RESUMEN
Alternative transcription start sites (TSS) are widespread in eukaryotes and can alter the 5' UTR length and coding potential of transcripts. Here we show that inorganic phosphate (Pi) availability regulates the usage of several alternative TSS in Arabidopsis (Arabidopsis thaliana). In comparison to phytohormone treatment, Pi had a pronounced and specific effect on the usage of many alternative TSS. By combining short-read RNA sequencing with long-read sequencing of full-length mRNAs, we identified a set of 45 genes showing alternative TSS under Pi deficiency. Alternative TSS affected several processes, such as translation via the exclusion of upstream open reading frames present in the 5' UTR of RETICULAN LIKE PROTEIN B1 mRNA, and subcellular localization via removal of the plastid transit peptide coding region from the mRNAs of HEME OXYGENASE 1 and SULFOQUINOVOSYLDIACYLGLYCEROL 2. Several alternative TSS also generated shorter transcripts lacking the coding potential for important domains. For example, the EVOLUTIONARILY CONSERVED C-TERMINAL REGION 4 (ECT4) locus, which encodes an N6-methyladenosine (m6A) reader, strongly expressed under Pi deficiency a short noncoding transcript (named ALTECT4) ~550 nt long with a TSS in the penultimate intron. The specific and robust induction of ALTECT4 production by Pi deficiency led to the identification of a role for m6A readers in primary root growth in response to low phosphate that is dependent on iron and is involved in modulating cell division in the root meristem. Our results identify alternative TSS usage as an important process in the plant response to Pi deficiency.
RESUMEN
Plants have evolved finely regulated defense systems to counter biotic and abiotic threats. In the natural environment, plants are typically challenged by simultaneous stresses and, amid such conditions, crosstalk between the activated signaling pathways becomes evident, ultimately altering the outcome of the defense response. As an example of combined biotic and abiotic stresses, inorganic phosphate (Pi) deficiency, common in natural and agricultural environments, can occur along with attack by the fungus Botrytis cinerea, a devastating necrotrophic generalist pathogen responsible for massive crop losses. We report that Pi deficiency in Arabidopsis thaliana increases its susceptibility to infection by B. cinerea by influencing the early stages of pathogen infection, namely spore adhesion and germination on the leaf surface. Remarkably, Pi-deficient plants are more susceptible to B. cinerea despite displaying the appropriate activation of the jasmonic acid and ethylene signaling pathways, as well as producing secondary defense metabolites and reactive oxygen species. Conversely, the callose deposition in response to B. cinerea infection is compromised under Pi-deficient conditions. The levels of abscisic acid (ABA) are increased in Pi-deficient plants, and the heightened susceptibility to B. cinerea observed under Pi deficiency can be reverted by blocking ABA biosynthesis. Furthermore, high level of leaf ABA induced by overexpression of NCED6 in Pi-sufficient plants also resulted in greater susceptibility to B. cinerea infection associated with increased spore adhesion and germination, and reduced callose deposition. Our findings reveal a link between the enhanced accumulation of ABA induced by Pi deficiency and an increased sensitivity to B. cinerea infection.
Asunto(s)
Ácido Abscísico , Arabidopsis , Botrytis , Fosfatos , Enfermedades de las Plantas , Transducción de Señal , Botrytis/fisiología , Ácido Abscísico/metabolismo , Arabidopsis/microbiología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/inmunología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Fosfatos/metabolismo , Fosfatos/deficiencia , Hojas de la Planta/microbiología , Hojas de la Planta/metabolismo , Etilenos/metabolismo , Ciclopentanos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Oxilipinas/metabolismo , Esporas Fúngicas/fisiología , Regulación de la Expresión Génica de las Plantas , Especies Reactivas de Oxígeno/metabolismo , Susceptibilidad a EnfermedadesRESUMEN
Arabidopsis primary root growth response to phosphate (Pi) deficiency is mainly controlled by changes in apoplastic iron (Fe). Upon Pi deficiency, apoplastic Fe deposition in the root apical meristem activates pathways leading to the arrest of meristem maintenance and inhibition of cell elongation. Here, we report that a member of the uncharacterized cytochrome b561 and DOMON domain (CYBDOM) protein family, named CRR, promotes iron reduction in an ascorbate-dependent manner and controls apoplastic iron deposition. Under low Pi, the crr mutant shows an enhanced reduction of primary root growth associated with increased apoplastic Fe in the root meristem and a reduction in meristematic cell division. Conversely, CRR overexpression abolishes apoplastic Fe deposition rendering primary root growth insensitive to low Pi. The crr single mutant and crr hyp1 double mutant, harboring a null allele in another member of the CYDOM family, shows increased tolerance to high-Fe stress upon germination and seedling growth. Conversely, CRR overexpression is associated with increased uptake and translocation of Fe to the shoot and results in plants highly sensitive to Fe excess. Our results identify a ferric reductase implicated in Fe homeostasis and developmental responses to abiotic stress, and reveal a biological role for CYBDOM proteins in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Homeostasis , Hierro/metabolismo , Fosfatos/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Efficiently regulating growth to adapt to varying resource availability is crucial for organisms, including plants. In particular, the acquisition of essential nutrients is vital for plant development, as a shortage of just one nutrient can significantly decrease crop yield. However, plants constantly experience fluctuations in the presence of multiple essential mineral nutrients, leading to combined nutrient stress conditions. Unfortunately, our understanding of how plants perceive and respond to these multiple stresses remains limited. Unlocking this mystery could provide valuable insights and help enhance plant nutrition strategies. This review focuses specifically on the regulation of phosphorous homeostasis in plants, with a primary emphasis on recent studies that have shed light on the intricate interactions between phosphorous and other essential elements, such as nitrogen, iron, and zinc, as well as non-essential elements like aluminum and sodium. By summarizing and consolidating these findings, this review aims to contribute to a better understanding of how plants respond to and cope with combined nutrient stress.
Asunto(s)
Minerales , Plantas , Hierro , Fósforo , NutrientesAsunto(s)
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Clatrina , Endocitosis , FosfatosRESUMEN
Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate-sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Desarrollo de la Planta , Nucleótidos/metabolismoRESUMEN
Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Calnexina/genética , Calnexina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Chaperonas Moleculares/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/genética , Fosfatos/metabolismo , Glicoproteínas/metabolismo , Adenosina Trifosfatasas/metabolismoRESUMEN
Plants need at least 13 different nutrients to maintain optimal growth. Nitrogen and phosphorus, from the Greek 'phôs' (meaning 'light') and 'phoros' (meaning 'bearer'), are the main nutrients limiting plant growth in both agricultural and natural ecosystems. Agriculture has relied heavily since the mid 1950s on the use of synthetic ammonium- and phosphorus-based fertilizers to increase crop productivity. While industrial synthesis of ammonium relies on the chemical conversion of atmospheric nitrogen, phosphorus is mined from finite reserves concentrated in a few countries. Considering our current dependence on phosphorus fertilizers for food production and the geopolitical aspects associated with current resources, it will be important to develop technologies enabling the maintenance of high crop yield with reduced fertilizer input. This will require an in-depth knowledge on the various pathways that enable plants to acquire phosphorus from the soil and maximize its economical use for growth and reproduction. In this primer, we give an overview of the factors limiting phosphorus acquisition by plants and highlight various pathways and strategies plants have evolved at the level of development, metabolism and signal transduction to adapt to phosphorus deficiency.
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Compuestos de Amonio , Fertilizantes , Agricultura , Compuestos de Amonio/metabolismo , Ecosistema , Fertilizantes/análisis , Nitrógeno/metabolismo , Fosfatos/metabolismo , Fósforo/metabolismo , Plantas/metabolismo , SueloRESUMEN
In plants, thousands of genes are associated with antisense transcription, which often produces noncoding RNAs. Although widespread, sense-antisense pairs have been implicated in a limited variety of functions in plants and are often thought to form extensive dsRNA stretches triggering gene silencing. In this opinion, we show that evidence does not support gene silencing as a major role for antisense transcription. In fact, it is more likely that antisense transcripts play diverse functions in gene regulation. We propose a general framework for the initial functional dissection of antisense transcripts, suggesting testable hypotheses relying on an experiment-based decision tree. By moving beyond the gene silencing paradigm, we argue that a broad and diverse role for natural antisense transcription will emerge.
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Regulación de la Expresión Génica , ARN sin Sentido , ARN sin Sentido/genética , ARN no Traducido , Transcripción GenéticaRESUMEN
A large portion of eukaryotic genes are associated with noncoding, natural antisense transcripts (NATs). Despite sharing extensive sequence complementarity with their sense mRNAs, mRNA-NAT pairs elusively often evade dsRNA-cleavage and siRNA-triggered silencing. More surprisingly, some NATs enhance translation of their sense mRNAs by yet unknown mechanism(s). Here, we show that translation enhancement of the rice (Oryza sativa) PHOSPHATE1.2 (PHO1.2) mRNA is enabled by specific structural rearrangements guided by its noncoding antisense RNA (cis-NATpho1.2). Their interaction in vitro revealed no evidence of widespread intermolecular dsRNA formation, but rather specific local changes in nucleotide base pairing, leading to higher flexibility of PHO1.2 mRNA at a key high guanine-cytosine�(GC) regulatory region inhibiting translation, â¼350-nt downstream of the start codon. Sense-antisense RNA interaction increased formation of the 80S complex in PHO1.2, possibly by inducing structural rearrangement within this inhibitory region, thus making this mRNA more accessible to 60S. This work presents a framework for nucleotide resolution studies of functional mRNA-antisense pairs.
Asunto(s)
Oryza/genética , ARN sin Sentido/genética , ARN Mensajero/genética , ARN no Traducido/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Biosíntesis de Proteínas , ARN Bicatenario , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN no Traducido/químicaRESUMEN
Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.
Asunto(s)
Medicago truncatula/genética , Fijación del Nitrógeno/fisiología , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/fisiología , Nódulos de las Raíces de las Plantas/fisiología , Sinorhizobium meliloti/fisiología , Simbiosis/fisiología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Fijación del Nitrógeno/genética , Nódulos de las Raíces de las Plantas/genética , Simbiosis/genéticaRESUMEN
Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Terminación de la Transcripción Genética , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Inorganic orthophosphate (Pi) is an essential nutrient for plant growth, and its availability strongly impacts crop yield. PHOSPHATE1 (PHO1) transfers Pi from root to shoot via Pi export into root xylem vessels. In this work, we demonstrate that an upstream open reading frame (uORF) present in the 5' untranslated region of the Arabidopsis (Arabidopsis thaliana) PHO1 inhibits its translation and influences Pi homeostasis. The presence of the uORF strongly inhibited the translation of a PHO1 5'UTR-luciferase construct in protoplasts. A point mutation removing the PHO1 uORF (ΔuORF) in transgenic Arabidopsis resulted in increased association of its mRNA with polysomes and led to higher PHO1 protein levels, independent of Pi availability. Interestingly, deletion of the uORF led to higher shoot Pi content and was associated with improved shoot growth under low external Pi supply and no deleterious effects under Pi-sufficient conditions. We further show that natural accessions lacking the PHO1 uORF exhibit higher PHO1 protein levels and shoot Pi content. Increased shoot Pi content was linked to the absence of the PHO1 uORF in a population of F2 segregants. We identified the PHO1 uORF in genomes of crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aesativum), and we verified the inhibitory effect of the rice PHO1 uORF on translation in protoplasts. Our work suggests that regulation of PHO1 expression via its uORF might be a genetic resource useful-both in natural populations and in the context of genome editing-toward improving plant growth under Pi-deficient conditions.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Sistemas de Lectura Abierta/genética , Fosfatos/metabolismo , Brotes de la Planta/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Ecotipo , Eliminación de Gen , Regulación de la Expresión Génica de las Plantas , Patrón de Herencia/genética , Fosfatos/deficiencia , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to control the efficiency of mRNA synthesis and consequently modulate plant growth and development. Encountering obstacles during transcription such as nucleosomes or particular DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant (TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable Arabidopsis (Arabidopsis thaliana) tfIIs plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic changes and massive, transcription-related redistribution of elongating RNAPII within transcribed regions toward the transcriptional start site. The predominant site of RNAPII accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest that polymerase backtracking/arrest frequently occurs in plant cells, and RNAPII-reactivation is essential for correct transcriptional output and proper growth/development.
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Arabidopsis/enzimología , Arabidopsis/genética , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Núcleo Celular/metabolismo , Proliferación Celular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Transcriptoma/genéticaRESUMEN
BACKGROUND: Long intergenic non-coding RNAs (lincRNAs) can act as regulators of expression of protein-coding genes. Trans-natural antisense transcripts (trans-NATs) are a type of lincRNAs that contain sequence complementary to mRNA from other loci. The regulatory potential of trans-NATs has been poorly studied in eukaryotes and no example of trans-NATs regulating gene expression in plants are reported. The goal of this study was to identify lincRNAs, and particularly trans-NATs, in Arabidopsis thaliana that have a potential to regulate expression of target genes in trans at the transcriptional or translational level. RESULTS: We identified 1001 lincRNAs using an RNAseq dataset from total polyA+ and polysome-associated RNA of seedlings grown under high and low phosphate, or shoots and roots treated with different phytohormones, of which 550 were differentially regulated. Approximately 30% of lincRNAs showed conservation amongst Brassicaceae and 25% harbored transposon element (TE) sequences. Gene co-expression network analysis highlighted a group of lincRNAs associated with the response of roots to low phosphate. A total of 129 trans-NATs were predicted, of which 88 were significantly differentially expressed under at least one pairwise comparison. Five trans-NATs showed a positive correlation between their expression and target mRNA steady-state levels, and three showed a negative correlation. Expression of four trans-NATs positively correlated with a change in target mRNA polysome association. The regulatory potential of these trans-NATs did not implicate miRNA mimics nor siRNAs. We also looked for lincRNAs that could regulate gene expression in trans by Watson-Crick DNA:RNA base pairing with target protein-encoding loci. We identified 100 and 81 with a positive or negative correlation, respectively, with steady-state level of their predicted target. The regulatory potential of one such candidate lincRNA harboring a SINE TE sequence was validated in a protoplast assay on three distinct genes containing homologous TE sequence in their promoters. Construction of networks highlighted other putative lincRNAs with multiple predicted target loci for which expression was positively correlated with target gene expression. CONCLUSIONS: This study identified lincRNAs in Arabidopsis with potential in regulating target gene expression in trans by both RNA:RNA and RNA:DNA base pairing and highlights lincRNAs harboring TE sequences in such activity.
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Emparejamiento Base , ARN sin Sentido/genética , ARN Largo no Codificante/genética , Cromatina/genética , Elementos Transponibles de ADN/genética , Redes Reguladoras de Genes , Sitios Genéticos/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Cis-Natural Antisense Transcripts (cis-NATs), which overlap protein coding genes and are transcribed from the opposite DNA strand, constitute an important group of noncoding RNAs. Whereas several examples of cis-NATs regulating the expression of their cognate sense gene are known, most cis-NATs function by altering the steady-state level or structure of mRNA via changes in transcription, mRNA stability, or splicing, and very few cases involve the regulation of sense mRNA translation. This study was designed to systematically search for cis-NATs influencing cognate sense mRNA translation in Arabidopsis (Arabidopsis thaliana). Establishment of a pipeline relying on sequencing of total polyA+ and polysomal RNA from Arabidopsis grown under various conditions (i.e. nutrient deprivation and phytohormone treatments) allowed the identification of 14 cis-NATs whose expression correlated either positively or negatively with cognate sense mRNA translation. With use of a combination of cis-NAT stable over-expression in transgenic plants and transient expression in protoplasts, the impact of cis-NAT expression on mRNA translation was confirmed for 4 out of 5 tested cis-NAT:sense mRNA pairs. These results expand the number of cis-NATs known to regulate cognate sense mRNA translation and provide a foundation for future studies of their mode of action. Moreover, this study highlights the role of this class of noncoding RNAs in translation regulation.
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Arabidopsis/genética , Biosíntesis de Proteínas , ARN sin Sentido/genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN de Planta , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Factores de Transcripción/genéticaRESUMEN
Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.
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Ácido Anhídrido Hidrolasas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regiones no Traducidas 5' , Ácido Anhídrido Hidrolasas/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Sistemas CRISPR-Cas , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Sistemas de Lectura Abierta , Plantas Modificadas Genéticamente , Polirribosomas/genética , Polirribosomas/metabolismoRESUMEN
Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input.
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Fosfatos de Inositol/metabolismo , Plantas/química , Transporte Biológico , Homeostasis , Fosfatos/metabolismo , Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Seed production requires the transfer of nutrients from the maternal seed coat to the filial endosperm and embryo. Because seed coat and filial tissues are symplasmically isolated, nutrients arriving in the seed coat via the phloem must be exported to the apoplast before reaching the embryo. Proteins implicated in the transfer of inorganic phosphate (Pi) from the seed coat to the embryo are unknown despite seed P content being an important agronomic trait. Here we show that the Arabidopsis Pi exporters PHO1 and PHOH1 are expressed in the chalazal seed coat (CZSC) of developing seeds. PHO1 is additionally expressed in developing ovules. Phosphorus (P) content and Pi flux between the seed coat and embryo were analyzed in seeds from grafts between WT roots and scions from either pho1, phoh1, or the pho1 phoh1 double mutant. Whereas P content and distribution between the seed coat and embryo in fully mature dry seeds of these mutants are similar to the WT, at the mature green stage of seed development the seed coat of the pho1 and pho1 phoh1 mutants, but not of the phoh1 mutant, retains approximately 2-fold more P than its WT control. Expression of PHO1 under a CZSC-specific promoter complemented the seed P distribution phenotype of the pho1 phoh1 double mutant. CZSC-specific down-expression of PHO1 also recapitulated the seed P distribution phenotype of pho1. Together, these experiments show that PHO1 expression in the CZSC is important for the transfer of P from the seed coat to the embryo in developing seeds.