A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Independent parental contributions initiate zygote polarization in Arabidopsis thaliana.

Embryogenesis of flowering plants is initiated by polarization of the zygote, a prerequisite for correct axis formation in the embryo. The daughter cells of the asymmetric zygote division form the pro-embryo and the mostly extra-embryonic suspensor.1 The suspensor plays a pivotal role in nutrient and hormone transport and rapid growth of the embryo.2,3 Zygote polarization is controlled by a MITOGEN-ACTIVATING PROTEIN (MAP) kinase signaling pathway including the MAPKK kinase (MAP3K) YODA (YDA)4 and the upstream membrane-associated proteins BRASINOSTEROID SIGNALING KINASE 1 (BSK1) and BSK2.5,6 Furthermore, suspensor development is controlled by cysteine-rich peptides of the EMBRYO SURROUNDING FACTOR 1 (ESF1) family.7 While they act genetically upstream of YDA, the corresponding receptor to perceive these potential ligands is unknown. In other developmental processes, such as stomata development, YDA activity is controlled by receptor kinases of the ERECTA family (ERf).8-12 While the receptor kinases upstream of BSK1/2 in the embryo have so far not been identified,1 YDA is in part activated by the sperm cell-derived BSK family member SHORT SUSPENSOR (SSP) that represents a naturally occurring, constitutively active variant of BSK1.5,13 It has been speculated that SSP might be a paternal component of a parental tug-of-war controlling resource allocation toward the embryo.2,13 Here, we show that in addition to SSP, the receptor kinase ERECTA plays a crucial role in zygote polarization as a maternally contributed part of the embryonic YDA pathway. We conclude that two independent parental contributions initiate zygote polarization and control embryo development.

Transcriptome Sequence Reveals Candidate Genes Involving in the Post-Harvest Hardening of Trifoliate Yam Dioscorea dumetorum.

Storage ability of trifoliate yam (Dioscorea dumetorum) is restricted by a severe post-harvest hardening (PHH) phenomenon, which starts within the first 24 h after harvest and renders tubers inedible. Previous work has only focused on the biochemical changes affecting PHH in D. dumetorum. To the best of our knowledge, the candidate genes responsible for the hardening of D. dumetorum have not been identified. Here, transcriptome analyses of D. dumetorum tubers were performed in yam tubers of four developmental stages: 4 months after emergence (4MAE), immediately after harvest (AH), 3 days after harvest (3DAH) and 14 days after harvest (14DAH) of four accessions (Bangou 1, Bayangam 2, Fonkouankem 1, and Ibo sweet 3) using RNA-Seq. In total, between AH and 3DAH, 165, 199, 128 and 61 differentially expressed genes (DEGs) were detected in Bayangam 2, Fonkouankem 1, Bangou 1 and Ibo sweet 3, respectively. Functional analysis of DEGs revealed that genes encoding for CELLULOSE SYNTHASE A (CESA), XYLAN O-ACETYLTRANSFERASE (XOAT), CHLOROPHYLL A/B BINDING PROTEIN1, 2, 3, 4 (LHCB1, LHCB2, LHCB3, and LCH4) and an MYB transcription factor were predominantly and significantly up-regulated 3DAH, implying that these genes were potentially involved in the PHH as confirmed by qRT-PCR. A hypothetical mechanism of this phenomenon and its regulation has been proposed. These findings provide the first comprehensive insights into gene expression in yam tubers after harvest and valuable information for molecular breeding against the PHH.

Marchantia TCP transcription factor activity correlates with three-dimensional chromatin structure.

Information in the genome is not only encoded within sequence or epigenetic modifications, but is also found in how it folds in three-dimensional space. The formation of self-interacting genomic regions, named topologically associated domains (TADs), is known as a key feature of genome organization beyond the nucleosomal level. However, our understanding of the formation and function of TADs in plants is extremely limited. Here we show that the genome of Marchantia polymorpha, a member of a basal land plant lineage, exhibits TADs with epigenetic features similar to those of higher plants. By analysing various epigenetic marks across Marchantia TADs, we find that these regions generally represent interstitial heterochromatin and their borders are enriched with Marchantia transcription factor TCP1. We also identify a type of TAD that we name 'TCP1-rich TAD', in which genomic regions are highly accessible and are densely bound by TCP1 proteins. Transcription of TCP1 target genes differs on the basis gene location, and those in TCP1-rich TADs clearly show a lower expression level. In tcp1 mutant lines, neither TCP1-bound TAD borders nor TCP1-rich TADs display drastically altered chromatin organization patterns, suggesting that, in Marchantia, TCP1 is dispensable for TAD formation. However, we find that in tcp1 mutants, genes residing in TCP1-rich TADs have a greater extent of expression fold change as opposed to genes that do not belong to these TADs. Our results suggest that, besides standing as spatial chromatin-packing modules, plant TADs function as nuclear microcompartments associated with transcription factor activities.

A novel isoform of cryptochrome 4 (Cry4b) is expressed in the retina of a night-migratory songbird.

The primary sensory molecule underlying light-dependent magnetic compass orientation in migratory birds has still not been identified. The cryptochromes are the only known class of vertebrate proteins which could mediate this mechanism in the avian retina. Cryptochrome 4 of the night-migratory songbird the European robin (Erithacus rubecula; erCry4) has several of the properties needed to be the primary magnetoreceptor in the avian eye. Here, we report on the identification of a novel isoform of erCry4, which we named erCry4b. Cry4b includes an additional exon of 29 amino acids compared to the previously described form of Cry4, now called Cry4a. When comparing the retinal circadian mRNA expression pattern of the already known isoform erCry4a and the novel erCry4b isoform, we find that erCry4a is stably expressed throughout day and night, whereas erCry4b shows a diurnal mRNA oscillation. The differential characteristics of the two erCry4 isoforms regarding their 24-h rhythmicity in mRNA expression leads us to suggest that they might have different functions. Based on the 24-h expression pattern, erCry4a remains the more likely cryptochrome to be involved in radical-pair-based magnetoreception, but at the present time, an involvement of erCry4b cannot be excluded.

Metabolic Labeling of RNAs Uncovers Hidden Features and Dynamics of the Arabidopsis Transcriptome.

Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants. Here, we demonstrate that RNA labeling with the modified, nontoxic uridine analog 5-ethynyl uridine (5-EU) in Arabidopsis (Arabidopsis thaliana) seedlings provides insight into plant transcriptome dynamics. Pulse labeling with 5-EU revealed nascent and unstable RNAs, RNA processing intermediates generated by splicing, and chloroplast RNAs. Pulse-chase experiments with 5-EU allowed us to determine RNA stabilities without the need for chemical transcription inhibitors such as actinomycin and cordycepin. Inhibitor-free, genome-wide analysis of polyadenylated RNA stability via 5-EU pulse-chase experiments revealed RNAs with shorter half-lives than those reported after chemical inhibition of transcription. In summary, our results indicate that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates and suggest that polyadenylated RNAs have low stability in plants. Our technique lays the foundation for easy, affordable, nascent transcriptome analysis and inhibitor-free analysis of RNA stability in plants.

The U1 snRNP Subunit LUC7 Modulates Plant Development and Stress Responses via Regulation of Alternative Splicing.

Introns are removed by the spliceosome, a large macromolecular complex composed of five ribonucleoprotein subcomplexes (U snRNPs). The U1 snRNP, which binds to 5' splice sites, plays an essential role in early steps of the splicing reaction. Here, we show that Arabidopsis thaliana LETHAL UNLESS CBC7 (LUC7) proteins, which are encoded by a three-member gene family in Arabidopsis, are important for plant development and stress resistance. We show that LUC7 is a U1 snRNP accessory protein by RNA immunoprecipitation experiments and LUC7 protein complex purifications. Transcriptome analyses revealed that LUC7 proteins are not only important for constitutive splicing, but also affect hundreds of alternative splicing events. Interestingly, LUC7 proteins specifically promote splicing of a subset of terminal introns. Splicing of LUC7-dependent introns is a prerequisite for nuclear export, and some splicing events are modulated by stress in a LUC7-dependent manner. Taken together, our results highlight the importance of the U1 snRNP component LUC7 in splicing regulation and suggest a previously unrecognized role of a U1 snRNP accessory factor in terminal intron splicing.

Arabidopsis RNA processing factor SERRATE regulates the transcription of intronless genes.

Intron splicing increases proteome complexity, promotes RNA stability, and enhances transcription. However, introns and the concomitant need for splicing extend the time required for gene expression and can cause an undesirable delay in the activation of genes. Here, we show that the plant microRNA processing factor SERRATE (SE) plays an unexpected and pivotal role in the regulation of intronless genes. Arabidopsis SE associated with more than 1000, mainly intronless, genes in a transcription-dependent manner. Chromatin-bound SE liaised with paused and elongating polymerase II complexes and promoted their association with intronless target genes. Our results indicate that stress-responsive genes contain no or few introns, which negatively affects their expression strength, but that some genes circumvent this limitation via a novel SE-dependent transcriptional activation mechanism. Transcriptome analysis of a Drosophila mutant defective in ARS2, the metazoan homologue of SE, suggests that SE/ARS2 function in regulating intronless genes might be conserved across kingdoms.

PORCUPINE regulates development in response to temperature through alternative splicing.

Recent findings suggest that alternative splicing has a critical role in controlling the responses of plants to temperature variations. However, alternative splicing factors in plants are largely uncharacterized. Here we establish the putative splice regulator, PORCUPINE (PCP), as temperature-specific regulator of development in Arabidopsis thaliana. Our findings point to the misregulation of WUSCHEL and CLAVATA3 as the possible cause for the meristem defects affecting the pcp-1 loss-of-function mutants at low temperatures.

Rapid Assessment of DNA Methylation Changes in Response to Salicylic Acid by Chop-qPCR.

Methylation of cytosines plays an important role in epigenetic regulation of gene expression. Several methods exist to determine the methylation status of DNA. Here, we describe a rapid and cost-effective method called Chop-qPCR to determine dynamic changes in the DNA methylation patterns, as they occur for instance in response to environmental stresses.

Determining Nucleosome Position at Individual Loci After Biotic Stress Using MNase-qPCR.

Nucleosome occupancy in promoter and genic regions can severely influence the transcription levels. Few methods have been established to investigate the nucleosome occupancy along the DNA. In this chapter we describe a detailed protocol to analyze the nucleosome occupancy at a specific locus using MNase-pPCR.

Rapid and parallel quantification of small and large RNA species.

Quantitative real-time PCR (qRT-PCR) is a common technique for mRNA quantification. Several methods have been developed in the past few years in order to adapt qRT-PCR also for small non-coding RNAs (sRNA). We here provide a simple and sensitive protocol that allows quantification of mRNAs, selected sRNAs, and long non-coding RNAs (lncRNA) in one cDNA sample by qRT-PCR.

Immunoprecipitation-based analysis of protein-protein interactions.

Several techniques allow the detection of protein-protein interactions. In vivo co-immunoprecipitation (Co-IP) studies are an important complement to other commonly used techniques such as yeast two-hybrid or fluorescence complementation, as they reveal interactions between functional proteins at physiological relevant concentrations. Here, we describe an in vivo Co-IP approach using either GFP affinity matrix or specific antibodies to purify proteins of interests and their interacting partners.

RACK1 and the microRNA pathway: is it déjà-vu all over again?

MicroRNAs (miRNAs) control many aspects of development and adaption in plants and in animals by post-transcriptional control of mRNA stability and translatability. Over the last years numerous proteins have been identified in the miRNA pathway. The versatile scaffold protein RACK1 has been associated with efficient miRNA production and function in plants and metazoans. Here, we briefly summarize the differences of RACK1 function in the plant and animal miRNA pathways and discuss putative mechanisms and functional roles of RACK1 in miRNA biogenesis and action.

RACK1 scaffold proteins influence miRNA abundance in Arabidopsis.

MicroRNAs (miRNAs) regulate plant development by post-transcriptional regulation of target genes. In Arabidopsis thaliana, DCL1 processes precursors (pri-miRNAs) to miRNA duplexes, which associate with AGO1. Additional proteins act in concert with DCL1 (e.g. HYL1 and SERRATE) or AGO1 to facilitate efficient and precise pri-miRNA processing and miRNA loading, respectively. In this study, we show that the accumulation of plant microRNAs depends on RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1), a scaffold protein that is found in all higher eukaryotes. miRNA levels are reduced in rack1 mutants, and our data suggest that RACK1 affects the microRNA pathway via several distinct mechanisms involving direct interactions with known microRNA factors: RACK1 ensures the accumulation and processing of some pri-miRNAs, directly interacts with SERRATE and is part of an AGO1 complex. As a result, mutations in RACK1 lead to over-accumulation of miRNA target mRNAs, which are important for ABA responses and phyllotaxy, for example. In conclusion, our study identified complex functioning of RACK1 proteins in the Arabidopsis miRNA pathway; these proteins are important for miRNA production and therefore plant development.

Enhanced microRNA accumulation through stemloop-adjacent introns.

MicroRNAs (miRNAs) originate from stemloop-forming precursor RNAs found in longer primary transcripts that often contain introns. We show that in plants, those introns, when located 3' of the stemloop, can promote mature miRNA accumulation, through a mechanism that likely operates at the level of miRNA processing or stability. Reversely, when miRNA production is reduced such as in dicer-like 1 mutants, splicing of introns that promote miRNA processing is considerably increased, pointing to a tight physical and temporal coordination of intron splicing and miRNA processing in plants. Our findings further suggest that miRNA transcripts without introns generated through alternative polyA-site usage might contribute to the differential adjustment of miRNA levels, possibly at a tissue-specific level.

Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1.

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.

RNA 3' processing functions of Arabidopsis FCA and FPA limit intergenic transcription.

The RNA-binding proteins FCA and FPA were identified based on their repression of the flowering time regulator FLC but have since been shown to have widespread roles in the Arabidopsis thaliana genome. Here, we use whole-genome tiling arrays to show that a wide spectrum of genes and transposable elements are misexpressed in the fca-9 fpa-7 (fcafpa) double mutant at two stages of seedling development. There was a significant bias for misregulated genomic segments mapping to the 3' region of genes. In addition, the double mutant misexpressed a large number of previously unannotated genomic segments corresponding to intergenic regions. We characterized a subset of these misexpressed unannotated segments and established that they resulted from extensive transcriptional read-through, use of downstream polyadenylation sites, and alternative splicing. In some cases, the transcriptional read-through significantly reduced expression of the associated genes. FCA/FPA-dependent changes in DNA methylation were found at several loci, supporting previous associations of FCA/FPA function with chromatin modifications. Our data suggest that FCA and FPA play important roles in the A. thaliana genome in RNA 3' processing and transcription termination, thus limiting intergenic transcription.

Support vector machines-based identification of alternative splicing in Arabidopsis thaliana from whole-genome tiling arrays.

BACKGROUND: Alternative splicing (AS) is a process which generates several distinct mRNA isoforms from the same gene by splicing different portions out of the precursor transcript. Due to the (patho-)physiological importance of AS, a complete inventory of AS is of great interest. While this is in reach for human and mammalian model organisms, our knowledge of AS in plants has remained more incomplete. Experimental approaches for monitoring AS are either based on transcript sequencing or rely on hybridization to DNA microarrays. Among the microarray platforms facilitating the discovery of AS events, tiling arrays are well-suited for identifying intron retention, the most prevalent type of AS in plants. However, analyzing tiling array data is challenging, because of high noise levels and limited probe coverage. RESULTS: In this work, we present a novel method to detect intron retentions (IR) and exon skips (ES) from tiling arrays. While statistical tests have typically been proposed for this purpose, our method instead utilizes support vector machines (SVMs) which are appreciated for their accuracy and robustness to noise. Existing EST and cDNA sequences served for supervised training and evaluation. Analyzing a large collection of publicly available microarray and sequence data for the model plant A. thaliana, we demonstrated that our method is more accurate than existing approaches. The method was applied in a genome-wide screen which resulted in the discovery of 1,355 IR events. A comparison of these IR events to the TAIR annotation and a large set of short-read RNA-seq data showed that 830 of the predicted IR events are novel and that 525 events (39%) overlap with either the TAIR annotation or the IR events inferred from the RNA-seq data. CONCLUSIONS: The method developed in this work expands the scarce repertoire of analysis tools for the identification of alternative mRNA splicing from whole-genome tiling arrays. Our predictions are highly enriched with known AS events and complement the A. thaliana genome annotation with respect to AS. Since all predicted AS events can be precisely attributed to experimental conditions, our work provides a basis for follow-up studies focused on the elucidation of the regulatory mechanisms underlying tissue-specific and stress-dependent AS in plants.