Arabidopsis noncoding RNA mediates control of photomorphogenesis by red light Yuqiu 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, Beijing 100871, China; bDepartment of Biology, Wilkes University, Wilkes-Barre, PA 18766; and cDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520 Contributed by Xing Wang Deng, May 23, 2014 (sent for review April 23, 2014) Seedling photomorphogenesis is a sophisticated developmental (pifq) has been shown to display a constitutive photomorphogenic process that is controlled by both the transcriptional and post- phenotype in darkness (10–14). Recent studies have revealed that transcriptional regulation of gene expression. Here, we identify these PIFs are targeted for rapid degradation via the ubiquitin– an Arabidopsis noncoding RNA, designated HIDDEN TREASURE 1 proteasome pathway by photo-activated phytochromes in light (15). (HID1), as a factor promoting photomorphogenesis in continuous Genome-wide transcriptomic and ChIP-sequence analyses have red light (cR). We show that HID1 acts through PHYTOCHROME- identified numerous genes regulated by PIFs. Many targets of PIFs INTERACTING FACTOR 3 (PIF3), which encodes a basic helix–loop– encode transcription factors, suggesting that PIFs act early in and helix transcription factor known to be a key repressor of photo- define a central hub in the phytochrome-mediated light-signaling morphogenesis. Knockdown of HID1 in hid1 mutants leads to a sig- pathways controlling seedling photomorphogenesis (3, 16, 17). However, the way in which these PIF genes are regulated at the nificant increase in the expression of PIF3, which in turn drives the transcriptional level is still the subject of intense investigation. development of elongated hypocotyls in cR. We identified two HID1 Recent genome-wide studies have shown that noncoding RNAs major stem-loops in that are essential for its modulation of (ncRNAs) comprise a significant portion of the transcriptome in hypocotyl growth in cR-grown seedlings. Furthermore, our data animals and plants. Despite their lack of protein-coding potential, HID1 – reveal that is assembled into large nuclear protein RNA com- many ncRNAs have been recognized as essential regulators of gene plex(es) and that it associates with the chromatin of the first intron expression (18, 19). Long ncRNAs (lncRNAs), which vary in length of PIF3 to repress its transcription. Strikingly, phylogenetic analysis from 200 nt to dozens of kilobases, are an important class of reveals that many land plants have conserved homologs of HID1 ncRNAs that recently have been shown to possess a diverse set of and that its rice homolog can rescue the mutant phenotype when functions in eukaryotes (20). Although thousands of lncRNAs have expressed in Arabidopsis hid1 mutants. We thus concluded that been systematically identified or predicted in silico in Arabidopsis, HID1 is a previously uncharacterized noncoding RNA whose func- wheat, and maize (21–23), very few have been characterized tion represents another layer of regulation in the precise control of functionally (24–29). Specifically, no report to date has outlined seedling photomorphogenesis. the function of lncRNAs in photomorphogenesis. Building on our recent global annotation of Arabidopsis 50- to light signaling | transcriptional regulation 300-nt ncRNAs and our large-scale reverse genetic analysis (30), here we report the identification and characterization of an evolutionarily conserved ncRNA of 236 nt in land plants, HID1 ight is one of the most important environmental cues influ- (HIDDEN TREASURE 1), that modulates red-light–mediated Lencing the growth and development of plants throughout seedling photomorphogenesis in Arabidopsis. Knocking down theentireplantlifecycle(1).Oneof the best-characterized light- HID1 led to increased levels of PIF3 mRNA, which in turn controlled developmental processes is seedling morphogenesis: pho- correlated directly with the elongated hypocotyl phenotype tomorphogenesis (de-etiolation) in light and skotomorphogenesis (etiolation) in darkness. Photomorphogenesis is characterized by the Significance development of short hypocotyls, opened cotyledons, and chlo- rophyll synthesis, whereas skotomorphogenesis is characterized The dynamic regulation of gene-expression programs is both by the development of long hypocotyls, closed cotyledons with critical to and regulated precisely in the light-mediated seed- apical hooks, and undifferentiated plastids (2). The switch from ling photomorphogenesis of higher plants. Our work adds skotomorphogenesis to photomorphogenesis is critical for seed- HIDDEN TREASURE 1 (HID1), a noncoding RNA that acts as a ling survival and is dependent on the precise control of gene- – positive regulator of photomorphogenesis, to the current expression patterns by genetic and epigenetic pathways (3 6). group of pivotal genetic factors known to control photomorpho- Plants have evolved multiple photoreceptors that are capable of genesis. Specifically, our data obtained by numerous approaches perceiving and propagating a variety of light signals. For example, reveal that HID1 modulates red light-mediated photomorpho- five phytochromes (phyA–phyE) that perceive far-red and red light, genesis by directly repressing PHYTOCHROME-INTERACTING two cryptochromes (CRY1 and CRY2) and two phototropins FACTOR 3, which encodes a key transcription factor that inhibits (PHOT1 and PHOT2) that sense blue/UV-A light, and a UV-B HID1 photoreceptor (UVR8) have been identified in the model plant red light responses. appears to be highly conserved Arabidopsis thaliana (7, 8). Traditional genetic and molecular among higher plants. analyses combined with recent genomic studies have identified a Author contributions: Y.W., D.Z., and X.W.D. designed research; Y.W., X.F., F.L., and D.Z. number of protein-coding genes that function as positive or negative performed research; Y.W., G.H., W.T., D.Z., and X.W.D. analyzed data; and Y.W., W.T., PLANT BIOLOGY regulators of seedling photomorphogenesis under different light D.Z., and X.W.D. wrote the paper. – conditions (1, 2, 9). Among these genes, a family of basic helix The authors declare no conflict of interest. loop–helix (bHLH) transcription factors, designated “phytochrome- ” The data reported in this paper have been deposited in GenBank database (accession interacting factors (PIFs), has been shown to repress seedling no. KM044009) and in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm. photomorphogenesis in the dark. PIF1, PIF3 (the founding mem- nih.gov/geo (accession no. GSE57806). ber), PIF4, and PIF5 are the most extensively characterized mem- 1To whom correspondence may be addressed. E-mail: [email protected], bers of this family. Specifically, pif3, pif4,andpif5 mutants have [email protected]. been shown to exhibit hyper-photomorphogenic phenotypes in re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sponse to continuous red (cR) light, whereas the quadruple mutant 1073/pnas.1409457111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1409457111 PNAS | July 15, 2014 | vol. 111 | no. 28 | 10359–10364 Downloaded by guest on September 24, 2021 cause of the elongated hypocotyl phenotype observed in the hid1 seedlings grown under cR, we transformed a DNA fragment encoding all four ncRNAs driven by the CaMV 35S promoter into the hid1 mutant background (Fig. 2A). The resulting transgenic plants (35S:A/hid1) expressed all four ncRNAs at levels slightly higher than WT and completely rescued the hid1 phenotype in cR (Fig. 2 B–D), indicating that the decreased expression of these noncoding genes was responsible for the observed hid1 phenotype in cR. Next, to determine which of these ncRNAs was responsible for the hid1 phenotype, we made several constructs expressing subsets of these ncRNAs under the control of the 35S promoter and transformed them into the hid1 mutant background. We found that the transgenic line (35S:B/hid1) harboring the construct expressing nc3019, nc3018,andnc3017 at WT levels still exhibited the hid1 phenotype in cR (Fig. 2 B–D), thus indicating that these three ncRNAs are not essential for cR-medi- ated photomorphogenesis. Therefore we reasoned that the reduced expression of nc3020 might be responsible for the hid1 phenotype. We tested this hypothesis by expressing nc3020 from the nc3020 promoter in the hid1 mutant background and found that the hypocotyls of 5-d-old transgenic seedlings grown in cR were the same 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 is necessary for cR-mediated seedling photomorphogenesis. Given that HID1 is a 236-nt ncRNA that had been identified Fig. 1. The ncRNA mutant hid1 exhibits hyposensitivity to cR. (A) Pheno- and verified in our previous genomic annotation (30), we next types of 5-d-old seedlings grown under various light qualities. (Scale bar: attempted to determine independently whether it acts directly or 1mm.)(B–E) Hypocotyl lengths of seedlings grown in cFr (B), cR (C), or cB (D) via a translational product. HID1 has a potential ORF encoding over a range of fluence rates or in darkness (E). Data are mean ± SD (n ≥ 20). a 44-aa peptide.