signals is therefore fundamental for plant development. In previous photomorphogenic studies, the expression patterns of HY5 and HYH were not adequately dissected, since both the roots and shoots were exposed to light. In the present study, we have revealed a tissue-specific HY5-mediated pathway that regulates lightinduced root growth independently of light signaling in the shoot. Further work is needed to reveal the molecular components involved in this process.