RESUMEN
The phytohormone auxin, and its directional transport through tissues, plays a fundamental role in the development of higher plants. This polar auxin transport predominantly relies on PIN-FORMED (PIN) auxin exporters. Hence, PIN polarization is crucial for development, but its evolution during the rise of morphological complexity in land plants remains unclear. Here, we performed a cross-species investigation by observing the trafficking and localization of endogenous and exogenous PINs in two bryophytes, Physcomitrium patens and Marchantia polymorpha, and in the flowering plant Arabidopsis thaliana. We confirmed that the GFP fusion did not compromise the auxin export function of all examined PINs by using a radioactive auxin export assay and by observing the phenotypic changes in transgenic bryophytes. Endogenous PINs polarize to filamentous apices, while exogenous Arabidopsis PINs distribute symmetrically on the membrane in both bryophytes. In the Arabidopsis root epidermis, bryophytic PINs have no defined polarity. Pharmacological interference revealed a strong cytoskeletal dependence of bryophytic but not Arabidopsis PIN polarization. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues of individual species. These results collectively reveal the divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and the co-evolution of PIN sequence-based and cell-based polarity mechanisms.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Indolacéticos , Raíces de Plantas/metabolismo , Proteínas de Transporte de Membrana/genéticaRESUMEN
Plants require light for carbon fixation in photosynthesis and activate a suite of signal-transducing photoreceptors that regulate plant development, ranging from seed germination to flowering and fruiting. Light perception by these photoreceptors triggers massive alterations of gene expression patterns and alternative splicing (AS) of many genes in plants. RNA sequencing (RNA-seq) is a powerful tool to study the full-length transcriptomes and AS of many model organisms, including the moss Physcomitrium patens. RNA-Seq has been applied successfully in transcriptome profiling of plants' developmental processes and responses to various environmental perturbations. Studies using this method provide valuable insights into the genetic networks of plants. Here we describe the use of a high-throughput Illumina sequencing system together with bioinformatics analysis software for transcriptome and AS analysis of Physcomitrium patens in response to red light (RL).
Asunto(s)
Precursores del ARN , Empalme del ARN , Precursores del ARN/genética , Empalme Alternativo , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Plantas/metabolismoAsunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Silenciador del Gen , Empalme Alternativo , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
The superior photosynthetic efficiency of C4 leaves over C3 leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta , Transcriptoma , Zea mays , Ácidos Indolacéticos/metabolismo , Captura por Microdisección con Láser , Fotosíntesis/genética , Hojas de la Planta/embriología , Hojas de la Planta/genética , Zea mays/enzimología , Zea mays/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis , Transcriptoma , Empalme Alternativo , Arabidopsis/genética , Perfilación de la Expresión Génica/métodos , RNA-Seq , Análisis de Secuencia de ARN/métodosRESUMEN
The establishment of dorsal-ventral (DV) petal asymmetry is accompanied by differential growth of DV petal size, shape, and color differences, which enhance ornamental values. Genes involved in flower symmetry in Sinningia speciosa have been identified as CYCLOIDEA (SsCYC), but which gene regulatory network (GRN) is associated with SsCYC to establish DV petal asymmetry is still unknown. To uncover the GRN of DV petal asymmetry, we identified 630 DV differentially expressed genes (DV-DEGs) from the RNA-Seq of dorsal and ventral petals in the wild progenitor, S. speciosa 'ES'. Validated by qRT-PCR, genes in the auxin signaling transduction pathway, SsCYC, and a major regulator of anthocyanin biosynthesis were upregulated in dorsal petals. These genes correlated with a higher endogenous auxin level in dorsal petals, with longer tube length growth through cell expansion and a purple dorsal color. Over-expression of SsCYC in Nicotiana reduced petal size by regulating cell growth, suggesting that SsCYC also controls cell expansion. This suggests that auxin and SsCYC both regulate DV petal asymmetry. Transiently over-expressed SsCYC, however, could not activate most major auxin signaling genes, suggesting that SsCYC may not trigger auxin regulation. Whether auxin can activate SsCYC or whether they act independently to regulate DV petal asymmetry remains to be explored in the future.
Asunto(s)
Flores/genética , Ácidos Indolacéticos/metabolismo , Lamiales/genética , Transcriptoma/genética , Flores/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/genética , Lamiales/metabolismo , Transducción de Señal/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Precursor messenger RNA (Pre-mRNA) splicing is a crucial step in gene expression whereby the spliceosome produces constitutively and alternatively spliced transcripts. These transcripts not only diversify the transcriptome, but also play essential roles in plant development and responses to environmental changes. Much evidence indicates that regulation at the pre-mRNA splicing step is important for flowering time control; however, the components and detailed mechanism underlying this process remain largely unknown. Here, we identified the splicing factor RNA BINDING PROTEIN 45d (RBP45d), a member of the RBP45/47 family in Arabidopsis thaliana. Using sequence comparison and biochemical analysis, we determined that RBP45d is a component of the U1 small nuclear ribonucleoprotein (U1 snRNP) with functions distinct from other family members. RBP45d associates with the U1 snRNP by interacting with pre-mRNA-processing factor 39a (PRP39a) and directly regulates alternative splicing (AS) for a specific set of genes. Plants with loss of RBP45d and PRP39a function exhibited defects in temperature-induced flowering, potentially due to the misregulation of temperature-sensitive AS of FLOWERING LOCUS M as well as the accumulation of the flowering repressor FLOWERING LOCUS C. Taken together, RBP45d is a U1 snRNP component in plants that functions with PRP39a in temperature-mediated flowering.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Flores , Ribonucleoproteína Nuclear Pequeña U1 , Empalme Alternativo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Evolución Molecular , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Mutación , Filogenia , Plantas Modificadas Genéticamente , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMEN
Plants perceive dynamic light conditions and optimize their growth and development accordingly by regulating gene expression at multiple levels. Alternative splicing (AS), a widespread mechanism in eukaryotes that post-transcriptionally generates two or more messenger RNAs (mRNAs) from the same pre-mRNA, is rapidly controlled by light. However, a detailed mechanism of light-regulated AS is still not clear. In this study, we demonstrate that histone 3 lysine 36 trimethylation (H3K36me3) rapidly and differentially responds to light at specific gene loci with light-regulated intron retention (IR) of their transcripts in the moss Physcomitrella patens. However, the level of H3K36me3 following exposure to light is inversely related to that of IR events. Physcomitrella patens MORF-related gene 1 (PpMRG1), a chromatin adaptor, bound with higher affinity to H3K36me3 in light conditions than in darkness and was differentially targeted to gene loci showing light-responsive IR. Transcriptome analysis indicated that PpMRG1 functions in the regulation of light-mediated AS. Furthermore, PpMRG1 was also involved in red light-mediated phototropic responses. Our results suggest that light regulates histone methylation, which leads to alterations of AS patterns. The chromatin adaptor PpMRG1 potentially participates in light-mediated AS, revealing that chromatin-coupled regulation of pre-mRNA splicing is an important aspect of the plant's response to environmental changes.
Asunto(s)
Empalme Alternativo/fisiología , Bryopsida/metabolismo , Cromatina/metabolismo , Empalme Alternativo/genética , Bryopsida/genética , Cromatina/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Intrones/genética , Empalme del ARN/genética , Empalme del ARN/fisiologíaRESUMEN
Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1, GNC, and AN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis.
Asunto(s)
Translocador 1 del Nucleótido Adenina/metabolismo , Zea mays/crecimiento & desarrollo , Zea mays/genética , Translocador 1 del Nucleótido Adenina/fisiología , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cloroplastos/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Mijos/genética , Mijos/metabolismo , Organogénesis de las Plantas/genética , Fotosíntesis/genética , Fotosíntesis/fisiología , Desarrollo de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Plants perceive environmental light conditions and optimize their growth and development accordingly by regulating gene activity at multiple levels. Photoreceptors are important for light sensing and downstream gene regulation. Phytochromes, red/far-red light receptors, are believed to regulate light-responsive alternative splicing, but little is known about the underlying mechanism. Alternative splicing is primarily regulated by transacting factors, such as splicing regulators, and by cis-acting elements in precursor mRNA. In the moss Physcomitrella patens, we show that phytochrome 4 (PpPHY4) directly interacts with a splicing regulator, heterogeneous nuclear ribonucleoprotein F1 (PphnRNP-F1), in the nucleus to regulate light-responsive alternative splicing. RNA sequencing analysis revealed that PpPHY4 and PphnRNP-F1 coregulate 70% of intron retention (IR) events in response to red light. A repetitive GAA motif was identified to be an exonic splicing silencer that controls red light-responsive IR. Biochemical studies indicated that PphnRNP-F1 is recruited by the GAA motif to form RNA-protein complexes. Finally, red light elevates PphnRNP-F1 protein levels via PpPHY4, increasing levels of IR. We propose that PpPHY4 and PphnRNP-F1 regulate alternative splicing through an exonic splicing silencer to control splicing machinery activity in response to light.
Asunto(s)
Empalme Alternativo/fisiología , Bryopsida/metabolismo , Exones/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Fitocromo/metabolismo , Empalme Alternativo/genética , Bryopsida/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Plant photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although gene expression is modulated by photoreceptors at various levels, the regulatory mechanism at the pre-mRNA splicing step remains unclear. Alternative splicing, a widespread mechanism in eukaryotes that generates two or more mRNAs from the same pre-mRNA, is largely controlled by splicing regulators, which recruit spliceosomal components to initiate pre-mRNA splicing. The red/far-red light photoreceptor phytochrome participates in light-mediated splicing regulation, but the detailed mechanism remains unclear. Here, using protein-protein interaction analysis, we demonstrate that in the moss Physcomitrella patens, phytochrome4 physically interacts with the splicing regulator heterogeneous nuclear ribonucleoprotein H1 (PphnRNP-H1) in the nucleus, a process dependent on red light. We show that PphnRNP-H1 is involved in red light-mediated phototropic responses in P. patens and that it binds with higher affinity to the splicing factor pre-mRNA-processing factor39-1 (PpPRP39-1) in the presence of red light-activated phytochromes. Furthermore, PpPRP39-1 associates with the core component of U1 small nuclear RNP in P. patens Genome-wide analyses demonstrated the involvement of both PphnRNP-H1 and PpPRP39-1 in light-mediated splicing regulation. Our results suggest that phytochromes target the early step of spliceosome assembly via a cascade of protein-protein interactions to control pre-mRNA splicing and photomorphogenic responses.
Asunto(s)
Empalme Alternativo/efectos de la radiación , Bryopsida/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Fotorreceptores de Plantas/metabolismo , Fitocromo/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Empalme Alternativo/genética , Bryopsida/genética , Bryopsida/efectos de la radiación , Ontología de Genes , Estudio de Asociación del Genoma Completo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Luz , Fitocromo/efectos de la radiación , Unión Proteica/efectos de la radiación , Mapeo de Interacción de Proteínas , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Empalmosomas/metabolismoRESUMEN
Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.
Asunto(s)
Redes Reguladoras de Genes/genética , Fotosíntesis/genética , Hojas de la Planta/genética , Transcriptoma/genética , Regulación de la Expresión Génica de las Plantas/genética , Oryza/genética , Fotoperiodo , Proteínas de Plantas , Ribulosa-Bifosfato Carboxilasa/genética , Zea mays/genéticaRESUMEN
Alternative splicing (AS) is the main source of proteome diversity that in large part contributes to the complexity of eukaryotes. Recent global analysis of AS with RNA sequencing has revealed that AS is prevalent in plants, particularly when responding to environmental changes. Light is one of the most important environmental factors for plant growth and development. To optimize light absorption, plants evolve complex photoreceptors and signaling systems to regulate gene expression and biological processes in the cell. Genome-wide analyses have shown that light induces intensive AS in plants. However, the biochemical mechanisms of light regulating AS remain poorly understood. In this review, we aim to discuss recent progress in investigating the functions of AS, discovery of cross-talk between AS and light signaling, and the potential mechanism of light-regulated AS. Understanding how light signaling regulates the efficiency of AS and the biological significance of light-regulated AS in plant systems will provide new insights into the adaptation of plants to their environment and, ultimately, crop improvement.
Asunto(s)
Empalme Alternativo , Plantas/genética , Transducción de Señal , Luz , Plantas/efectos de la radiaciónRESUMEN
High vein density, a distinctive trait of C4 leaves, is central to both C3-to-C4 evolution and conversion of C3 to C4-like crops. We tested the hypothesis that high vein density in C4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. Up-regulation of genes in auxin biosynthesis pathways and higher auxin content were found in developing C4 leaves compared with developing C3 leaves. The same observation held for maize foliar (C4) and husk (C3) leaf primordia. Moreover, auxin content and vein density were increased in loss-of-function mutants of Arabidopsis MYC2, a suppressor of auxin biosynthesis. Treatment with an auxin biosynthesis inhibitor or an auxin transport inhibitor led to much fewer veins in new leaves. Finally, both Arabidopsis thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. Thus, development of high leaf vein density requires elevated auxin biosynthesis and transport.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Hojas de la Planta/genética , Plantas/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Transporte Biológico/genética , Vías Biosintéticas/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Desarrollo de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/clasificación , Plantas/metabolismo , Especificidad de la Especie , Zea mays/genética , Zea mays/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
C4 photosynthesis is more efficient than C3 photosynthesis for two reasons. First, C4 plants have evolved a repertoire of C4 enzymes to enhance CO2 fixation. Second, C4 leaves have Kranz anatomy with a high vein density in which the veins are surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. The BS and M cells are not only functionally well differentiated, but also well-coordinated for rapid transport of photo-assimilates between the two types of photosynthetic cells. Recent comparative transcriptomic and anatomical analyses of C3 and C4 leaves have revealed early onset of C4-related processes in leaf development, suggesting that delayed mesophyll differentiation contributes to higher C4 vein density, and have identified some candidate regulators for the higher vein density in C4 leaves. Moreover, comparative transcriptomics of maize husk (C3) and foliar leaves (C4) has identified a cohort of candidate regulators of Kranz anatomy development. In addition, there has been major progress in the identification of transcription factor binding sites, greatly increasing our knowledge of gene regulation in plants.
Asunto(s)
Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Transcriptoma/genética , Flaveria/citología , Flaveria/genética , Flaveria/metabolismo , Magnoliopsida/citología , Magnoliopsida/genética , Magnoliopsida/metabolismo , Modelos Biológicos , Fotosíntesis/genética , Fotosíntesis/fisiología , Hojas de la Planta/genética , Proteínas de Plantas/genéticaRESUMEN
Plant growth and development are constantly influenced by temperature fluctuations. To respond to temperature changes, different levels of gene regulation are modulated in the cell. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns in plants, whether AS impacts the stress defense of plants is not known. We used heat shock (HS) treatments at nondamaging temperature and messenger RNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis identified a significant number of novel AS events in the moss protonema. Nearly 50% of genes are alternatively spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site and exon skipping are mainly induced, indicating differential regulation of AS in response to heat stress. Transcripts undergoing heat-sensitive IR are mostly involved in specific functions, which suggests that plants regulate AS with transcript specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory cis-element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in P. patens and Arabidopsis (Arabidopsis thaliana) reveals that heat regulation for AS evolved early during land colonization of green plants. Our results support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to modulate gene regulation and reorganize metabolic processes.
RESUMEN
BACKGROUND: Light is one of the most important factors regulating plant growth and development. Light-sensing photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although many levels of gene expression are modulated by photoreceptors, regulation at the mRNA splicing step remains unclear. RESULTS: We performed high-throughput mRNA sequencing to analyze light-responsive changes in alternative splicing in the moss Physcomitrella patens, and found that a large number of alternative splicing events were induced by light in the moss protonema. Light-responsive intron retention preferentially occurred in transcripts involved in photosynthesis and translation. Many of the alternatively spliced transcripts were expressed from genes with a function relating to splicing or light signaling, suggesting a potential impact on pre-mRNA splicing and photomorphogenic gene regulation in response to light. Moreover, most light-regulated intron retention was induced immediately upon light exposure, while motif analysis identified a repetitive GAA motif that may function as an exonic regulatory cis element in light-mediated alternative splicing. Further analysis in gene-disrupted mutants was consistent with a function for multiple red-light photoreceptors in the upstream regulation of light-responsive alternative splicing. CONCLUSIONS: Our results indicate that intensive alternative splicing occurs in non-vascular plants and that, during photomorphogenesis, light regulates alternative splicing with transcript selectivity. We further suggest that alternative splicing is rapidly fine-tuned by light to modulate gene expression and reorganize metabolic processes, and that pre-mRNA cis elements are involved in photoreceptor-mediated splicing regulation.
Asunto(s)
Empalme Alternativo/genética , Bryopsida/genética , Regulación de la Expresión Génica de las Plantas , Fotorreceptores de Plantas/metabolismo , Bryopsida/metabolismo , Exones , Intrones , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/aislamiento & purificación , Análisis de Secuencia de ARNRESUMEN
The red/far-red light photoreceptor phytochrome mediates photomorphological responses in plants. For light sensing and signaling, phytochromes need to associate with open-chain tetrapyrrole molecules as the chromophore. Biosynthesis of tetrapyrrole chromophores requires members of ferredoxin-dependent bilin reductases (FDBRs). It was shown that LONG HYPOCOTYL 2 (HY2) is the only FDBR in flowering plants producing the phytochromobilin (PΦB) for phytochromes. However, in the moss Physcomitrella patens, we found a second FDBR that catalyzes the formation of phycourobilin (PUB), a tetrapyrrole pigment usually found as the protein-bound form in cyanobacteria and red algae. Thus, we named the enzyme PUB synthase (PUBS). Severe photomorphogenic phenotypes, including the defect of phytochrome-mediated phototropism, were observed in Physcomitrella patens when both HY2 and PUBS were disrupted by gene targeting. This indicates HY2 and PUBS function redundantly in phytochrome-mediated responses of nonvascular plants. Our studies also show that functional PUBS orthologs are found in selected lycopod and chlorophyte genomes. Using mRNA sequencing for transcriptome profiling, we demonstrate that expression of the majority of red-light-responsive genes are misregulated in the pubs hy2 double mutant. These studies showed that moss phytochromes rapidly repress expression of genes involved in cell wall organization, transcription, hormone responses, and protein phosphorylation but activate genes involved in photosynthesis and stress signaling during deetiolation. We propose that, in nonvascular plants, HY2 and PUBS produce structurally different but functionally similar chromophore precursors for phytochromes. Holophytochromes regulate biological processes through light signaling to efficiently reprogram gene expression for vegetative growth in the light.
Asunto(s)
Bryopsida/enzimología , Oxidorreductasas/metabolismo , Ficobilinas/biosíntesis , Ficoeritrina/biosíntesis , Proteínas de Plantas/metabolismo , Plastidios/fisiología , Urobilina/análogos & derivados , Bryopsida/genética , Bryopsida/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Técnicas de Inactivación de Genes , Luz , Datos de Secuencia Molecular , Oxidorreductasas/genética , Fotoperiodo , Fitocromo/genética , Fitocromo/metabolismo , Proteínas de Plantas/genética , Tetrapirroles/biosíntesis , Transcriptoma/fisiología , Urobilina/biosíntesisRESUMEN
In plants, phytochromobilin synthase (HY2) synthesize the open chain tetrapyrrole chromophore for light-sensing phytochromes. It catalyzes the double bond reduction of a heme-derived tetrapyrrole intermediate biliverdin IXalpha (BV) at the A-ring diene system. HY2 is a member of ferredoxin-dependent bilin reductases (FDBRs), which require ferredoxins (Fds) as the electron donors for double bond reductions. In this study, we investigated the interaction mechanism of FDBRs and Fds by using HY2 and Fd from Arabidopsis thaliana as model proteins. We found that one of the six Arabidopsis Fds, AtFd2, was the preferred electron donor for HY2. HY2 and AtFd2 formed a heterodimeric complex that was stabilized by chemical cross-linking. Surface-charged residues on HY2 and AtFd2 were important in the protein-protein interaction as well as BV reduction activity of HY2. These surface residues are close to the iron-sulfur center of Fd and the HY2 active site, implying that the interaction promotes direct electron transfer from the Fd to HY2-bound BV. In addition, the C12 propionate group of BV is important for HY2-catalyzed BV reduction. A possible role for this functional group is to mediate the electron transfer by interacting directly with AtFd2. Together, our biochemical data suggest a docking mechanism for HY2:BV and AtFd2.