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.

Vernalization, the promotion of flowering by cold, involves Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC). Cold progressively promotes cell-autonomous switching to a silenced state. Here, we used live-cell imaging of FLC-lacO to monitor changes in nuclear organization during vernalization. FLC-lacO alleles physically cluster during the cold and generally remain so after plants are returned to warm. Clustering is dependent on the Polycomb trans-factors necessary for establishment of the FLC silenced state but not on LIKE HETEROCHROMATIN PROTEIN 1, which functions to maintain silencing. These data support the view that physical clustering may be a common feature of Polycomb-mediated epigenetic switching mechanisms.

Precursor mRNA (pre-mRNA) splicing is essential for gene expression in most eukaryotic organisms. Previous studies from mammals, Drosophila, and yeast show that the majority of splicing events occurs co-transcriptionally. In plants, however, the features of co-transcriptional splicing (CTS) and its regulation still remain largely unknown. Here, we used chromatin-bound RNA sequencing to study CTS in Arabidopsis thaliana. We found that CTS is widespread in Arabidopsis seedlings, with a large proportion of alternative splicing events determined co-transcriptionally. CTS efficiency correlated with gene expression level, the chromatin landscape and, most surprisingly, the number of introns and exons of individual genes, but is independent of gene length. In combination with enhanced crosslinking and immunoprecipitation sequencing analysis, we further showed that the hnRNP-like proteins RZ-1B and RZ-1C promote efficient CTS globally through direct binding, frequently to exonic sequences. Notably, this general effect of RZ-1B/1C on splicing promotion is mainly observed at the chromatin level, not at the mRNA level. RZ-1C promotes CTS of multiple-exon genes in association with its binding to regions both proximal and distal to the regulated introns. We propose that RZ-1C promotes efficient CTS of genes with multiple exons through cooperative interactions with many exons, introns, and splicing factors. Our work thus reveals important features of CTS in plants and provides methodologies for the investigation of CTS and RNA-binding proteins in plants.

As a cell cycle regulator, the Myb-related CDC5 protein was reported to be essential for the G2 phase of the cell cycle in yeast and animals, but little is known about its function in plants. Here we report the functional characterization of the CDC5 gene in Arabidopsis thaliana. Arabidopsis CDC5 (AtCDC5) is mainly expressed in tissues with high cell division activity, and is expressed throughout the entire process of embryo formation. The AtCDC5 loss-of-function mutant is embryonic lethal. In order to investigate the function of AtCDC5 in vivo, we generated AtCDC5-RNAi plants in which the expression of AtCDC5 was reduced by RNA interference. We found that the G2 to M (G2/M) phase transition was affected in the AtCDC5-RNAi plants, and that endoreduplication was increased. Additionally, the maintenance of shoot apical meristem (SAM) function was disturbed in the AtCDC5-RNAi plants, in which both the WUSCHEL (WUS)-CLAVATA (CLV) and the SHOOT MERISTEMLESS (STM) pathways were impaired. In situ hybridization analysis showed that the expression of STM was greatly reduced in the shoot apical cells of the AtCDC5-RNAi plants. Moreover, cyclinB1 or Histone4 was found to be expressed in some of these cells when the transcript of STM was undetectable. These results suggest that AtCDC5 is essential for the G2/M phase transition and may regulate the function of SAM by controlling the expression of STM and WUS.

The timing of flowering is vital for plant reproductive success and is therefore tightly regulated by endogenous and exogenous cues. In summer annual Arabidopsis (Arabidopsis thaliana) accessions, like Columbia-0, rapid flowering is promoted by repression of the floral repressor FLOWERING LOCUS C (FLC). This is through the activity of the autonomous pathway, a group of proteins with diverse functions including RNA 39-end processing factors, spliceosome components, a transcription elongation factor, and chromatin modifiers. These factors function at the FLC locus linking alternative processing of an antisense long noncoding RNA, called COOLAIR, with delivery of a repressive chromatin environment that affects the transcriptional output. The transcriptional output feeds back to influence the chromatin environment, reinforcing and stabilizing that state. This review summarizes our current knowledge of the autonomous pathway and compares it with similar cotranscriptional mechanisms in other organisms.

Water is the most abundant molecule in almost all living organisms. Aquaporins are channel proteins that play critical roles in controlling the water content of cells. Here, we report the identification of an AP2/EREBP family transcription factor in Arabidopsis thaliana, TRANSLUCENT GREEN (TG), whose overexpression in transgenic plants gave enhanced drought tolerance and vitrified leaves. TG protein is localized in the nucleus, binds DRE and GCC elements in vitro, and acts as a transcriptional activator in yeast cells. Microarray analysis revealed a total of 330 genes regulated by TG, among which five genes encode aquaporins. A transient expression assay showed that TG directly binds to the promoters of three aquaporin genes, such as AtTIP1;1, AtTIP2;3, and AtPIP2;2, indicating that TG directly regulates the expression of these genes. Moreover, overexpression of AtTIP1;1 resulted in vitrified phenotypes in transgenic Arabidopsis plants, similar to those observed in TG overexpression lines. Water injection into wild-type leaves recapitulated the vitrified leaf phenotypes, which was reversed by cutting off the water supply from vascular bundles. Taken together, our data support that TG controls water balance in Arabidopsis through directly activating the expression of aquaporin genes.

Changes in nuclear organization are considered an important complement to trans-acting factors, histone modifications and non-coding RNAs in robust and stable epigenetic silencing. However, how these multiple layers interconnect mechanistically to reinforce each other's activity is still unclear. A system providing long timescales facilitating analysis of these interconnections is vernalization. This involves the Polycomb-mediated epigenetic silencing of flowering locus C (FLC) that occurs as Arabidopsis plants are exposed to prolonged cold. Analysis of changes in nuclear organization during vernalization has revealed that disruption of a gene loop and physical clustering of FLC loci are part of the vernalization mechanism. These events occur at different times and thus contribute to distinct aspects of the silencing mechanism. The physical clustering of FLC loci is tightly correlated with the accumulation of specific Polycomb complexes/H3K27me3 at a localized intragenic site during the cold. Since the quantitative nature of vernalization is a reflection of a bistable cell autonomous switch in an increasing number of cells, this correlation suggests a tight connection between the switching mechanism and changes in nuclear organization. This integrated picture is likely to be informative for many epigenetic mechanisms.

Seed germination represents a major developmental switch in plants that is vital to agriculture, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis thaliana requires a chromatin mechanism that progressively silences 9-CIS-EPOXYCAROTENOID DIOXYGENASE 6 (NCED6), which encodes a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocked germination and synergistically derepressed NCEDs and hundreds of genes. At NCED6, in part by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also an H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis revealed that RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination, when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate for and synergize with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via the RNA-binding protein RZ1 and PRC2 acting in synergy, a process that is vital for seed germination.