Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect~150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using highresolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (~40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally,~51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation. [Supplemental material is available for this article.]Alternative splicing (AS) is a widespread mechanism which increases transcriptome and proteome complexity and controls developmental programs and responses to the environment in higher eukaryotes. The splicing process, removal of introns and ligation of exons, is performed by a large RNA-protein complex, the spliceosome, consisting of five small nuclear RNAs (snRNAs) and about 180 proteins with different functions (Wahl et al. 2009). Assembly of the spliceosome on introns in a precursor messenger RNA (pre-mRNA) is directed by cis elements and trans-acting factors (Black 2003;Stamm et al. 2005). The cis sequences include the splice sites, branchpoint, and polypyrimidine tract which have degenerate consensus sequences in higher eukaryotes. While many splice sites are selected in all transcripts (constitutive splicing), others are used to various levels, resulting in alternative transcripts. Selection of such alternative splice sites is affected by auxiliary cis elements located within exonic and intronic sequences, termed splicing enhancers and silencers. These elements are binding sites for trans-acting splicing factors, for example, hnRNP and SR proteins. These proteins, in addition to their functions in constitutive splicing, play a key role in AS by inhibition or ...

Alternative splicing (AS) coupled to nonsense-mediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT–PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5′ or 3′UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3′-UTRs were >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes.

PRMT5Premature Stop Codon (prmt5-5) PRMT5 PRMT5Premature

Alternative splicing (AS) of pre-mRNA represents a major mechanism underlying increased transcriptome and proteome complexity. Here, we show that the nuclear speckle RNA-binding protein (NSR) and the AS competitor long noncoding RNA (or ASCO-lncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change in AS patterns of many genes, a response largely dependent on NSRs. RNA immunoprecipitation assays demonstrate that AtNSRs interact not only with their alternatively spliced mRNA targets but also with the ASCO-RNA in vivo. The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. Expression of ASCO-RNA in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, lncRNA can hijack nuclear AS regulators to modulate AS patterns during development.

The domestication of cereals has involved common changes in morphological features, such as seed size, seed retention and modification of vegetative and inflorescence architecture that ultimately contributed to an increase in harvested yield. In barley, this process has resulted in two different cultivated types, two-rowed and six-rowed forms, both derived from the wild two-rowed ancestor, with archaeo-botanical evidence indicating the origin of six-rowed barley early in the domestication of the species, some 8,600-8,000 years ago. Variation at SIX-ROWED SPIKE 1 (VRS1) is sufficient to control this phenotype. However, phenotypes imposed by VRS1 alleles are modified by alleles at the INTERMEDIUM-C (INT-C) locus. Here we show that INT-C is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1 (TB1) and identify 17 coding mutations in barley TB1 correlated with lateral spikelet fertility phenotypes.

Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.Light regulates approximately 20% of the transcriptome in Arabidopsis thaliana and rice (1, 2). Alternative splicing has been shown to modulate gene expression during plant development and in response to environmental cues (3). We observed that the alternative splicing of At-RS31 ( Figure 1A), encoding a Ser-Arg-rich splicing factor (4), changed in different light regimes, which led us to investigate how light regulates alternative splicing in plants. Figure 1B). This effect was rapidly reversed when seedlings were placed back in light, with total recovery of the original SI in about 3 hr ( Figure 1C), indicating that the kinetics of the splicing response is slower from light to dark than from dark to light.The light effect is gene-specific ( Figure S1) and is also observed in diurnal cycles under short day conditions ( Figures 1D and S2). Furthermore, three circadian clock mutants behaved like the wild type (WT) in the response of At-RS31 alternative splicing to light/dark ( Figure S3). Changes in At-RS31 splicing are proportional to light intensity both under constant light or in short day grown seedlings ( Figure S4).Both red (660 nm) and blue (470 nm) lights produced similar results as white light ( Figure 1E). Moreover, At-RS31 splicing is not affected in phytochrome and cryptochrome signaling mutants (5, 6) behave as WT seedlings, ruling out photosensory pathways in this light regulation (Figures 1F, S5 and S6).Light-triggered changes in At-RS31 mRNA patterns are not due to differential mRNA degradation. First, the light effect is not observed in the presence of the transcription inhibitor actinomycin D ( Figure 1G). Second, the effects are still observed in upf mutants, defective in the nonsense-mediated mRNA decay (NMD) pathway (7) mRNA1 is the only isoform encoding a full-length At-RS31 protein (9). mRNA3 and mRNA2 are almost fully retained in the nucleus ( Figure S8). mRNA1 levels decrease considerably in dark without significant changes in the total amount of At-RS31 transcripts (Figures 2A and S9) which suggests that alternative splicing is instrumental in the control of mRNA1 cellular levels and, consequently, At-RS31 protein abundance. To assess how interference with At-RS31 alternative splicing regulation could affect Arabidopsis phenotype we ...

Although significant work has been undertaken regarding the response of model and crop plants to heat shock during the acclimatory phase, few studies have examined the steadystate response to the mild heat stress encountered in temperate agriculture. In the present work, we therefore exposed tuberizing potato plants to mildly elevated temperatures (30/ 20°C, day/night) for up to 5 weeks and compared tuber yield, physiological and biochemical responses, and leaf and tuber metabolomes and transcriptomes with plants grown under optimal conditions (22/16°C). Growth at elevated temperature reduced tuber yield despite an increase in net foliar photosynthesis. This was associated with major shifts in leaf and tuber metabolite profiles, a significant decrease in leaf glutathione redox state and decreased starch synthesis in tubers. Furthermore, growth at elevated temperature had a profound impact on leaf and tuber transcript expression with large numbers of transcripts displaying a rhythmic oscillation at the higher growth temperature. RT-PCR revealed perturbation in the expression of circadian clock transcripts including StSP6A, previously identified as a tuberization signal. Our data indicate that potato plants grown at moderately elevated temperatures do not exhibit classic symptoms of abiotic stress but that tuber development responds via a diversity of biochemical and molecular signals.

SummaryAlternative splicing (AS) increases the proteomic and functional capacity of genomes through the generation of alternative mRNA transcripts from the same gene. AS is now estimated to occur in a third of Arabidopsis and rice genes, and includes genes involved in the control of growth and development, responses to stress and signalling. Regulation of AS reflects the interactions between positive and negative cis sequences in the precursor messenger RNA and a range of trans-acting factors. The levels and activities of these factors differ in different cells and growth conditions. To identify changes in AS in multiple genes simultaneously, we have established a reproducible RT-PCR panel that can analyse 96 alternative splicing events and accurately measure the ratio of alternatively spliced products. This procedure detected statistically significant changes in AS in different plant organs, in plants grown under different light and day-length conditions, and in plants overexpressing splicing factors. The system provides a convenient, medium-throughput means of monitoring changes in AS in multiple genes. It can readily be applied to much larger or targeted sets of gene transcripts to generate information on the significance and regulation of AS in plant growth and development, specific processes and responses to external stimuli.