Arabidopsis noncoding RNA mediates control ofphotomorphogenesis by red lightYuqiu Wanga, Xiuduo Fana, Fang Lina, Guangming Hea, William Terzaghib,c, Danmeng Zhua,1, and Xing Wang Denga,c,1

aState Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, College of Life Sciences, Peking University, Beijing100871, China; bDepartment of Biology, Wilkes University, Wilkes-Barre, PA 18766; and cDepartment of Molecular, Cellular and Developmental Biology, YaleUniversity, New Haven, CT 06520

Contributed by Xing Wang Deng, May 23, 2014 (sent for review April 23, 2014)

Seedling photomorphogenesis is a sophisticated developmentalprocess that is controlled by both the transcriptional and post-transcriptional regulation of gene expression. Here, we identifyan Arabidopsis noncoding RNA, designated HIDDEN TREASURE 1(HID1), as a factor promoting photomorphogenesis in continuousred light (cR). We show that HID1 acts through PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), which encodes a basic helix–loop–helix transcription factor known to be a key repressor of photo-morphogenesis. Knockdown of HID1 in hid1mutants leads to a sig-nificant increase in the expression of PIF3, which in turn drives thedevelopment of elongated hypocotyls in cR. We identified twomajor stem-loops in HID1 that are essential for its modulation ofhypocotyl growth in cR-grown seedlings. Furthermore, our datareveal that HID1 is assembled into large nuclear protein–RNA com-plex(es) and that it associates with the chromatin of the first intronof PIF3 to repress its transcription. Strikingly, phylogenetic analysisreveals that many land plants have conserved homologs of HID1and that its rice homolog can rescue the mutant phenotype whenexpressed in Arabidopsis hid1 mutants. We thus concluded thatHID1 is a previously uncharacterized noncoding RNA whose func-tion represents another layer of regulation in the precise control ofseedling photomorphogenesis.

light signaling | transcriptional regulation

Light is one of the most important environmental cues influ-encing the growth and development of plants throughout

the entire plant life cycle (1). One of the best-characterized light-controlled developmental processes is seedling morphogenesis: pho-tomorphogenesis (de-etiolation) in light and skotomorphogenesis(etiolation) in darkness. Photomorphogenesis is characterized by thedevelopment of short hypocotyls, opened cotyledons, and chlo-rophyll synthesis, whereas skotomorphogenesis is characterizedby the development of long hypocotyls, closed cotyledons withapical hooks, and undifferentiated plastids (2). The switch fromskotomorphogenesis to photomorphogenesis is critical for seed-ling survival and is dependent on the precise control of gene-expression patterns by genetic and epigenetic pathways (3–6).Plants have evolved multiple photoreceptors that are capable of

perceiving and propagating a variety of light signals. For example,five phytochromes (phyA–phyE) that perceive far-red and red light,two cryptochromes (CRY1 and CRY2) and two phototropins(PHOT1 and PHOT2) that sense blue/UV-A light, and a UV-Bphotoreceptor (UVR8) have been identified in the model plantArabidopsis thaliana (7, 8). Traditional genetic and molecularanalyses combined with recent genomic studies have identified anumber of protein-coding genes that function as positive or negativeregulators of seedling photomorphogenesis under different lightconditions (1, 2, 9). Among these genes, a family of basic helix–loop–helix (bHLH) transcription factors, designated “phytochrome-interacting factors” (PIFs), has been shown to repress seedlingphotomorphogenesis in the dark. PIF1, PIF3 (the founding mem-ber), PIF4, and PIF5 are the most extensively characterized mem-bers of this family. Specifically, pif3, pif4, and pif5 mutants havebeen shown to exhibit hyper-photomorphogenic phenotypes in re-sponse to continuous red (cR) light, whereas the quadruple mutant

(pifq) has been shown to display a constitutive photomorphogenicphenotype in darkness (10–14). Recent studies have revealed thatthese PIFs are targeted for rapid degradation via the ubiquitin–proteasome pathway by photo-activated phytochromes in light (15).Genome-wide transcriptomic and ChIP-sequence analyses haveidentified numerous genes regulated by PIFs. Many targets of PIFsencode transcription factors, suggesting that PIFs act early in anddefine a central hub in the phytochrome-mediated light-signalingpathways controlling seedling photomorphogenesis (3, 16, 17).However, the way in which these PIF genes are regulated at thetranscriptional level is still the subject of intense investigation.Recent genome-wide studies have shown that noncoding RNAs

(ncRNAs) comprise a significant portion of the transcriptome inanimals and plants. Despite their lack of protein-coding potential,many ncRNAs have been recognized as essential regulators of geneexpression (18, 19). Long ncRNAs (lncRNAs), which vary in lengthfrom 200 nt to dozens of kilobases, are an important class ofncRNAs that recently have been shown to possess a diverse set offunctions in eukaryotes (20). Although thousands of lncRNAs havebeen systematically identified or predicted in silico in Arabidopsis,wheat, and maize (21–23), very few have been characterizedfunctionally (24–29). Specifically, no report to date has outlinedthe function of lncRNAs in photomorphogenesis.Building on our recent global annotation of Arabidopsis 50- to

300-nt ncRNAs and our large-scale reverse genetic analysis (30),here we report the identification and characterization of anevolutionarily conserved ncRNA of 236 nt in land plants, HID1(HIDDEN TREASURE 1), that modulates red-light–mediatedseedling photomorphogenesis in Arabidopsis. Knocking downHID1 led to increased levels of PIF3 mRNA, which in turncorrelated directly with the elongated hypocotyl phenotype

Significance

The dynamic regulation of gene-expression programs is bothcritical to and regulated precisely in the light-mediated seed-ling photomorphogenesis of higher plants. Our work addsHIDDEN TREASURE 1 (HID1), a noncoding RNA that acts as apositive regulator of photomorphogenesis, to the currentgroup of pivotal genetic factors known to control photomorpho-genesis. Specifically, our data obtained by numerous approachesreveal that HID1 modulates red light-mediated photomorpho-genesis by directly repressing PHYTOCHROME-INTERACTINGFACTOR 3, which encodes a key transcription factor that inhibitsred light responses. HID1 appears to be highly conservedamong higher plants.

Author contributions: Y.W., D.Z., and X.W.D. designed research; Y.W., X.F., F.L., and D.Z.performed research; Y.W., G.H., W.T., D.Z., and X.W.D. analyzed data; and Y.W., W.T.,D.Z., and X.W.D. wrote the paper.

The authors declare no conflict of interest.

The data reported in this paper have been deposited in GenBank database (accessionno. KM044009) and in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE57806).1To whom correspondence may be addressed. E-mail: xingwang.deng@yale.edu,zhudanmeng@pku.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1409457111/-/DCSupplemental.

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observed in the hid1 mutant seedlings grown under cR. Wefurther demonstrated that HID1 is a chromatin-bound RNA andfunctions as a direct repressor of PIF3 transcription in cR. Thus,to our knowledge, HID1 appears to be the first lncRNA identi-fied as being involved in the precise control of light-mediatedseedling development.

Resultshid1 Exhibits a Hypo-Photomorphogenic Phenotype Under cR. Toidentify RNA molecules that function in the light-signaling path-ways, we screened a collection of Agrobacterium transferred DNA(T-DNA) insertion mutants that were compromised in either theexpression or structure of specific ncRNAs for photomorphogenicphenotypes (30). One such mutant, hid1, was identified and foundto express defectively a polycistronic cluster of four ncRNAs (Fig.S1). Upon comparing the hypocotyl lengths of hid1 and WT seed-lings grown in continuous far-red (cFr), cR, or continuous blue (cB)light over a range of fluence rates, we observed a significant re-duction in the inhibition of hypocotyl growth among the hid1mutants specifically under cR (Fig. 1A). Moreover, the difference inhypocotyl length observed between the hid1 and WT seedlings wasmore apparent under higher cR fluence rates (Fig. 1C). In otherlight conditions and in darkness, hid1 mutants were indistinguish-able from WT plants (Fig. 1 B–E). The hid1mutant cotyledons alsowere less open than their WT counterparts over the tested range ofcR fluence rates (Fig. 1F), and, hid1 seedlings grown under cRcontained less chlorophyll than their WT counterparts (Fig. 1G).Overall, our results demonstrate that hid1 mutants exhibit a de-creased responsiveness to cR.

hid1 is Defective in the ncRNA HID1. In the hid1mutant, the T-DNAinserted into a polycistronic cluster of four noncoding genes,causing a dramatic reduction in the expression of all fourncRNAs as verified by Northern blot analysis (Fig. S1B). Todetermine whether the lack of these ncRNAs was the direct

cause of the elongated hypocotyl phenotype observed in the hid1seedlings grown under cR, we transformed a DNA fragmentencoding all four ncRNAs driven by the CaMV 35S promoterinto the hid1 mutant background (Fig. 2A). The resultingtransgenic plants (35S:A/hid1) expressed all four ncRNAs atlevels slightly higher than WT and completely rescued the hid1phenotype in cR (Fig. 2 B–D), indicating that the decreasedexpression of these noncoding genes was responsible for theobserved hid1 phenotype in cR. Next, to determine which ofthese ncRNAs was responsible for the hid1 phenotype, we madeseveral constructs expressing subsets of these ncRNAs under thecontrol of the 35S promoter and transformed them into the hid1mutant background. We found that the transgenic line (35S:B/hid1)harboring the construct expressing nc3019, nc3018, and nc3017 atWT levels still exhibited the hid1 phenotype in cR (Fig. 2 B–D), thusindicating that these three ncRNAs are not essential for cR-medi-ated photomorphogenesis. Therefore we reasoned that the reducedexpression of nc3020 might be responsible for the hid1 phenotype.We tested this hypothesis by expressing nc3020 from the nc3020promoter in the hid1 mutant background and found that thehypocotyls of 5-d-old transgenic seedlings grown in cR were thesame length as those of WT seedlings (Fig. 2 B–D). Therefore,our data demonstrated that nc3020, hereafter referred to as“HID1,” is a negative regulator of hypocotyl elongation and isnecessary for cR-mediated seedling photomorphogenesis.Given that HID1 is a 236-nt ncRNA that had been identified

and verified in our previous genomic annotation (30), we nextattempted to determine independently whether it acts directly orvia a translational product. HID1 has a potential ORF encodinga 44-aa peptide. However, no homolog of this peptide was foundin the annotated peptides or proteins of Arabidopsis. To inves-tigate whether this predicted peptide was needed for HID1function, we prepared two constructs and transformed them intohid1: M1, which contained two mutations changing ATG to ATAin the first predicted ORF, and M2, which changed ATG to AG.This latter mutation broke the predicted ORF but kept thepredicted RNA secondary structure intact (Fig. 3A). The trans-genic lines harboring either M1 or M2 expressed mutated HID1at a level comparable to that observed in the WT plants and alsofully rescued the reduced inhibition of hypocotyl elongationobserved in hid1 under cR (Fig. 3 B–D). Therefore we concludedthat HID1 most likely is a bona fide regulatory ncRNA in Ara-bidopsis that does not need a translational product to exertits function.

Fig. 1. The ncRNA mutant hid1 exhibits hyposensitivity to cR. (A) Pheno-types of 5-d-old seedlings grown under various light qualities. (Scale bar:1 mm.) (B–E) Hypocotyl lengths of seedlings grown in cFr (B), cR (C), or cB (D)over a range of fluence rates or in darkness (E). Data are mean ± SD (n ≥ 20).(F and G) Cotyledon opening angles (in degrees) (F) and total chlorophyllcontents (in milligrams per gram) (G) of 5-d-old seedlings grown in cR. *P <0.05, **P < 0.01 (t test).

Fig. 2. HID1 is the predominant player promoting cR-mediated seedlingphotomorphogenesis in the hid1 mutant. (A) Schematic illustration of con-structs containing different members of the ncRNA gene cluster. (B) Phe-notypes of 5-d-old WT, hid1, 35S:A/hid1, 35S:B/hid1, and pHID1:HID1/hid1seedlings grown in cR. (Scale bar: 1 mm.) (C) Northern blot analysis showingthe expression levels of nc3020 (HID1), nc3019, nc3018, and nc3017 in WT,hid1, and indicated transgenic lines, with 5S rRNA as the loading control. (D)Hypocotyl lengths of seedlings grown under the conditions in B. Data aremean ± SD (n ≥ 20). **P < 0.01 (t test).

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HID1 Is Constitutively Expressed and Contains Two Essential Stem-Loops. We next examined the steady-state expression levels ofHID1 in eight different organs/developmental stages by North-ern blot analysis. We observed that HID1 was comparablyabundant in all the organs and developmental stages analyzed,suggesting that HID1 was expressed ubiquitously throughout theArabidopsis development processes (Fig. S2A). In addition, weobserved that the expression of HID1 did not vary betweenseedlings grown in darkness and those grown under the variouslight conditions included in this study. Similarly, HID1 expres-sion was not altered in mutants lacking the photoreceptor thatdetects the tested wavelength (e.g., phyA mutant grown in cFr)(Fig. S2B) under any of our light conditions. This result indicatesthat the abundance of HID1 is not regulated by light.We next examined the role of secondary structure in HID1

function. HID1 was predicted to fold into four stem-loops by themfold Web Server (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) (Fig. 3A). To test the functional importance ofeach of these motifs, we generated four constructs containing theendogenous promoter driving HID1 mutants in which one motif,i.e., one of the major stem-loops 1, 2, 3, or 4 (SL1–SL4), wasdeleted. Each of these deletions removed only an individualmotif and thus did not change the secondary structures of theremaining motifs predicted by mfold (Fig. 3E). After each con-struct was introduced into the hid1 background, the expression ofeach mutant was confirmed by Northern blot analysis in at leasttwo independent transgenic lines. Then the hypocotyl growth ofthese two transgenic lines was examined under cR. As shown inFig. 3 F and G, cR-grown mutants lacking either SL1 or SL3 hadhypocotyls similar in length to those in WT plants. In contrast,mutants lacking SL2 or SL4 had elongated hypocotyls similar tocR-grown hid1 seedlings, suggesting that both SL2 and SL4 areessential for HID1-regulated hypocotyl elongation under cR.Thus, our results suggest that HID1’s function in response to cRis not limited to a particular submotif. Instead, it requires a com-plex structural organization of the HID1 molecule.

HID1 Acts Through PIF3 to Modulate Hypocotyl Elongation. To in-vestigate the mode of HID1 action, we analyzed the expression ofgenes located at either end of the HID1-coding sequence us-ing quantitative RT-PCR (qRT-PCR). Our results showedthat the expression of the tested neighboring genes in hid1 or35S:A/hid1 mutants was comparable to the levels observed inWT plants, or was not changed by defective HID1 (Fig. S3). Inaddition, we did not find any known light-signaling mediatorswithin 10 kb of either end of the sequence encoding HID1.Thus, our data did not support the hypothesis that the elon-gated hypocotyl growth observed in cR-grown hid1 seedlingswas the consequence of the in-cis regulation of expression ofneighboring genes by HID1.To elucidate further the function of HID1 under cR, we com-

pared global changes in gene expression in 5-d-old cR-grown WTand hid1 seedlings by RNA-seq. We found ∼635 genes that dis-played statistically significant twofold changes in expression in hid1mutants as compared with WT plants. Gene Ontology analysis(http://bioinfo.cau.edu.cn/agriGO/) showed that these genes pri-marily were enriched in Gene Ontology terms related to “responseto stimulus” (P = 4.19e−16) (Fig. S4A). Among those genes, a smallset of red light–responsive genes that were up-regulated more thantwofold in hid1 mutants compared with WT plants particularlycaught our attention (Fig. S4B). Notably, PIF3, a bHLH tran-scription factor that functions antagonistically in photomorpho-genesis under prolonged cR and is one of the known positiveregulators of hypocotyl elongation, was included in this group.To confirm further the role of HID1 in regulating PIF3 in vivo,

we performed qRT-PCR to validate the mRNA levels of PIF3and other PIFs in WT and hid1 seedlings grown under cR. Ourdata showed that the mRNA levels of PIF3, but not of the otherPIFs, were increased notably in hid1 mutants (Fig. 4A). The in-crease in PIF3 expression observed in hid1 mutants was con-firmed under cR over a wide range of fluence rates (Fig. 4 B and C)and was correlated with the longer hypocotyls observed in thecR-grown hid1 seedlings. In addition, our data showed that incR-grown 35S:A/hid1 and pHID1:HID1/hid1 seedlings in whichthe expression of HID1 was equal to or greater than the WTlevels, the PIF3 transcript levels declined to WT levels (Fig. 4D),supporting the negative correlation between HID1 and PIF3mRNA levels.We therefore examined the genetic interaction between HID1

and PIF3 by generating hid1pif3 double mutants. As shown inFig. 4 E and F, the hypocotyl lengths of the hid1pif3 seedlingsclosely resembled those of the pif3 single-mutant seedlings, in-dicating that PIF3 acts downstream of HID1 genetically.

HID1 Negatively Regulates PIF3 Gene Expression. To determinewhether the increased expression of PIF3 in hid1 mutants isregulated at the transcriptional level, we first treated WT andhid1 seedlings with a transcriptional inhibitor. As shown in Fig.4G, the rate of PIF3 mRNA decay after the arrest of transcrip-tion by actinomycin D did not differ between 5-d-old cR-grownWT and hid1 seedlings, suggesting that PIF3 mRNA stability wasnot affected by the hid1 mutation.Next, we used qRT-PCR to analyze the transcript levels of

PIF3 in WT and hid1 seedlings subjected to cR-to-dark treat-ments. Intriguingly, PIF3 expression continued to be about two-fold greater in hid1 mutants than in WT seedlings during thetransfer from 5-d cR to dark for 3–12 h, confirming our hy-pothesis that HID1 played a role in downregulating PIF3 tran-script levels (Fig. 4H). We then examined PIF3 protein levelsover the same cR-to-dark time course. Our immunoblot analysisshowed that PIF3 proteins accumulated rapidly in hid1 mutants3 h after the plants were transferred from cR to dark and con-tinued to increase over the course of the subsequent darktreatment (Fig. 4I). In contrast, only a mild increase in PIF3protein was observed in WT seedlings over the course of thesame period; but this increase could be detected clearly until 12 hof dark treatment. Consistent with the induction of PIF3 proteinlevels, three representative genes known to be direct targets of

Fig. 3. HID1 contains two essential stem-loops. (A) The predicted secondarystructure of HID1 showing four major stem-loops, SL1–4. M1 and M2 representtwo mutated HID1 derivatives. SL1 is shaded pink, SL2 is blue, SL3 is lightgreen, and SL4 is yellow. (B) Phenotypes of 5-d-old cR-grown WT, pHID1:M1/hid1 (M1/hid1), and pHID1:M2/hid1 (M2/hid1) seedlings. (Scale bar: 1 mm.) (Cand D) The expression levels of HID1 examined by Northern blot (C) and hy-pocotyl lengths (D) determined in WT, M1/hid1, and M2/hid1 seedlings grownunder the conditions in B. Data are mean ± SD (n ≥ 20). (E) Secondary struc-tures of Arabidopsis HID1-deletion derivatives predicted by the mfold webserver (Upper) and Northern blot analysis showing their expression in the cor-responding transgenic lines as indicated by the black arrowheads (Lower). 5SrRNA was used as the loading control. (F and G) Phenotypes (F) and hypocotyllengths (G) of 5-d-old cR-grown seedlings of WT, hid1, and transgenic linesexpressing the deletion derivatives ofHID1. (Scale bar: 1 mm.) **P < 0.01 (t test).

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PIF3—SNRK2.5, XTR7, and AT4G35720 (16)—also showedsignificant induction in hid1 compared with WT plants duringour dark treatment (Fig. 4 J–L). Thus, our data suggest thatHID1 mediates the transcriptional control of PIF3 under cR.

HID1 Is Part of Large Nuclear Protein–RNA Complex(es) and Is Capableof Associating with Chromatin. To gain further insights into theHID1 regulatory mechanism, we determined its cellular locali-zation by biochemical fractionation. Notably, the majority ofHID1 was detected in the nuclear fraction (Fig. 5A). Similarly,through the use of an alternative cellular fractionation method(31), we found that ∼90% of HID1 (the P1 fraction) was ex-tracted by mild detergent treatment, suggesting that it is sol-uble in the nucleoplasm (Fig. 5B). Furthermore, most of theremaining HID1 (the S2 fraction) was associated with thechromatin fraction, suggesting that HID1 may regulate gene

expression directly. Because ncRNAs usually function in theform of RNA–protein complexes, we performed a gel filtrationanalysis on 5-d-old cR-grown WT and hid1 seedlings. We detectedmost HID1 in the fractions around 500 kDa in size, suggestingthat it is assembled into large complex(es) (Fig. 5C).

HID1 Associates with the PIF3 Promoter and Modulates Its TranscriptionalActivity.Our detection of the majority of solubleHID1 on chromatinsuggested that HID1 could be closely associated with genomicDNA. Therefore we investigated whether HID1 is associated withthe promoter of PIF3. To this end, we incorporated the S1 strep-tavidin-binding RNA aptamer tag into the SL3 stem-loop of HID1(Fig. 5D). To examine the in vivo activity of this S1-taggedHID1, wetransformed S1-HID1 driven by the HID1 promoter into the hid1mutant background. This construct rescued the hid1 mutant phe-notype in cR (Fig. 5 E–G), suggesting that S1-HID1 is biologicallyfunctional. Moreover, we were able to enrich S1-HID1 by immu-noprecipitation with streptavidin-conjugated Sepharose beads (Fig.5H). We then used this stable transgenic line to perform ChIP-qPCR analysis to determine if and where HID1 binds to the PIF3locus. Intriguingly, compared with ACT7 controls, we observed asignificant enrichment in the first intron of the PIF3 5′UTR(amplicon 3 and 4) in pHID1:S1-HID1/hid1 seedlings relative tohid1 seedlings, suggesting that HID1 associates with the proximalpromoter of PIF3 (Fig. 5 I and J). Next, to test the functionalsignificance of this association, we examined the activity of thefull-length PIF3 promoter sequence (WT-p) and the same se-quence lacking the HID1-binding region (dI-p) using firefly lu-ciferase as reporter in the WT and hid1 mutant plants. Fireflyluciferase activity driven by the intact promoter was found to betwo times greater in hid1 than in WT plants kept under cR (Fig.5K). In contrast, no difference was observed in hid1 and WTseedlings kept under cFr or cB (Fig. 5K). Likewise, no difference infirefly luciferase activity was detected between WT and hid1 plantstreated with the dI-p construct, suggesting that HID1 binding in thePIF3 promoter is required for the transcriptional regulation ofPIF3 in response to cR (Fig. 5L).

HID1 Is Evolutionarily Conserved in Land Plants. Interestingly, whenwe used the entire primary sequence of HID1 for BLASTNsearches of the National Center for Biotechnology Information(www.ncbi.nih.gov) and Phytozome databases (www.phytozome.net), orthologs of Arabidopsis HID1 (AtHID1) were found only inthe plant kingdom, ranging from moss to Arabidopsis (E < 10−5).ClustalW alignment of these sequences in five representativeplant species from disparate taxa identified a highly conservedregion (∼74 nt) near the 3′ end of AtHID1. The resulting sec-ondary structures predicted by RNAalifold (http://rna.tbi.univie.ac.at/cgi-bin/RNAalifold.cgi) also were well conserved (Fig. 6A).The full-length transcripts of AtHID1 and of HID1 in rice(OsHID1) had been experimentally identified previously (32),and their expression was confirmed by Northern blot analysis(Fig. 6C). Moreover, the entire predicted secondary structures ofOsHID1 and AtHID1 are notably similar. Specifically, SL4 washighly conserved, whereas SL2 was relatively conserved, furthersuggesting the functional significance of HID1 (Fig. 6B). Thereforewe tested whether OsHID1 was functional in Arabidopsis by ex-pressing OsHID1 from its own promoter in the hid1 mutantbackground. Interestingly, OsHID1 was highly expressed from itsown promoter in Arabidopsis and rescued the hid1 elongatedhypocotyl phenotype (Fig. 6 D–F). This finding suggests that thefunction of this structurally conserved ncRNA may be conservedin monocots and dicots.

DiscussionPhotomorphogenic development in seedlings has been studiedextensively for decades, and the underlying mechanism has beenestablished as primarily regulated proteolysis by the ubiquitin–proteasome pathway. In this study we introduced a lncRNA,HID1,as another layer of regulator and demonstrated that it functions incR by regulating the transcription of key light intermediates in light

Fig. 4. HID1 represses PIF3 expression to modulate hypocotyl elongation incR. (A) qRT-PCR analysis showing the transcript levels of PIFs in 5-d-old cR-grown WT and hid1 seedlings. Expression of PIF1 in WT seedlings was setas 1. Error bars represent SD of triplicate biological replicates. **P < 0.01(t test). (B and C) qRT-PCR analysis showing the transcript levels of PIF3 in5-d-old WT and hid1 seedlings grown in various light conditions and dark-ness (B) and under a range of R light intensities (C). (D) PIF3 transcript levelsin 5-d-old cR-grown WT, hid1, 35S:A/hid1, and pHID1:HID1 seedlings. (E andF) Phenotypes (E) and hypocotyl lengths (F) of 5-d-old WT, hid1, pif3, andhid1pif3 seedlings grown in cR. (Scale bar: 1 mm.) (G) Transcript abundancesof PIF3 following actinomycin D treatment in WT and hid1 seedlings. (H)qRT-PCR analysis showing the expression of PIF3 in WT and hid1 seedlingsgrown in cR for 5 d and then transferred to dark for the indicated times. (I)Immunoblots showing PIF3 protein levels in WT and hid1 seedlings collectedfrom the time-course in H. (J–L) qRT-PCR analysis showing the transcriptlevels of SNRK2.5 (J), XTR7 (K), and AT4G35720 (L) in WT and hid1 seedlingscollected from the time-course in H. CSN6 was used as the loading control.*P < 0.05, **P < 0.01 (t test).

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signaling exemplified by PIF3. To our knowledge, this report is thefirst example of an ncRNA functioning in light-mediated plantdevelopment.Mutant hid1 seedlings were observed to be hyposensitive to

cR, exhibiting elongated hypocotyls, reduced cotyledon angles,and reduced chlorophyll accumulation (Fig. 1). Several lines ofevidence indicate that HID1 acts as an in trans transcriptionalrepressor that associates with the PIF3 promoter and negativelyregulates PIF3 transcription in Arabidopsis. This process, in turn,may explain how the red light–dependent function of HID1 isachieved. Disruption of HID1 results in a mild PIF3 overex-pression phenotype, as shown by the elongated hypocotyls of theseedlings grown in cR (Fig. 4). The transcriptional regulation ofPIF3 by HID1 represents both an elaborate control of the ex-pression of a key transcription factor and an additional mechanisticlayer of the light-signaling pathway. This function may be a way tocoordinate transcription with the translation of PIF3 to maintainproper abundance of a key signaling mediator under cR.

Knowledge of lncRNAs’ functional motifs is essential to un-cover their regulatory mechanisms. Genomic analyses of a vari-ety of animal species have revealed that lncRNAs are signifi-cantly less conserved than protein-coding genes in animals.Furthermore, some, but not all, have clearly conserved regionsthat are both under selective pressure and functionally important(33, 34). To date, however, we have only a limited understandingof the structure and function of the lncRNAs found in plants.Phylogenetic analyses showed that HID1 occurs only in plants, in-dicating that it evolved after plants diverged from other eukaryotesand thus functions in processes unique to plants. Two piecesof evidence suggest that HID1 functions as part of a complexstructure rather than as a precursor of a small RNA. First, ourresearch has demonstrated that two unique modules of HID1,SL2 and SL4, are both essential for its function in cR. Thisfinding, in turn, suggests that more than one structural feature ofHID1 is required for its function (Fig. 3). Second, our BLASTsearches of the available Arabidopsis small RNA databases didnot reveal the existence of any small RNA that could be derivedfrom SL2 or SL4, suggesting that HID1 is unlikely to function asa precursor of a small RNA. Thus, it appears that HID1 may bea good place to begin an investigation of the structures andfunctions of lncRNAs in plants. Further study of the structure ofHID1 should yield new insights into the mechanisms by whichlncRNAs function.Case studies of lncRNAs in animals have shown that these

RNAs regulate transcription by binding specific sites in trans inthe nucleus. Many lncRNAs that bind chromatin to regulate thechromatin functional state have been identified in animals, in-cluding Drosophila roX1 and roX2 RNA (35), human HOTAIR(36), XIST (37), and mammalian pRNA (38). However, ouridentification of Arabidopsis HID1 is, to our knowledge, the first

Fig. 5. Nuclear-localized HID1 associates with the PIF3 promoter to repressPIF3 expression. (A) Northern blot analysis showing HID1 expression levels inpurified nuclear (N) and nuclear-depleted (C) fractions extracted from 5-d-old cR-grown seedlings. T, total extract. tRNA0119 was used as the markerfor the cytoplasmic fraction. (B) Northern blot analysis showing the chro-matin-bound fractions of HID1 (P1 or S2). tRNA0119 served as a marker forRNAs unbound to chromatin. (C) Gel filtration profiles of HID1 in cR-grownWT (Upper) and hid1 seedlings (Lower). The fraction numbers and molecularweights are indicated. T, total soluble extracts used for gel filtration. (D)Schematic illustration of HID1 tagged with the minimal S1 aptamer. SA.streptavidin Sepharose beads. (E and F) Phenotypes (E) and hypocotyllengths (F) of 5-d-old cR-grown WT, hid1, and indicated transgenic lines.(Scale bar: 1 mm.) Data are mean ± SD (n ≥ 20). (G) Northern blot analysisshowing the expression levels of S1-HID1 in two independent transgeniclines, with 5.8S rRNA as the loading control. (H) Northern blot analysisshowing the expression levels of S1-HID1 in input and immunoprecipitation(IP) samples. (I) Diagram of the PIF3 amplicons used in the ChIP assays. (J)ChIP-qPCR analysis of S1-HID1 at the PIF3 locus. Data represent means offour biological replicates ± SD. *P < 0.05 (t test). (K) Transient dual-luciferase(Dual-LUC) transcription assay showing PIF3 promoter activity in WT andhid1 seedlings grown under the indicated light conditions. (L) TransientDual-LUC transcription assay showing the differences between WT andtruncated PIF3 promoter activity in WT and hid1 seedlings grown under cR.Luciferase/Renilla ratios in WT were normalized to 1. Data are means oftriplicates ± SD. *P < 0.05, **P < 0.01 (t test).

Fig. 6. HID1 is a plant-specific ncRNA with conserved structure and functionin monocots and dicots. (A) Sequence and structural alignment of AtHID1 inthe five representative organisms prepared using RNAalifold showing a con-served region near its 3′ end. (B) Predicted secondary structures of AtHID1and OsHID1. Conserved sequences are indicated in red. (C) Northern blotanalysis of HID1 showing its expression in seedlings and leaves of Arabidopsisand rice respectively, with 5S rRNA as the loading control. (D) Northernblot analysis showing the expression of OsHID1 in pOsHID1:OsHID1/hid1(OsHID1/hid1) transgenic plants. (E and F ) Phenotypes (E ) and hypocotyllengths (F) of 5-d-old cR-grownWT, hid1, and pOsHID1:OsHID1/hid1 seedlings.(Scale bar: 2 mm.) Data are mean ± SD (n ≥ 20). **P < 0.01 (t test).

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example of an lncRNA that associates with chromatin at a distantgene locus in land plants. Given both the abundant expression ofHID1 detected in numerous tissues during plant development andthe pleiotropic phenotypes observed in hid1 mutants, we postu-late that HID1may have other targets in addition to PIF3. In fact,it seems likely that HID1 may associate with multiple chromatintargets. Identification of HID1’s chromatin occupancy and that ofits partners in the RNA–protein nuclear complex(es) undoubtedlywill provide new insights into the details of this specific lncRNA’sfunction. More broadly, this identification may uncover evolu-tionarily divergent regulation of lncRNAs in plants and animals.In addition, we anticipate that further studies connecting theRNA-based gene-regulatory network to the known protein-basedmechanisms of gene regulation should lead to a better under-standing of both RNA biology and the regulation of gene ex-pression in general.

Materials and MethodsPlant materials and growth conditions, phenotype analyses, plasmid construc-tion, and the generation of transgenic plants are described in SI Materials andMethods. The detailed procedures of Northern blot analysis, qRT-PCR, RNA-Seq,mRNA decay assay, Western blot analysis, biochemical fractionation assay, gelfiltration chromatography, RNA immunoprecipitation assay, ChIP assay, andtransient transcription dual-luciferase (Dual-LUC) assay are provided in SIMaterials and Methods. The sequences used for probing ncRNAs in this studyare listed in Table S1.

ACKNOWLEDGMENTS. We thank Dr. Haiyang Wang, Dr. Yijun Qi, and Dr.LigengMa for their helpful discussions and comments on this project; Wei Chenand Xuncheng Wang for bioinformatics assistance; Huikun Duan and JunjieLing for technical assistance; and Abigail Coplin for critical reading of the man-uscript. This work was supported by National Natural Science Foundationof China Grant 31171156, National Basic Research Program of China (973Program) Grant 2012CB910900, and in part by the State Key Laboratoryof Protein and Plant Gene Research at Peking University and the Peking-Tsinghua Center for Life Sciences.

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