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Variation in gene expression levels is pervasive among individuals and races or varieties, and has substantial agronomic consequences, for example, by contributing to hybrid vigor. Gene expression level variation results from mutations in regulatory sequences (cis) and/or transcription factor (TF) activity (trans), but the mechanisms underlying cis and/or trans-regulatory variation of complex phenotypes remain largely unknown. Here, we investigated gene expression variation mechanisms underlying the differential accumulation of the insecticidal compounds maysin and chlorogenic acid in silks of two widely used maize (Zea mays) inbreds, B73 and A632. By combining transcriptomics and cistromics, we identified 1,338 silk direct targets of the maize R2R3-MYB TF Pericarp color1 (P1), consistent with it being a regulator of maysin and chlorogenic acid biosynthesis. Among these P1 targets, 464 showed allele-specific expression (ASE) between B73 and A632 silks. Allelic DNA-affinity purification sequencing identified 34 examples in which P1 allelic specific binding (ASB) correlated with cis-expression variation. From previous yeast one-hybrid studies, we identified nine TFs potentially implicated in the control of P1 targets, with ASB to 83 out of 464 ASE genes (cis) and differential expression of 4 out of 9 TFs between B73 and A632 silks (trans). These results provide a molecular framework for understanding universal mechanisms underlying natural variation of gene expression levels, and how the regulation of metabolic diversity is established.
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Elucidating gene regulatory networks is a major area of study within plant systems biology. Phenotypic traits are intricately linked to specific gene expression profiles. These expression patterns arise primarily from regulatory connections between sets of transcription factors (TFs) and their target genes. Here, we integrated 46 co-expression networks, 283 protein-DNA interaction (PDI) assays, and 16 million SNPs used to identify expression quantitative trait loci (eQTL) to construct TF-target networks. In total, we analyzed â¼4.6M interactions to generate four distinct types of TF-target networks: co-expression, PDI, trans -eQTL, and cis -eQTL combined with PDIs. To functionally annotate TFs based on their target genes, we implemented three different network integration strategies. We evaluated the effectiveness of each strategy through TF loss-of function mutant inspection and random network analyses. The multi-network integration allowed us to identify transcriptional regulators of several biological processes. Using the topological properties of the fully integrated network, we identified potential functionally redundant TF paralogs. Our findings retrieved functions previously documented for numerous TFs and revealed novel functions that are crucial for informing the design of future experiments. The approach here-described lays the foundation for the integration of multi-omic datasets in maize and other plant systems.
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Arabidopsis , Flavonoides , Flavonoides/metabolismo , Flavonoides/biosíntesis , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genéticaRESUMEN
At the heart of all biological processes lies the control of nuclear gene expression, which is primarily achieved through the action of transcription factors (TFs) that generally contain a nuclear localization signal (NLS) to facilitate their transport into the nucleus. However, some TFs reside in the cytoplasm in a transcriptionally inactive state and only enter the nucleus in response to specific signals, which in plants include biotic or abiotic stresses. These extra-nuclear TFs can be found in the cytosol or associated with various membrane systems, including the endoplasmic reticulum and plasma membrane. They may be integral proteins with transmembrane domains or associate peripherally with the lipid bilayer via acylation or membrane-binding domains. Although over 30 plant TFs, most of them involved in stress responses, have been experimentally shown to reside outside the nucleus, computational predictions suggest that this number is much larger. Understanding how extra-nuclear TFs are trafficked into the nucleus is essential for reconstructing transcriptional regulatory networks that govern major cellular pathways in response to biotic and abiotic signals. Here, we provide a perspective on what is known on plant extranuclear-nuclear TF retention, nuclear trafficking, and the post-translational modifications that ultimately enable them to regulate gene expression upon entering the nucleus.
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Núcleo Celular , Proteínas de Plantas , Factores de Transcripción , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Núcleo Celular/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Transporte de Proteínas , Procesamiento Proteico-Postraduccional , Regulación de la Expresión Génica de las Plantas , Plantas/metabolismo , Plantas/genética , Señales de Localización NuclearRESUMEN
Triple-negative breast cancer (TNBC) is characterized by its aggressiveness and resistance to cancer-specific transcriptome alterations. Alternative splicing (AS) is a major contributor to the diversification of cancer-specific transcriptomes. The TNBC transcriptome landscape is characterized by aberrantly spliced isoforms that promote tumor growth and resistance, underscoring the need to identify approaches that reprogram AS circuitry towards transcriptomes, favoring a delay in tumorigenesis or responsiveness to therapy. We have previously shown that flavonoid apigenin is associated with splicing factors, including heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2). Here, we showed that apigenin reprograms TNBC-associated AS transcriptome-wide. The AS events affected by apigenin were statistically enriched in hnRNPA2 substrates. Comparative transcriptomic analyses of human TNBC tumors and non-tumor tissues showed that apigenin can switch cancer-associated alternative spliced isoforms (ASI) to those found in non-tumor tissues. Apigenin preferentially affects the splicing of anti-apoptotic and proliferation factors, which are uniquely observed in cancer cells, but not in non-tumor cells. Apigenin switches cancer-associated aberrant ASI in vivo in TNBC xenograft mice by diminishing proliferation and increasing pro-apoptotic ASI. In accordance with these findings, apigenin increased apoptosis and reduced tumor proliferation, thereby halting TNBC growth in vivo. Our results revealed that apigenin reprograms transcriptome-wide TNBC-specific AS, thereby inducing apoptosis and hindering tumor growth. These findings underscore the impactful effects of nutraceuticals in altering cancer transcriptomes, offering new options to influence outcomes in TNBC treatments.
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Empalme Alternativo , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Empalme Alternativo/genética , Transcriptoma/genética , Línea Celular Tumoral , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Apigenina/farmacología , Apoptosis/genética , Isoformas de Proteínas/metabolismo , Proliferación Celular/genéticaRESUMEN
Maize (Zea mays) production systems are heavily reliant on the provision of managed inputs such as fertilizers to maximize growth and yield. Hence, the effective use of N fertilizer is crucial to minimize the associated financial and environmental costs, as well as maximize yield. However, how to effectively utilize N inputs for increased grain yields remains a substantial challenge for maize growers that requires a deeper understanding of the underlying physiological responses to N fertilizer application. We report a multi-scale investigation of five field-grown maize hybrids under low or high N supplementation regimes that includes the quantification of phenolic and prenyl-lipid compounds, cellular ultrastructural features, and gene expression traits at three developmental stages of growth. Our results reveal that maize perceives the lack of supplemented N as a stress and, when provided with additional N, will prolong vegetative growth. However, the manifestation of the stress and responses to N supplementation are highly hybrid-specific. Eight genes were differentially expressed in leaves in response to N supplementation in all tested hybrids and at all developmental stages. These genes represent potential biomarkers of N status and include two isoforms of Thiamine Thiazole Synthase involved in vitamin B1 biosynthesis. Our results uncover a detailed view of the physiological responses of maize hybrids to N supplementation in field conditions that provides insight into the interactions between management practices and the genetic diversity within maize.
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The highly active family of Mutator (Mu) DNA transposons has been widely used for forward and reverse genetics in maize. There are examples of Mu-suppressible alleles that result in conditional phenotypic effects based on the activity of Mu. Phenotypes from these Mu-suppressible mutations are observed in Mu-active genetic backgrounds, but absent when Mu activity is lost. For some Mu-suppressible alleles, phenotypic suppression likely results from an outward-reading promoter within Mu that is only active when the autonomous Mu element is silenced or lost. We isolated 35 Mu alleles from the UniformMu population that represent insertions in 24 different genes. Most of these mutant alleles are due to insertions within gene coding sequences, but several 5' UTR and intron insertions were included. RNA-seq and de novo transcript assembly were utilized to document the transcripts produced from 33 of these Mu insertion alleles. For 20 of the 33 alleles, there was evidence of transcripts initiating within the Mu sequence reading through the gene. This outward-reading promoter activity was detected in multiple types of Mu elements and does not depend on the orientation of Mu. Expression analyses of Mu-initiated transcripts revealed the Mu promoter often provides gene expression levels and patterns that are similar to the wild-type gene. These results suggest the Mu promoter may represent a minimal promoter that can respond to gene cis-regulatory elements. Findings from this study have implications for maize researchers using the UniformMu population, and more broadly highlight a strategy for transposons to co-exist with their host.
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Zea mays , Secuencia de Bases , Elementos Transponibles de ADN , Mutación , Zea mays/genéticaRESUMEN
The amount of biological data is growing at a rapid pace as many high-throughput omics technologies and data pipelines are developed. This is resulting in the growth of databases for DNA and protein sequences, gene expression, protein accumulation, structural, and localization information. The diversity and multi-omics nature of such bioinformatic data requires well-designed databases for flexible organization and presentation. Besides general-purpose online bioinformatic databases, users need narrowly focused online databases to quickly access a meaningful collection of related data for their research. Here, we describe the methodology used to implement a plant gene regulatory knowledgebase, with data, query, and tool features, as well as the ability to expand to accommodate future datasets. We exemplify this methodology for the GRASSIUS knowledgebase, but it is applicable to developing and updating similar plant gene regulatory knowledgebases. GRASSIUS organizes and presents gene regulatory data from grass species with a central focus on maize (Zea mays). The main class of data presented include not only the families of transcription factors (TFs) and co-regulators (CRs) but also protein-DNA interaction data, where available.
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Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Secuencia de Aminoácidos , Biología Computacional , Bases del Conocimiento , Zea maysRESUMEN
Recent advances in our understanding of plant metabolism have highlighted the significance of specialized metabolites in the regulation of gene expression associated with biosynthetic networks. This opinion article focuses on the molecular mechanisms of small-molecule-mediated feedback regulation at the transcriptional level and its potential modes of action, including metabolite signal perception, the nature of the sensor, and the signaling transduction mechanisms leading to transcriptional and post-transcriptional regulation, based on evidence available from plants and other kingdoms of life. We also discuss the challenges associated with identifying the occurrences, effects, and localization of small molecule-protein interactions. Further understanding of small-molecule-controlled metabolic fluxes will enable rational design of transcriptional regulation systems in metabolic engineering to produce high-value specialized metabolites.
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Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co-expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.
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Brassicaceae , Transcriptoma , Transcriptoma/genética , Ácidos Erucicos/metabolismo , Fitomejoramiento , Brassicaceae/genética , Brassicaceae/metabolismo , Aceites de Plantas/metabolismo , Semillas/genéticaRESUMEN
Basic helix-loop-helix (bHLH) proteins are one of the largest families of transcription factor (TF) in eukaryotes, and ~30% of all flowering plants' bHLH TFs contain the aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain at variable distances C-terminal from the bHLH. However, the evolutionary history and functional consequences of the bHLH/ACT-like domain association remain unknown. Here, we show that this domain association is unique to the plantae kingdom with green algae (chlorophytes) harboring a small number of bHLH genes with variable frequency of ACT-like domain's presence. bHLH-associated ACT-like domains form a monophyletic group, indicating a common origin. Indeed, phylogenetic analysis results suggest that the association of ACT-like and bHLH domains occurred early in Plantae by recruitment of an ACT-like domain in a common ancestor with widely distributed ACT DOMAIN REPEAT (ACR) genes by an ancestral bHLH gene. We determined the functional significance of this association by showing that Chlamydomonas reinhardtii ACT-like domains mediate homodimer formation and negatively affect DNA binding of the associated bHLH domains. We show that, while ACT-like domains have experienced faster selection than the associated bHLH domain, their rates of evolution are strongly and positively correlated, suggesting that the evolution of the ACT-like domains was constrained by the bHLH domains. This study proposes an evolutionary trajectory for the association of ACT-like and bHLH domains with the experimental characterization of the functional consequence in the regulation of plant-specific processes, highlighting the impacts of functional domain coevolution.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Plantas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Filogenia , Plantas/genética , Factores de Transcripción/metabolismo , Secuencias Hélice-Asa-HéliceRESUMEN
Pennycress is a potentially lucrative biofuel crop due to its high content of long-chain unsaturated fatty acids, and because it uses non-conventional pathways to achieve efficient oil production. However, metabolic engineering is required to improve pennycress oilseed content and make it an economically viable source of aviation fuel. Research is warranted to determine if further upregulation of these non-conventional pathways could improve oil production within the species even more, which would indicate these processes serve as promising metabolic engineering targets and could provide the improvement necessary for economic feasibility of this crop. To test this hypothesis, we performed a comparative biomass, metabolomic, and transcriptomic analyses between a high oil accession (HO) and low oil accession (LO) of pennycress to assess potential factors required to optimize oil content. An evident reduction in glycolysis intermediates, improved oxidative pentose phosphate pathway activity, malate accumulation in the tricarboxylic acid cycle, and an anaplerotic pathway upregulation were noted in the HO genotype. Additionally, higher levels of threonine aldolase transcripts imply a pyruvate bypass mechanism for acetyl-CoA production. Nucleotide sugar and ascorbate accumulation also were evident in HO, suggesting differential fate of associated carbon between the two genotypes. An altered transcriptome related to lipid droplet (LD) biosynthesis and stability suggests a contribution to a more tightly-packed LD arrangement in HO cotyledons. In addition to the importance of central carbon metabolism augmentation, alternative routes of carbon entry into fatty acid synthesis and modification, as well as transcriptionally modified changes in LD regulation, are key aspects of metabolism and storage associated with economically favorable phenotypes of the species.
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In biological research domains, liquid chromatography-mass spectroscopy (LC-MS) has prevailed as the preferred technique for generating high quality metabolomic data. However, even with advanced instrumentation and established data acquisition protocols, technical errors are still routinely encountered and can pose a significant challenge to unveiling biologically relevant information. In large-scale studies, signal drift and batch effects are how technical errors are most commonly manifested. We developed pseudoDrift, an R package with capabilities for data simulation and outlier detection, and a new training and testing approach that is implemented to capture and to optionally correct for technical errors in LC-MS metabolomic data. Using data simulation, we demonstrate here that our approach performs equally as well as existing methods and offers increased flexibility to the researcher. As part of our study, we generated a targeted LC-MS dataset that profiled 33 phenolic compounds from seedling stem tissue in 602 genetically diverse non-transgenic maize inbred lines. This dataset provides a unique opportunity to investigate the dynamics of specialized metabolism in plants.
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The Brassicaceae family comprises more than 3,700 species with a diversity of phenotypic characteristics, including seed oil content and composition. Recently, the global interest in Thlaspi arvense L. (pennycress) has grown as the seed oil composition makes it a suitable source for biodiesel and aviation fuel production. However, many wild traits of this species need to be domesticated to make pennycress ideal for cultivation. Molecular breeding and engineering efforts require the availability of an accurate genome sequence of the species. Here, we describe pennycress genome annotation improvements, using a combination of long- and short-read transcriptome data obtained from RNA derived from embryos of 22 accessions, in addition to public genome and gene expression information. Our analysis identified 27,213 protein-coding genes, as well as on average 6,188 biallelic SNPs. In addition, we used the identified SNPs to evaluate the population structure of our accessions. The data from this analysis support that the accession Ames 32872, originally from Armenia, is highly divergent from the other accessions, while the accessions originating from Canada and the United States cluster together. When we evaluated the likely signatures of natural selection from alternative SNPs, we found 7 candidate genes under likely recent positive selection. These genes are enriched with functions related to amino acid metabolism and lipid biosynthesis and highlight possible future targets for crop improvement efforts in pennycress.
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Thlaspi , Biocombustibles , Aceites de Plantas/metabolismo , Semillas/genética , Thlaspi/genética , Thlaspi/metabolismo , TranscriptomaRESUMEN
The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.
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Camelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Here, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co-expressed with lipid-related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well-known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA-binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP-seq). Enrichment analyses of targets supported the co-expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1-2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1-2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co-expression data and DNA-binding assays permitted us to generate a high-confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.
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Arabidopsis , Brassicaceae , Arabidopsis/genética , Brassicaceae/genética , Humanos , Metabolismo de los Lípidos/genética , Semillas/metabolismo , Triglicéridos/metabolismoRESUMEN
Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.
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Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Reguladores del Crecimiento de las Plantas/fisiología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Ciclopentanos/farmacología , Redes y Vías Metabólicas , Modelos Biológicos , Oxilipinas/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Plantas Modificadas Genéticamente , Secuencias Reguladoras de Ácidos Nucleicos , Reproducibilidad de los Resultados , Factores de Transcripción/genéticaRESUMEN
The identification and characterization of cis-regulatory DNA sequences and how they function to coordinate responses to developmental and environmental cues is of paramount importance to plant biology. Key to these regulatory processes are cis-regulatory modules (CRMs), which include enhancers and silencers. Despite the extraordinary advances in high-quality sequence assemblies and genome annotations, the identification and understanding of CRMs, and how they regulate gene expression, lag significantly behind. This is especially true for their distinguishing characteristics and activity states. Here, we review the current knowledge on CRMs and breakthrough technologies enabling identification, characterization, and validation of CRMs; we compare the genomic distributions of CRMs with respect to their target genes between different plant species, and discuss the role of transposable elements harboring CRMs in the evolution of gene expression. This is an exciting time to study cis-regulomes in plants; however, significant existing challenges need to be overcome to fully understand and appreciate the role of CRMs in plant biology and in crop improvement.
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Genoma de Planta , Plantas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Sitios de Unión , Cromatina/genética , Regulación de la Expresión Génica de las Plantas , Tamaño del Genoma , Estudio de Asociación del Genoma Completo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.
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Respuesta al Choque por Frío/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas , Respuesta al Choque Térmico/genética , Transcriptoma , Zea mays/fisiología , Perfilación de la Expresión Génica , Zea mays/genéticaRESUMEN
About a third of the plant basic helix-loop-helix (bHLH) transcription factors harbor a C-terminal aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain, which was originally identified in the maize R regulator of anthocyanin biosynthesis, where it modulates the ability of the bHLH to dimerize and bind DNA. Characterization of other bHLH ACT-like domains, such as the one in the Arabidopsis R ortholog, GL3, has not definitively confirmed dimerization, raising the question of the overall role of this potential regulatory domain. To learn more, we compared the dimerization of the ACT-like domains of R (RACT) and GL3 (GL3ACT). We show that RACT dimerizes with a dissociation constant around 100 nM, over an order of magnitude stronger than GL3ACT. Structural predictions combined with mutational analyses demonstrated that V568, located in a hydrophobic pocket in RACT, is important: when mutated to the Ser residue present in GL3ACT, dimerization affinity dropped by almost an order of magnitude. The converse S595V mutation in GL3ACT significantly increased the dimerization strength. We cloned and assayed dimerization for all identified maize ACT-like domains and determined that 12 of 42 formed heterodimers in yeast two-hybrid assays, irrespective of whether they harbored V568, which was often replaced by other aliphatic amino acids. Moreover, we determined that the presence of polar residues at that position occurs only in a small subset of anthocyanin regulators. The combined results provide new insights into possibly regulatory mechanisms and suggest that many of the other plant ACT-like domains associate to modulate fundamental cellular processes.