The noncoding RNA HIDDEN TREASURE 1 promotes phytochrome B-dependent seed germination by repressing abscisic acid biosynthesis.

Light is a major environmental factor for seed germination. Red light-activated phytochrome B (phyB) promotes seed germination by modulating the dynamic balance of two phytohormones, gibberellic acid (GA) and abscisic acid (ABA). How phyB modulates ABA biosynthesis after perceiving a light signal is not yet well understood. Here, we identified the noncoding RNA HIDDEN TREASURE 1 (HID1) as a repressor of ABA biosynthesis acting downstream of phyB during Arabidopsis thaliana seed germination. Loss of HID1 function led to delayed phyB-dependent seed germination. Photoactivated phyB promoted the accumulation of HID1 in the radicle within 48 h of imbibition. Our transcriptomics analysis showed that HID1 and phyB co-regulate the transcription of a common set of genes involved in ABA and GA metabolism. Through a forward genetic screen, we identified three ABA biosynthesis genes, ABA DEFICIENT 1 (ABA1), ABA2, and ABA3, as suppressors of HID1. We further demonstrated that HID1 directly inhibits the transcription of 9-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED9), a gene encoding a key rate-limiting enzyme of ABA biosynthesis. HID1 interacts with ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7), an H3K4me3 methyltransferase, inhibiting its occupancy and H3K4me3 modification at the NCED9 locus. Our study reveals a nuclear mechanism of phyB signaling transmitted through HID1 to control the internal homeostasis of ABA and GA, which gradually optimizes the transcriptional network during seed germination.

Chromatin remodeling complexes regulate genome architecture in Arabidopsis.

In eukaryotes, three-dimensional (3D) chromatin architecture maintains genome stability and is important in regulating gene transcription. However, little is known about the mechanisms by which diverse ATP-dependent chromatin remodeling complexes regulate the 3D chromatin structure in plants. We examined the 3D chromatin structure within the ATPase subunit of the SWI/SNF, ISWI, INO80, and CHD remodeling complexes in wild-type (WT) and mutant Arabidopsis thaliana plants by combining high-throughput sequencing with in situ Hi-C, the enrichment of histone marks, nucleosome density, and gene expression. We found that compartment regions switched and compartmental strength was significantly weakened in all four enzyme mutants. Chromatin remodeling complexes differentially regulated the nucleosome distribution pattern and density within the switching compartments. Alterations of nucleosome distribution pattern and density were associated with a reduction in H3K27me3 levels in the chromatin remodeling enzyme mutants and led to compartment switching. Our data show that chromatin remodeling complexes regulate the linear nucleosome distribution pattern and density to promote H3K27me3 deposition, which in turn regulates 3D chromatin structure.

The small nucleolar RNA SnoR28 regulates plant growth and development by directing rRNA maturation.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs (ncRNAs) that guide chemical modifications of structural RNAs, which are essential for ribosome assembly and function in eukaryotes. Although numerous snoRNAs have been identified in plants by high-throughput sequencing, the biological functions of most of these snoRNAs remain unclear. Here, we identified box C/D SnoR28.1s as important regulators of plant growth and development by screening a CRISPR/Cas9-generated ncRNA deletion mutant library in Arabidopsis thaliana. Deletion of the SnoR28.1 locus, which contains a cluster of three genes producing SnoR28.1s, resulted in defects in root and shoot growth. SnoR28.1s guide 2'-O-ribose methylation of 25S rRNA at G2396. SnoR28.1s facilitate proper and efficient pre-rRNA processing, as the SnoR28.1 deletion mutants also showed impaired ribosome assembly and function, which may account for the growth defects. SnoR28 contains a 7-bp antisense box, which is required for 2'-O-ribose methylation of 25S rRNA at G2396, and an 8-bp extra box that is complementary to a nearby rRNA methylation site and is partially responsible for methylation of G2396. Both of these motifs are required for proper and efficient pre-rRNA processing. Finally, we show that SnoR28.1s genetically interact with HIDDEN TREASURE2 and NUCLEOLIN1. Our results advance our understanding of the roles of snoRNAs in Arabidopsis.

SlRBP1 promotes translational efficiency via SleIF4A2 to maintain chloroplast function in tomato.

Many glycine-rich RNA-binding proteins (GR-RBPs) have critical functions in RNA processing and metabolism. Here, we describe a role for the tomato (Solanum lycopersicum) GR-RBP SlRBP1 in regulating mRNA translation. We found that SlRBP1 knockdown mutants (slrbp1) displayed reduced accumulation of total chlorophyll and impaired chloroplast ultrastructure. These phenotypes were accompanied by deregulation of the levels of numerous key transcripts associated with chloroplast functions in slrbp1. Furthermore, native RNA immunoprecipitation-sequencing (nRIP-seq) recovered 61 SlRBP1-associated RNAs, most of which are involved in photosynthesis. SlRBP1 binding to selected target RNAs was validated by nRIP-qPCR. Intriguingly, the accumulation of proteins encoded by SlRBP1-bound transcripts, but not the mRNAs themselves, was reduced in slrbp1 mutants. Polysome profiling followed by RT-qPCR assays indicated that the polysome occupancy of target RNAs was lower in slrbp1 plants than in wild-type. Furthermore, SlRBP1 interacted with the eukaryotic translation initiation factor SleIF4A2. Silencing of SlRBP1 significantly reduced SleIF4A2 binding to SlRBP1-target RNAs. Taking these observations together, we propose that SlRBP1 binds to and channels RNAs onto the SleIF4A2 translation initiation complex and promotes the translation of its target RNAs to regulate chloroplast functions.

The Arabidopsis DREAM complex antagonizes WDR5A to modulate histone H3K4me2/3 deposition for a subset of genome repression.

The master transcriptional repressor DREAM (dimerization partner, RB-like, E2F and multivulval class B) complex regulates the cell cycle in eukaryotes, but much remains unknown about how it transmits repressive signals on chromatin to the primary transcriptional machinery (e.g., RNA polymerase II [Pol II]). Through a forward genetic screen, we identified BTE1 (barrier of transcription elongation 1), a plant-specific component of the DREAM complex. The subsequent characterization demonstrated that DREAM complex containing BTE1 antagonizes the activity of Complex Proteins Associated with Set1 (COMPASS)-like complex to repress H3K4me3 occupancy and inhibits Pol II elongation at DREAM target genes. We showed that BTE1 is recruited to chromatin at the promoter-proximal regions of target genes by E2F transcription factors. DREAM target genes exhibit characteristic enrichment of H2A.Z and H3K4me2 modification on chromatin. We further showed that BTE1 directly interacts with WDR5A, a core component of COMPASS-like complex, repressing WDR5A chromatin binding and the elongation of transcription on DREAM target genes. H3K4me3 is known to correlate with the Pol II transcription activation and promotes efficient elongation. Thus, our study illustrates a transcriptional repression mechanism by which the DREAM complex dampens H3K4me3 deposition at a set of genes through its interaction with WDR5A.

From molecular basics to agronomic benefits: Insights into noncoding RNA-mediated gene regulation in plants.

The development of plants is largely dependent on their growth environment. To better adapt to a particular habitat, plants have evolved various subtle regulatory mechanisms for altering gene expression. Non coding RNAs (ncRNAs) constitute a major portion of the transcriptomes of eukaryotes. Various ncRNAs have been recognized as important regulators of the expression of genes involved in essential biological processes throughout the whole life cycles of plants. In this review, we summarize the current understanding of the biogenesis and contributions of small nucle olar RNA (snoRNA)- and regulatory long non coding RNA (lncRNA)-mediated gene regulation in plant development and environmental responses. Many regulatory ncRNAs appear to be associated with increased yield, quality and disease resistance of various species and cultivars. These ncRNAs may potentially be used as genetic resources for improving agronomic traits and for molecular breeding. The challenges in understanding plant ncRNA biology and the possibilities to make better use of these valuable gene resources in the future are discussed in this review.

Hinge region of Arabidopsis phyA plays an important role in regulating phyA function.

Phytochrome A (phyA) is the only plant photoreceptor that perceives far-red light and then mediates various responses to this signal. Phosphorylation and dephosphorylation of oat phyA have been extensively studied, and it was shown that phosphorylation of a serine residue in the hinge region of oat phyA could regulate the interaction of phyA with its signal transducers. However, little is known about the role of the hinge region of Arabidopsis phyA. Here, we report that three sites in the hinge region of Arabidopsis phyA (i.e., S590, T593, and S602) are essential in regulating phyA function. Mutating all three of these sites to either alanines or aspartic acids impaired phyA function, changed the interactions of mutant phyA with FHY1 and FHL, and delayed the degradation of mutant phyA upon light exposure. Moreover, the in vivo formation of a phosphorylated phyA form was greatly affected by these mutations, while our data indicated that the abundance of this phosphorylated phyA form correlated well with the extent of phyA function, thus suggesting a pivotal role of the phosphorylated phyA in inducing the far-red light response. Taking these data together, our study reveals the important role of the hinge region of Arabidopsis phyA in regulating phyA phosphorylation and function, thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.

Noncanonical role of Arabidopsis COP1/SPA complex in repressing BIN2-mediated PIF3 phosphorylation and degradation in darkness.

The E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) has been known to mediate key signaling factors for degradation via the ubiquitin/26S proteasome pathway in both plants and animals. Here, we report a noncanonical function of Arabidopsis COP1, the central repressor of photomorphogenesis, in the form of a COP1/ SUPPRESSOR of phyA-105 (SPA) complex. We show that the COP1/SPA complex associates with and stabilizes PHYTOCHROME INTERACTING FACTOR 3 (PIF3) to repress photomorphogenesis in the dark. We identify the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) as a kinase of PIF3, which induces PIF3 degradation via 26S proteasome during skotomorphogenesis. Mutations on two typical BIN2 phosphorylation motifs of PIF3 lead to a strong stabilization of the protein in the dark. We further show that the COP1/SPA complex promotes PIF3 stability by repressing BIN2 activity. Intriguingly, without affecting BIN2 expression, the COP1/SPA complex modulates BIN2 activity through interfering with BIN2-PIF3 interaction, thereby inhibiting BIN2-mediated PIF3 phosphorylation and degradation. Taken together, our results suggest another paradigm for COP1/SPA complex action in the precise control of skotomorphogenesis.

RNA Binding Proteins RZ-1B and RZ-1C Play Critical Roles in Regulating Pre-mRNA Splicing and Gene Expression during Development in Arabidopsis.

Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here, we report the functions of two proteins containing RNA recognition motifs, RZ-1B and RZ-1C, in Arabidopsis thaliana. RZ-1B and RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C termini. RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA binding activity of RZ-1C with SR proteins through overexpression of the C terminus of RZ-1C conferred defective phenotypes similar to those observed in rz-1b rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of RZ-1B and RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that RZ-1C directly targeted FLOWERING LOCUS C (FLC), promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins.

Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis.

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA-rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.

Cryptochromes (CRYs) are blue-light photoreceptors that mediate various light responses in plants and animals. The signaling mechanism by which CRYs regulate light responses involves their physical interactions with COP1. Here, we report that CRY1 interacts physically with SPA1 in a blue-light-dependent manner. SPA acts genetically downstream from CRYs to regulate light-controlled development. Blue-light activation of CRY1 attenuates the association of COP1 with SPA1 in both yeast and plant cells. These results indicate that the blue-light-triggered CRY1-SPA1 interaction may negatively regulate COP1, at least in part, by promoting the dissociation of COP1 from SPA1. This interaction and consequent dissociation define a dynamic photosensory signaling mechanism.

Arabidopsis noncoding RNA mediates control of photomorphogenesis by red light.

Seedling photomorphogenesis is a sophisticated developmental process that is controlled by both the transcriptional and posttranscriptional regulation of gene expression. Here, we identify an Arabidopsis noncoding RNA, designated hidden treasure 1 (HID1), as a factor promoting photomorphogenesis in continuous red 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 photomorphogenesis. Knockdown of HID1 in hid1 mutants leads to a significant increase in the expression of PIF3, which in turn drives the development of elongated hypocotyls in cR. We identified two major stem-loops in HID1 that are essential for its modulation of hypocotyl growth in cR-grown seedlings. Furthermore, our data reveal that HID1 is assembled into large nuclear protein-RNA complex(es) and that it associates with the chromatin of the first intron of PIF3 to repress its transcription. Strikingly, phylogenetic analysis reveals that many land plants have conserved homologs of HID1 and that its rice homolog can rescue the mutant phenotype when expressed in Arabidopsis hid1 mutants. We thus concluded that HID1 is a previously uncharacterized noncoding RNA whose function represents another layer of regulation in the precise control of seedling photomorphogenesis.

Arabidopsis COP1/SPA1 complex and FHY1/FHY3 associate with distinct phosphorylated forms of phytochrome A in balancing light signaling.

Fine tuning of light signaling is crucial to plant development. Following light-triggered nuclear translocation, the photoreceptor phytochrome A (phyA) regulates gene expression under continuous far-red light and is rapidly destabilized upon red light irradiation by E3 ubiquitin ligases, including COP1. Here we provide evidence that the light signaling repressors SPA proteins contribute to COP1-mediated phyA degradation and that a COP1/SPA1 protein complex is tightly associated with phyA ubiquitination activity. Furthermore, a phosphorylated phyA form accumulates in the nucleus and preferentially associates with the COP1/SPA1 complex. In contrast, underphosphorylated phyA predominantly associates with the phyA-signaling intermediates FHY3 and FHY1. However, COP1 associates with underphosphorylated phyA in the absence of FHY3 or FHY1, suggesting that phyA associations with FHY3 and FHY1 protect underphosphorylated phyA from being recognized by the COP1/SPA complex. We propose that light-induced phyA phosphorylation acts as a switch controlling differential interactions of the photoreceptor with signal propagation or attenuation machineries.

Natural variation of H3K27me3 modification in two Arabidopsis accessions and their hybrid.

Histone modifications affect gene expression, but the mechanism and biological consequence of natural variation in histone modifications remain unclear. Here, we generated genome-wide integrated maps of H3K27me3 modification and transcriptome for Col, C24 and their F1 hybrid. A total of 1,828 genomic regions showing variation in H3K27me3 modification between Col and C24 were identified, most of which were associated with genic regions. Natural variation of H3K27me3 modification between parents could result in allelic bias of H3K27me3 in hybrids. Furthermore, we found that H3K27me3 variation between Col and C24 was negatively correlated with gene expression differences between two accessions, especially with those arising from the cis-effect. Importantly, mutation of CLF, an Arabidopsis methyltransferase for H3K27, altered gene expression patterns between the parents. Together, these data provide insights into natural variation of histone modifications and their association with gene expression differences between Arabidopsis ecotypes.

Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and flowering time.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1 (DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1 with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis and flowered early under short days. Early flowering of short day-grown cul4 mutants correlated with increased FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1 can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert. Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and, possibly, of flowering time.

Structural and Functional Analyses of Hub MicroRNAs in An Integrated Gene Regulatory Network of Arabidopsis.

MicroRNAs (miRNAs) are trans-acting small regulatory RNAs that work coordinately with transcription factors (TFs) to shape the repertoire of cellular mRNAs available for translation. Despite our growing knowledge of individual plant miRNAs, their global roles in gene regulatory networks remain mostly unassessed. Based on interactions obtained from public databases and curated from the literature, we reconstructed an integrated miRNA network in Arabidopsis that includes 66 core TFs, 318 miRNAs, and 1712 downstream genes. We found that miRNAs occupy distinct niches and enrich miRNA-containing feed-forward loops (FFLs), particularly those with miRNAs as intermediate nodes. Further analyses revealed that miRNA-containing FFLs coordinate TFs located in different hierarchical layers and that intertwined miRNA-containing FFLs are associated with party and date miRNA hubs. Using the date hub MIR858A as an example, we performed detailed molecular and genetic analyses of three interconnected miRNA-containing FFLs. These analyses revealed individual functions of the selected miRNA-containing FFLs and elucidated how the date hub miRNA fulfills multiple regulatory roles. Collectively, our findings highlight the prevalence and importance of miRNA-containing FFLs, and provide new insights into the design principles and control logics of miRNA regulatory networks governing gene expression programs in plants.

Modulation of BIN2 kinase activity by HY5 controls hypocotyl elongation in the light.

ELONGATED HYPOCOTYL 5 (HY5), a basic domain/leucine zipper (bZIP) transcription factor, acts as a master regulator of transcription to promote photomorphogenesis. At present, it's unclear whether HY5 uses additional mechanisms to inhibit hypocotyl elongation. Here, we demonstrate that HY5 enhances the activity of GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), a key repressor of brassinosteroid signaling, to repress hypocotyl elongation. We show that HY5 physically interacts with and genetically acts through BIN2 to inhibit hypocotyl elongation. The interaction of HY5 with BIN2 enhances its kinase activity possibly by the promotion of BIN2 Tyr200 autophosphorylation, and subsequently represses the accumulation of the transcription factor BRASSINAZOLE-RESISTANT 1 (BZR1). Leu137 of HY5 is found to be important for the HY5-BIN2 interaction and HY5-mediated regulation of BIN2 activity, without affecting the transcriptional activity of HY5. HY5 levels increase with light intensity, which gradually enhances BIN2 activity. Thus, our work reveals an additional way in which HY5 promotes photomorphogenesis, and provides an insight into the regulation of GSK3 activity.

Arabidopsis noncoding RNA modulates seedling greening during deetiolation.

Seedling greening is essential for the survival of plants emerging from the soil. The abundance of chlorophyll precursors, including protochlorophyllide (Pchlide), is precisely controlled during the dark-to-light transition, as over-accumulation of Pchlide can lead to cellular photooxidative damage. Previous studies have identified and characterized multiple regulators controlling this important process. HID1 (hidden treasure 1) is the first noncoding RNA (ncRNA) found in photomorphogenesis. Under continuous red light, HID1 has been shown to inhibit hypocotyl elongation by repressing the transcription of PIF3 (phytochrome interacting factor 3). Here, we report that HID1 acts as a negative regulator of cotyledon greening. Knockdown of HID1 resulted in an increased greening rate of etiolated seedlings relative to wild type when exposed to white light. Genetically, HID1 acts downstream of PIF3 during the dark-to-light transition. The expression of HID1 is not regulated by PIF3 in the dark. Molecularly, the Pchlide content was reduced in dark-grown hid1 mutants than WT. Meanwhile, transcript levels of the protochlorophyllide oxidoreductases known to catalyze Pchlide to chlorophyllide conversion were significantly increased in hid1 seedlings. Thus, our study reveals an additional role of HID1 in the dark-to-light transition in Arabidopsis. Moreover, these results suggest HID1 could regulate distinct targets in different light-mediated developmental processes, and thus is essential to the control of these mechanisms.

The role of COP1 in repression of photoperiodic flowering.

Plants use the circadian clock as a timekeeping mechanism to regulate photoperiodic flowering in response to the seasonal changes. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), initially identified as a central repressor of seedling photomorphogenesis, was recently shown to be involved in the regulation of light input to the circadian clock, modulating the circadian rhythm and flowering. COP1 encodes a RING-finger E3 ubiquitin ligase and works in concert with SUPPRESSOR of phyA-105 (SPA) proteins to repress photoperiodic flowering by regulating proteasome-mediated degradation of CONSTANS (CO), a central regulator of photoperiodic flowering. In addition, COP1 and EARLY FLOWERING 3 (ELF3) indirectly modulate CO expression via the degradation of GIGANTEA (GI). Here, we summarize the current understanding of the molecular mechanisms underlying COP1's role in controlling of photoperiodic flowering.

Genomic features and regulatory roles of intermediate-sized non-coding RNAs in Arabidopsis.

Recent advances in genome-wide techniques allowed the identification of thousands of non-coding RNAs with various sizes in eukaryotes, some of which have further been shown to serve important functions in many biological processes. However, in model plant Arabidopsis, novel intermediate-sized ncRNAs (im-ncRNAs) (50~300 nt) have very limited information. By using a modified isolation strategy combined with deep-sequencing technology, we identified 838 im-ncRNAs in Arabidopsis globally. More than half (58%) are new ncRNA species, mostly evolutionary divergent. Interestingly, annotated protein-coding genes with 5'-UTR-derived novel im-ncRNAs tend to be highly expressed. For intergenic im-ncRNAs, their average abundances were comparable to mRNAs in seedlings, but subsets exhibited significantly lower expression in senescing leaves. Further, intergenic im-ncRNAs were regulated by similar genetic and epigenetic mechanisms to those of protein-coding genes, and some showed developmentally regulated expression patterns. Large-scale reverse genetic screening showed that the down-regulation of a number of im-ncRNAs resulted in either obvious molecular changes or abnormal developmental phenotypes in vivo, indicating the functional importance of im-ncRNAs in plant growth and development. Together, our results demonstrate that novel Arabidopsis im-ncRNAs are developmentally regulated and functional components discovered in the transcriptome.