Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs

Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance

Insight on genes affecting tuber development in potato upon <i>Potato spindle tuber viroid</i> (PSTVd) infection

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection

Vernalization-Repression of Arabidopsis FLC Requires Promoter Sequences but Not Antisense Transcripts

The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts

Sense and Antisense Transcripts of Convergent Gene Pairs in Arabidopsis thaliana Can Share a Common Polyadenylation Region

The Arabidopsis genome contains a large number of gene pairs that encode sense and antisense transcripts with overlapping 3′ regions, indicative for a potential role of natural antisense transcription in regulating sense gene expression or transcript processing. When we mapped poly(A) transcripts of three plant gene pairs with long overlapping antisense transcripts, we identified an unusual transcript composition for two of the three gene pairs. Both genes pairs encoded a class of long sense transcripts and a class of short sense transcripts that terminate within the same polyadenylation region as the antisense transcripts encoded by the opposite strand. We find that the presence of the short sense transcript was not dependent on the expression of an antisense transcript. This argues against the assumption that the common termination region for sense and antisense poly(A) transcripts is the result of antisense-specific regulation. We speculate that for some genes evolution may have especially favoured alternative polyadenylation events that shorten transcript length for gene pairs with overlapping sense/antisense transcription, if this reduces the likelihood for dsRNA formation and transcript degradation

Antagonistic Roles of SEPALLATA3, FT and FLC Genes as Targets of the Polycomb Group Gene CURLY LEAF

In Arabidopsis, mutations in the Pc-G gene CURLY LEAF (CLF) give early flowering plants with curled leaves. This phenotype is caused by mis-expression of the floral homeotic gene AGAMOUS (AG) in leaves, so that ag mutations largely suppress the clf phenotype. Here, we identify three mutations that suppress clf despite maintaining high AG expression. We show that the suppressors correspond to mutations in FPA and FT, two genes promoting flowering, and in SEPALLATA3 (SEP3) which encodes a co-factor for AG protein. The suppression of the clf phenotype is correlated with low SEP3 expression in all case and reveals that SEP3 has a role in promoting flowering in addition to its role in controlling floral organ identity. Genetic analysis of clf ft mutants indicates that CLF promotes flowering by reducing expression of FLC, a repressor of flowering. We conclude that SEP3 is the key target mediating the clf phenotype, and that the antagonistic effects of CLF target genes masks a role for CLF in promoting flowering

Arabidopsis Homologs of Retinoblastoma-Associated Protein 46/48 Associate with a Histone Deacetylase to Act Redundantly in Chromatin Silencing

RNA molecules such as small-interfering RNAs (siRNAs) and antisense RNAs (asRNAs) trigger chromatin silencing of target loci. In the model plant Arabidopsis, RNA–triggered chromatin silencing involves repressive histone modifications such as histone deacetylation, histone H3 lysine-9 methylation, and H3 lysine-27 monomethylation. Here, we report that two Arabidopsis homologs of the human histone-binding proteins Retinoblastoma-Associated Protein 46/48 (RbAp46/48), known as MSI4 (or FVE) and MSI5, function in partial redundancy in chromatin silencing of various loci targeted by siRNAs or asRNAs. We show that MSI5 acts in partial redundancy with FVE to silence FLOWERING LOCUS C (FLC), which is a crucial floral repressor subject to asRNA–mediated silencing, FLC homologs, and other loci including transposable and repetitive elements which are targets of siRNA–directed DNA Methylation (RdDM). Both FVE and MSI5 associate with HISTONE DEACETYLASE 6 (HDA6) to form complexes and directly interact with the target loci, leading to histone deacetylation and transcriptional silencing. In addition, these two genes function in de novo CHH (H = A, T, or C) methylation and maintenance of symmetric cytosine methylation (mainly CHG methylation) at endogenous RdDM target loci, and they are also required for establishment of cytosine methylation in the previously unmethylated sequences directed by the RdDM pathway. This reveals an important functional divergence of the plant RbAp46/48 relatives from animal counterparts

A Companion Cell–Dominant and Developmentally Regulated H3K4 Demethylase Controls Flowering Time in Arabidopsis via the Repression of FLC Expression

Flowering time relies on the integration of intrinsic developmental cues and environmental signals. FLC and its downstream target FT are key players in the floral transition in Arabidopsis. Here, we characterized the expression pattern and function of JMJ18, a novel JmjC domain-containing histone H3K4 demethylase gene in Arabidopsis. JMJ18 was dominantly expressed in companion cells; its temporal expression pattern was negatively and positively correlated with that of FLC and FT, respectively, during vegetative development. Mutations in JMJ18 resulted in a weak late-flowering phenotype, while JMJ18 overexpressors exhibited an obvious early-flowering phenotype. JMJ18 displayed demethylase activity toward H3K4me3 and H3K4me2, and bound FLC chromatin directly. The levels of H3K4me3 and H3K4me2 in chromatins of FLC clade genes and the expression of FLC clade genes were reduced, whereas FT expression was induced and the protein expression of FT increased in JMJ18 overexpressor lines. The early-flowering phenotype caused by the overexpression of JMJ18 was mainly dependent on the functional FT. Our findings suggest that the companion cell–dominant and developmentally regulated JMJ18 binds directly to the FLC locus, reducing the level of H3K4 methylation in FLC chromatin and repressing the expression of FLC, thereby promoting the expression of FT in companion cells to stimulate flowering

Control of Flowering and Cell Fate by LIF2, an RNA Binding Partner of the Polycomb Complex Component LHP1

Polycomb Repressive Complexes (PRC) modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2). LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA processing and Polycomb regulation