Arabidopsis noncoding RNA mediates control of photomorphogenesis by red light

hid1 Exhibits a Hypo-Photomorphogenic Phenotype Under cR.

To identify RNA molecules that function in the light-signaling pathways, we screened a collection of Agrobacterium transferred DNA (T-DNA) insertion mutants that were compromised in either the expression or structure of specific ncRNAs for photomorphogenic phenotypes (30). One such mutant, hid1, was identified and found to express defectively a polycistronic cluster of four ncRNAs (Fig. S1). Upon comparing the hypocotyl lengths of hid1 and WT seedlings grown in continuous far-red (cFr), cR, or continuous blue (cB) light over a range of fluence rates, we observed a significant reduction in the inhibition of hypocotyl growth among the hid1 mutants specifically under cR (Fig. 1A). Moreover, the difference in hypocotyl length observed between the hid1 and WT seedlings was more apparent under higher cR fluence rates (Fig. 1C). In other light conditions and in darkness, hid1 mutants were indistinguishable from WT plants (Fig. 1 B–E). The hid1 mutant cotyledons also were less open than their WT counterparts over the tested range of cR fluence rates (Fig. 1F), and, hid1 seedlings grown under cR contained less chlorophyll than their WT counterparts (Fig. 1G). Overall, our results demonstrate that hid1 mutants exhibit a decreased responsiveness to cR.

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Fig. 1.

The ncRNA mutant hid1 exhibits hyposensitivity to cR. (A) Phenotypes 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 chlorophyll contents (in milligrams per gram) (G) of 5-d-old seedlings grown in cR. *P < 0.05, **P < 0.01 (t test).

hid1 is Defective in the ncRNA HID1.

In the hid1 mutant, the T-DNA inserted into a polycistronic cluster of four noncoding genes, causing a dramatic reduction in the expression of all four ncRNAs as verified by Northern blot analysis (Fig. S1B). To determine whether the lack of these ncRNAs was the direct 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, and nc3017 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-mediated 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.

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Fig. 2.

HID1 is the predominant player promoting cR-mediated seedling photomorphogenesis in the hid1 mutant. (A) Schematic illustration of constructs containing different members of the ncRNA gene cluster. (B) Phenotypes of 5-d-old WT, hid1, 35S:A/hid1, 35S:B/hid1, and pHID1:HID1/hid1 seedlings grown in cR. (Scale bar: 1 mm.) (C) Northern blot analysis showing the 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 are mean ± SD (n ≥ 20). **P < 0.01 (t test).

Given that HID1 is a 236-nt ncRNA that had been identified and verified in our previous genomic annotation (30), we next attempted to determine independently whether it acts directly or via a translational product. HID1 has a potential ORF encoding a 44-aa peptide. However, no homolog of this peptide was found in the annotated peptides or proteins of Arabidopsis. To investigate whether this predicted peptide was needed for HID1 function, we prepared two constructs and transformed them into hid1: M1, which contained two mutations changing ATG to ATA in the first predicted ORF, and M2, which changed ATG to AG. This latter mutation broke the predicted ORF but kept the predicted RNA secondary structure intact (Fig. 3A). The transgenic lines harboring either M1 or M2 expressed mutated HID1 at a level comparable to that observed in the WT plants and also fully rescued the reduced inhibition of hypocotyl elongation observed in hid1 under cR (Fig. 3 B–D). Therefore we concluded that HID1 most likely is a bona fide regulatory ncRNA in Arabidopsis that does not need a translational product to exert its function.

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Fig. 3.

HID1 contains two essential stem-loops. (A) The predicted secondary structure of HID1 showing four major stem-loops, SL1–4. M1 and M2 represent two mutated HID1 derivatives. SL1 is shaded pink, SL2 is blue, SL3 is light green, 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.) (C and D) The expression levels of HID1 examined by Northern blot (C) and hypocotyl lengths (D) determined in WT, M1/hid1, and M2/hid1 seedlings grown under the conditions in B. Data are mean ± SD (n ≥ 20). (E) Secondary structures of Arabidopsis HID1-deletion derivatives predicted by the mfold web server (Upper) and Northern blot analysis showing their expression in the corresponding transgenic lines as indicated by the black arrowheads (Lower). 5S rRNA was used as the loading control. (F and G) Phenotypes (F) and hypocotyl lengths (G) of 5-d-old cR-grown seedlings of WT, hid1, and transgenic lines expressing the deletion derivatives of HID1. (Scale bar: 1 mm.) **P < 0.01 (t test).

HID1 Is Constitutively Expressed and Contains Two Essential Stem-Loops.

We next examined the steady-state expression levels of HID1 in eight different organs/developmental stages by Northern blot analysis. We observed that HID1 was comparably abundant in all the organs and developmental stages analyzed, suggesting that HID1 was expressed ubiquitously throughout the Arabidopsis development processes (Fig. S2A). In addition, we observed that the expression of HID1 did not vary between seedlings grown in darkness and those grown under the various light conditions included in this study. Similarly, HID1 expression was not altered in mutants lacking the photoreceptor that detects the tested wavelength (e.g., phyA mutant grown in cFr) (Fig. S2B) under any of our light conditions. This result indicates that 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 the mfold Web Server (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) (Fig. 3A). To test the functional importance of each of these motifs, we generated four constructs containing the endogenous promoter driving HID1 mutants in which one motif, i.e., one of the major stem-loops 1, 2, 3, or 4 (SL1–SL4), was deleted. Each of these deletions removed only an individual motif and thus did not change the secondary structures of the remaining motifs predicted by mfold (Fig. 3E). After each construct was introduced into the hid1 background, the expression of each mutant was confirmed by Northern blot analysis in at least two independent transgenic lines. Then the hypocotyl growth of these two transgenic lines was examined under cR. As shown in Fig. 3 F and G, cR-grown mutants lacking either SL1 or SL3 had hypocotyls similar in length to those in WT plants. In contrast, mutants lacking SL2 or SL4 had elongated hypocotyls similar to cR-grown hid1 seedlings, suggesting that both SL2 and SL4 are essential for HID1-regulated hypocotyl elongation under cR. Thus, our results suggest that HID1’s function in response to cR is not limited to a particular submotif. Instead, it requires a complex structural organization of the HID1 molecule.

HID1 Acts Through PIF3 to Modulate Hypocotyl Elongation.

To investigate the mode of HID1 action, we analyzed the expression of genes located at either end of the HID1-coding sequence using quantitative RT-PCR (qRT-PCR). Our results showed that the expression of the tested neighboring genes in hid1 or 35S:A/hid1 mutants was comparable to the levels observed in WT plants, or was not changed by defective HID1 (Fig. S3). In addition, we did not find any known light-signaling mediators within 10 kb of either end of the sequence encoding HID1. Thus, our data did not support the hypothesis that the elongated hypocotyl growth observed in cR-grown hid1 seedlings was the consequence of the in-cis regulation of expression of neighboring genes by HID1.

To elucidate further the function of HID1 under cR, we compared global changes in gene expression in 5-d-old cR-grown WT and hid1 seedlings by RNA-seq. We found ∼635 genes that displayed statistically significant twofold changes in expression in hid1 mutants as compared with WT plants. Gene Ontology analysis (http://bioinfo.cau.edu.cn/agriGO/) showed that these genes primarily were enriched in Gene Ontology terms related to “response to stimulus” (P = 4.19e⁻¹⁶) (Fig. S4A). Among those genes, a small set of red light–responsive genes that were up-regulated more than twofold in hid1 mutants compared with WT plants particularly caught our attention (Fig. S4B). Notably, PIF3, a bHLH transcription factor that functions antagonistically in photomorphogenesis under prolonged cR and is one of the known positive regulators 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 PIF3 and other PIFs in WT and hid1 seedlings grown under cR. Our data showed that the mRNA levels of PIF3, but not of the other PIFs, were increased notably in hid1 mutants (Fig. 4A). The increase in PIF3 expression observed in hid1 mutants was confirmed under cR over a wide range of fluence rates (Fig. 4 B and C) and was correlated with the longer hypocotyls observed in the cR-grown hid1 seedlings. In addition, our data showed that in cR-grown 35S:A/hid1 and pHID1:HID1/hid1 seedlings in which the expression of HID1 was equal to or greater than the WT levels, the PIF3 transcript levels declined to WT levels (Fig. 4D), supporting the negative correlation between HID1 and PIF3 mRNA levels.

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Fig. 4.

HID1 represses PIF3 expression to modulate hypocotyl elongation in cR. (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 set as 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 in 5-d-old WT and hid1 seedlings grown in various light conditions and darkness (B) and under a range of R light intensities (C). (D) PIF3 transcript levels in 5-d-old cR-grown WT, hid1, 35S:A/hid1, and pHID1:HID1 seedlings. (E and F) Phenotypes (E) and hypocotyl lengths (F) of 5-d-old WT, hid1, pif3, and hid1pif3 seedlings grown in cR. (Scale bar: 1 mm.) (G) Transcript abundances of PIF3 following actinomycin D treatment in WT and hid1 seedlings. (H) qRT-PCR analysis showing the expression of PIF3 in WT and hid1 seedlings grown 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 collected from the time-course in H. (J–L) qRT-PCR analysis showing the transcript levels of SNRK2.5 (J), XTR7 (K), and AT4G35720 (L) in WT and hid1 seedlings collected from the time-course in H. CSN6 was used as the loading control. *P < 0.05, **P < 0.01 (t test).

We therefore examined the genetic interaction between HID1 and PIF3 by generating hid1pif3 double mutants. As shown in Fig. 4 E and F, the hypocotyl lengths of the hid1pif3 seedlings closely resembled those of the pif3 single-mutant seedlings, indicating that PIF3 acts downstream of HID1 genetically.

HID1 Negatively Regulates PIF3 Gene Expression.

To determine whether the increased expression of PIF3 in hid1 mutants is regulated at the transcriptional level, we first treated WT and hid1 seedlings with a transcriptional inhibitor. As shown in Fig. 4G, the rate of PIF3 mRNA decay after the arrest of transcription by actinomycin D did not differ between 5-d-old cR-grown WT and hid1 seedlings, suggesting that PIF3 mRNA stability was not 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 treatments. Intriguingly, PIF3 expression continued to be about twofold greater in hid1 mutants than in WT seedlings during the transfer from 5-d cR to dark for 3–12 h, confirming our hypothesis that HID1 played a role in downregulating PIF3 transcript levels (Fig. 4H). We then examined PIF3 protein levels over the same cR-to-dark time course. Our immunoblot analysis showed that PIF3 proteins accumulated rapidly in hid1 mutants 3 h after the plants were transferred from cR to dark and continued to increase over the course of the subsequent dark treatment (Fig. 4I). In contrast, only a mild increase in PIF3 protein was observed in WT seedlings over the course of the same period; but this increase could be detected clearly until 12 h of dark treatment. Consistent with the induction of PIF3 protein levels, three representative genes known to be direct targets of PIF3—SNRK2.5, XTR7, and AT4G35720 (16)—also showed significant induction in hid1 compared with WT plants during our dark treatment (Fig. 4 J–L). Thus, our data suggest that HID1 mediates the transcriptional control of PIF3 under cR.

HID1 Is Part of Large Nuclear Protein–RNA Complex(es) and Is Capable of Associating with Chromatin.

To gain further insights into the HID1 regulatory mechanism, we determined its cellular localization by biochemical fractionation. Notably, the majority of HID1 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 extracted by mild detergent treatment, suggesting that it is soluble in the nucleoplasm (Fig. 5B). Furthermore, most of the remaining HID1 (the S2 fraction) was associated with the chromatin fraction, suggesting that HID1 may regulate gene expression directly. Because ncRNAs usually function in the form of RNA–protein complexes, we performed a gel filtration analysis on 5-d-old cR-grown WT and hid1 seedlings. We detected most HID1 in the fractions around 500 kDa in size, suggesting that it is assembled into large complex(es) (Fig. 5C).

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Fig. 5.

Nuclear-localized HID1 associates with the PIF3 promoter to repress PIF3 expression. (A) Northern blot analysis showing HID1 expression levels in purified nuclear (N) and nuclear-depleted (C) fractions extracted from 5-d-old cR-grown seedlings. T, total extract. tRNA0119 was used as the marker for the cytoplasmic fraction. (B) Northern blot analysis showing the chromatin-bound fractions of HID1 (P1 or S2). tRNA0119 served as a marker for RNAs unbound to chromatin. (C) Gel filtration profiles of HID1 in cR-grown WT (Upper) and hid1 seedlings (Lower). The fraction numbers and molecular weights 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 hypocotyl lengths (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 analysis showing the expression levels of S1-HID1 in two independent transgenic lines, with 5.8S rRNA as the loading control. (H) Northern blot analysis showing 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 of four biological replicates ± SD. *P < 0.05 (t test). (K) Transient dual-luciferase (Dual-LUC) transcription assay showing PIF3 promoter activity in WT and hid1 seedlings grown under the indicated light conditions. (L) Transient Dual-LUC transcription assay showing the differences between WT and truncated PIF3 promoter activity in WT and hid1 seedlings grown under cR. Luciferase/Renilla ratios in WT were normalized to 1. Data are means of triplicates ± SD. *P < 0.05, **P < 0.01 (t test).

HID1 Associates with the PIF3 Promoter and Modulates Its Transcriptional Activity.

Our detection of the majority of soluble HID1 on chromatin suggested that HID1 could be closely associated with genomic DNA. Therefore we investigated whether HID1 is associated with the promoter of PIF3. To this end, we incorporated the S1 streptavidin-binding RNA aptamer tag into the SL3 stem-loop of HID1 (Fig. 5D). To examine the in vivo activity of this S1-tagged HID1, we transformed S1-HID1 driven by the HID1 promoter into the hid1 mutant background. This construct rescued the hid1 mutant phenotype in cR (Fig. 5 E–G), suggesting that S1-HID1 is biologically functional. Moreover, we were able to enrich S1-HID1 by immunoprecipitation 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 PIF3 locus. Intriguingly, compared with ACT7 controls, we observed a significant enrichment in the first intron of the PIF3 5′UTR (amplicon 3 and 4) in pHID1:S1-HID1/hid1 seedlings relative to hid1 seedlings, suggesting that HID1 associates with the proximal promoter of PIF3 (Fig. 5 I and J). Next, to test the functional significance of this association, we examined the activity of the full-length PIF3 promoter sequence (WT-p) and the same sequence lacking the HID1-binding region (dI-p) using firefly luciferase as reporter in the WT and hid1 mutant plants. Firefly luciferase activity driven by the intact promoter was found to be two times greater in hid1 than in WT plants kept under cR (Fig. 5K). In contrast, no difference was observed in hid1 and WT seedlings kept under cFr or cB (Fig. 5K). Likewise, no difference in firefly luciferase activity was detected between WT and hid1 plants treated with the dI-p construct, suggesting that HID1 binding in the PIF3 promoter is required for the transcriptional regulation of PIF3 in response to cR (Fig. 5L).

HID1 Is Evolutionarily Conserved in Land Plants.

Interestingly, when we used the entire primary sequence of HID1 for BLASTN searches 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 in the plant kingdom, ranging from moss to Arabidopsis (E < 10⁻⁵). ClustalW alignment of these sequences in five representative plant species from disparate taxa identified a highly conserved region (∼74 nt) near the 3′ end of AtHID1. The resulting secondary 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 of OsHID1 and AtHID1 are notably similar. Specifically, SL4 was highly conserved, whereas SL2 was relatively conserved, further suggesting the functional significance of HID1 (Fig. 6B). Therefore we tested whether OsHID1 was functional in Arabidopsis by expressing OsHID1 from its own promoter in the hid1 mutant background. Interestingly, OsHID1 was highly expressed from its own promoter in Arabidopsis and rescued the hid1 elongated hypocotyl phenotype (Fig. 6 D–F). This finding suggests that the function of this structurally conserved ncRNA may be conserved in monocots and dicots.

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Fig. 6.

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