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1.
Plant J ; 117(3): 818-839, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37947266

RESUMO

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ribossomos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo
2.
Plant Cell ; 34(1): 514-534, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34735005

RESUMO

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.


Assuntos
Resposta ao Choque Frio/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas , Resposta ao Choque Térmico/genética , Transcriptoma , Zea mays/fisiologia , Perfilação da Expressão Gênica , Zea mays/genética
3.
Proc Natl Acad Sci U S A ; 119(20): e2121362119, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35549553

RESUMO

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light­induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteólise , Proteostase , Transcrição Gênica , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Luz , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteólise/efeitos da radiação , Proteostase/genética , Proteostase/efeitos da radiação , Transcrição Gênica/efeitos da radiação
4.
Plant J ; 111(1): 103-116, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35436373

RESUMO

The DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element (TE) content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. Double mutant (drm1ab) plants exhibit some morphological abnormalities but are fully viable. Whole-genome methylation profiling provided evidence for the widespread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for the locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified genes with altered expression in the drm1ab mutants. However, the majority of genes with high levels of CHH methylation directly surrounding the transcription start site or in nearby promoter regions in wild-type plants do not have altered expression in the drm1ab mutant, even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of TEs identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons are likely to require active RdDM for the maintenance of transcriptional repression.


Assuntos
Setaria (Planta) , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Metiltransferases/genética , Setaria (Planta)/genética , Transcriptoma
5.
Plant Physiol ; 190(2): 1153-1164, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-35689624

RESUMO

CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.


Assuntos
Arabidopsis , Sistemas CRISPR-Cas , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Cromatina/genética , Epigenômica , Edição de Genes/métodos
6.
Plant Cell ; 32(5): 1377-1396, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32184350

RESUMO

The regulation of gene expression is central to many biological processes. Gene regulatory networks (GRNs) link transcription factors (TFs) to their target genes and represent maps of potential transcriptional regulation. Here, we analyzed a large number of publically available maize (Zea mays) transcriptome data sets including >6000 RNA sequencing samples to generate 45 coexpression-based GRNs that represent potential regulatory relationships between TFs and other genes in different populations of samples (cross-tissue, cross-genotype, and tissue-and-genotype samples). While these networks are all enriched for biologically relevant interactions, different networks capture distinct TF-target associations and biological processes. By examining the power of our coexpression-based GRNs to accurately predict covarying TF-target relationships in natural variation data sets, we found that presence/absence changes rather than quantitative changes in TF gene expression are more likely associated with changes in target gene expression. Integrating information from our TF-target predictions and previous expression quantitative trait loci (eQTL) mapping results provided support for 68 TFs underlying 74 previously identified trans-eQTL hotspots spanning a variety of metabolic pathways. This study highlights the utility of developing multiple GRNs within a species to detect putative regulators of important plant pathways and provides potential targets for breeding or biotechnological applications.


Assuntos
Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Zea mays/genética , Arabidopsis/genética , Bases de Dados Genéticas , Ontologia Genética , Anotação de Sequência Molecular , Filogenia , Locos de Características Quantitativas/genética , Fatores de Transcrição/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(38): 23991-24000, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32879011

RESUMO

The genomic sequences of crops continue to be produced at a frenetic pace. It remains challenging to develop complete annotations of functional genes and regulatory elements in these genomes. Chromatin accessibility assays enable discovery of functional elements; however, to uncover the full portfolio of cis-elements would require profiling of many combinations of cell types, tissues, developmental stages, and environments. Here, we explore the potential to use DNA methylation profiles to develop more complete annotations. Using leaf tissue in maize, we define ∼100,000 unmethylated regions (UMRs) that account for 5.8% of the genome; 33,375 UMRs are found greater than 2 kb from genes. UMRs are highly stable in multiple vegetative tissues, and they capture the vast majority of accessible chromatin regions from leaf tissue. However, many UMRs are not accessible in leaf, and these represent regions with potential to become accessible in specific cell types or developmental stages. These UMRs often occur near genes that are expressed in other tissues and are enriched for binding sites of transcription factors. The leaf-inaccessible UMRs exhibit unique chromatin modification patterns and are enriched for chromatin interactions with nearby genes. The total UMR space in four additional monocots ranges from 80 to 120 megabases, which is remarkably similar considering the range in genome size of 271 megabases to 4.8 gigabases. In summary, based on the profile from a single tissue, DNA methylation signatures provide powerful filters to distill large genomes down to the small fraction of putative functional genes and regulatory elements.


Assuntos
Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Sequências Reguladoras de Ácido Nucleico/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA de Plantas/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Zea mays/genética
8.
Biochem Soc Trans ; 50(1): 583-596, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-35212360

RESUMO

DNA methylation is a chromatin modification that plays an essential role in regulating gene expression and genome stability and it is typically associated with gene silencing and heterochromatin. Owing to its heritability, alterations in the patterns of DNA methylation have the potential to provide for epigenetic inheritance of traits. Contemporary epigenomic technologies provide information beyond sequence variation and could supply alternative sources of trait variation for improvement in crops such as sorghum. Yet, compared with other species such as maize and rice, the sorghum DNA methylome is far less well understood. The distribution of CG, CHG, and CHH methylation in the genome is different compared with other species. CG and CHG methylation levels peak around centromeric segments in the sorghum genome and are far more depleted in the gene dense chromosome arms. The genes regulating DNA methylation in sorghum are also yet to be functionally characterised; better understanding of their identity and functional analysis of DNA methylation machinery mutants in diverse genotypes will be important to better characterise the sorghum methylome. Here, we catalogue homologous genes encoding methylation regulatory enzymes in sorghum based on genes in Arabidopsis, maize, and rice. Discovering variation in the methylome may uncover epialleles that provide extra information to explain trait variation and has the potential to be applied in epigenome-wide association studies or genomic prediction. DNA methylation can also improve genome annotations and discover regulatory elements underlying traits. Thus, improving our knowledge of the sorghum methylome can enhance our understanding of the molecular basis of traits and may be useful to improve sorghum performance.


Assuntos
Arabidopsis , Oryza , Sorghum , Arabidopsis/genética , Metilação de DNA , Epigenoma , Epigenômica , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Oryza/genética , Sorghum/genética , Zea mays/genética
9.
Plant Physiol ; 186(1): 420-433, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33591319

RESUMO

Transposable elements (TEs) pervade most eukaryotic genomes. The repetitive nature of TEs complicates the analysis of their expression. Evaluation of the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress reveals no evidence for genome-wide activation of TEs; however, some specific TE families generate transcripts only in stress conditions. There is substantial variation for which TE families exhibit stress-responsive expression in the different genotypes. In order to understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. The stress-responsive activation of a TE family can often be attributed to a small number of elements in the family that contains regions lacking DNA methylation. Comparisons of the expression of TEs in different genotypes revealed both genetic and epigenetic variation. Many of the specific TEs that are activated in stress in one inbred are not present in the other inbred, explaining the lack of activation. Among the elements that are shared in both genomes but only expressed in one genotype, we found that many exhibit differences in DNA methylation such that the genotype without expression is fully methylated. This study provides insights into the regulation of expression of TEs in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression variation. The highly repetitive nature of many TEs complicates the analysis of their expression. Although most TEs are not expressed, some exhibits expression in certain tissues or conditions. We monitored the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress. While genome-wide activation of TEs did not occur, some TE families generated transcripts only in stress conditions with variation by genotype. To better understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. In most cases, stress-responsive activation of a TE family was attributed to a small number of elements in the family. The elements that contained small regions lacking DNA methylation regions showed enriched expression while fully methylated elements were rarely expressed in control or stress conditions. The cause of varied expression in the different genotypes was due to both genetic and epigenetic variation. Many specific TEs activated by stress in one inbred were not present in the other inbred. Among the elements shared in both genomes, full methylation inhibited expression in one of the genotypes. This study provides insights into the regulation of TE expression in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Expressão Gênica , Variação Genética , Estresse Fisiológico/genética , Zea mays/fisiologia , Zea mays/genética
10.
PLoS Genet ; 15(9): e1008291, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31498837

RESUMO

DNA methylation and epigenetic silencing play important roles in the regulation of transposable elements (TEs) in many eukaryotic genomes. A majority of the maize genome is derived from TEs that can be classified into different orders and families based on their mechanism of transposition and sequence similarity, respectively. TEs themselves are highly methylated and it can be tempting to view them as a single uniform group. However, the analysis of DNA methylation profiles in flanking regions provides evidence for distinct groups of chromatin properties at different TE families. These differences among TE families are reproducible in different tissues and different inbred lines. TE families with varying levels of DNA methylation in flanking regions also show distinct patterns of chromatin accessibility and modifications within the TEs. The differences in the patterns of DNA methylation flanking TE families arise from a combination of non-random insertion preferences of TE families, changes in DNA methylation triggered by the insertion of the TE and subsequent selection pressure. A set of nearly 70,000 TE polymorphisms among four assembled maize genomes were used to monitor the level of DNA methylation at haplotypes with and without the TE insertions. In many cases, TE families with high levels of DNA methylation in flanking sequence are enriched for insertions into highly methylated regions. The majority of the >2,500 TE insertions into unmethylated regions result in changes in DNA methylation in haplotypes with the TE, suggesting the widespread potential for TE insertions to condition altered methylation in conserved regions of the genome. This study highlights the interplay between TEs and the methylome of a major crop species.


Assuntos
Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Zea mays/genética , Epigênese Genética/genética , Epigenômica/métodos , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Genótipo , Haplótipos/genética , Polimorfismo Genético/genética , Análise de Sequência de DNA/métodos
11.
Plant J ; 104(3): 828-838, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32786122

RESUMO

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Setaria (Planta)/genética , Exodesoxirribonucleases/genética , Técnicas de Inativação de Genes , Genoma de Planta , Mutagênese , Mutação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Protoplastos/fisiologia
12.
Plant Physiol ; 182(1): 318-331, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575624

RESUMO

Small RNAs (sRNAs) regulate gene expression, play important roles in epigenetic pathways, and are hypothesized to contribute to hybrid vigor in plants. Prior investigations have provided valuable insights into associations between sRNAs and heterosis, often using a single hybrid genotype or tissue, but our understanding of the role of sRNAs and their potential value to plant breeding are limited by an incomplete picture of sRNA variation between diverse genotypes and development stages. Here, we provide a deep exploration of sRNA variation and inheritance among a panel of 108 maize (Zea mays) samples spanning five tissues from eight inbred parents and 12 hybrid genotypes, covering a spectrum of heterotic groups, genetic variation, and levels of heterosis for various traits. We document substantial developmental and genotypic influences on sRNA expression, with varying patterns for 21-nucleotide (nt), 22-nt, and 24-nt sRNAs. We provide a detailed view of the distribution of sRNAs in the maize genome, revealing a complex makeup that also shows developmental plasticity, particularly for 22-nt sRNAs. sRNAs exhibited substantially more variation between inbreds as compared with observed variation for gene expression. In hybrids, we identify locus-specific examples of nonadditive inheritance, mostly characterized as partial or complete dominance, but rarely outside the parental range. However, the global abundance of 21-nt, 22-nt, and 24-nt sRNAs varies very little between inbreds and hybrids, suggesting that hybridization affects sRNA expression principally at specific loci rather than on a global scale. This study provides a valuable resource for understanding the potential role of sRNAs in hybrid vigor.


Assuntos
RNA de Plantas/genética , Zea mays/genética , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Vigor Híbrido/genética , Hibridização Genética
13.
Plant Cell Environ ; 43(3): 594-610, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31860752

RESUMO

To further our understanding of how sustained changes in temperature affect the carbon economy of rice (Oryza sativa), hydroponically grown plants of the IR64 cultivar were developed at 30°C/25°C (day/night) before being shifted to 25/20°C or 40/35°C. Leaf messenger RNA and protein abundance, sugar and starch concentrations, and gas-exchange and elongation rates were measured on preexisting leaves (PE) already developed at 30/25°C or leaves newly developed (ND) subsequent to temperature transfer. Following a shift in growth temperature, there was a transient adjustment in metabolic gene transcript abundance of PE leaves before homoeostasis was reached within 24 hr, aligning with Rdark (leaf dark respiratory CO2 release) and An (net CO2 assimilation) changes. With longer exposure, the central respiratory protein cytochrome c oxidase (COX) declined in abundance at 40/35°C. In contrast to Rdark , An was maintained across the three growth temperatures in ND leaves. Soluble sugars did not differ significantly with growth temperature, and growth was fastest with extended exposure at 40/35°C. The results highlight that acclimation of photosynthesis and respiration is asynchronous in rice, with heat-acclimated plants exhibiting a striking ability to maintain net carbon gain and growth when exposed to heat-wave temperatures, even while reducing investment in energy-conserving respiratory pathways.


Assuntos
Aclimatação/fisiologia , Oryza/genética , Oryza/fisiologia , Fotossíntese/fisiologia , Folhas de Planta/fisiologia , Temperatura , Aclimatação/efeitos da radiação , Biomassa , Dióxido de Carbono/metabolismo , Respiração Celular/genética , Respiração Celular/efeitos da radiação , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Transporte de Elétrons/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Ontologia Genética , Luz , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Oryza/efeitos da radiação , Fotossíntese/efeitos da radiação , Folhas de Planta/efeitos da radiação , Análise de Componente Principal , Ribulose-Bifosfato Carboxilase/metabolismo , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
14.
Plant Cell ; 29(8): 1836-1863, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28705956

RESUMO

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.


Assuntos
Arabidopsis/genética , Arabidopsis/efeitos da radiação , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas , Luz , Estresse Fisiológico/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metilação de DNA/genética , Perfilação da Expressão Gênica , Inativação Gênica , Loci Gênicos , Meia-Vida , Dinâmica não Linear , Biossíntese de Proteínas , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Estresse Fisiológico/efeitos da radiação , Fatores de Tempo , Transcrição Gênica/efeitos da radiação , Transcriptoma/genética
15.
Plant Physiol ; 178(4): 1614-1630, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30301775

RESUMO

In plants, the molecular function(s) of the nucleus-localized 5'-3' EXORIBONUCLEASES (XRNs) are unclear; however, their activity is reported to have a significant effect on gene expression and SAL1-mediated retrograde signaling. Using parallel analysis of RNA ends, we documented a dramatic increase in uncapped RNA substrates of the XRNs in both sal1 and xrn2xrn3 mutants. We found that a major consequence of reducing SAL1 or XRN activity was RNA Polymerase II 3' read-through. This occurred at 72% of expressed genes, demonstrating a major genome-wide role for the XRN-torpedo model of transcription termination in Arabidopsis (Arabidopsis thaliana). Read-through is speculated to have a negative effect on transcript abundance; however, we did not observe this. Rather, we identified a strong association between read-through and increased transcript abundance of tandemly orientated downstream genes, strongly correlated with the proximity (less than 1,000 bp) and expression of the upstream gene. We observed read-through in the proximity of 903 genes up-regulated in the sal1-8 retrograde signaling mutant; thus, this phenomenon may account directly for up to 23% of genes up-regulated in sal1-8 Using APX2 and AT5G43770 as exemplars, we genetically uncoupled read-through loci from downstream genes to validate the principle of read-through-mediated mRNA regulation, providing one mechanism by which an ostensibly posttranscriptional exoribonuclease that targets uncapped RNAs could modulate gene expression.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Monoéster Fosfórico Hidrolases/genética , RNA Polimerase II/metabolismo , Difosfato de Adenosina/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Polimerase II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais
16.
Plant Physiol ; 175(4): 1893-1912, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28986422

RESUMO

Improving the responsiveness, acclimation, and memory of plants to abiotic stress holds substantive potential for improving agriculture. An unresolved question is the involvement of chromatin marks in the memory of agriculturally relevant stresses. Such potential has spurred numerous investigations yielding both promising and conflicting results. Consequently, it remains unclear to what extent robust stress-induced DNA methylation variation can underpin stress memory. Using a slow-onset water deprivation treatment in Arabidopsis (Arabidopsis thaliana), we investigated the malleability of the DNA methylome to drought stress within a generation and under repeated drought stress over five successive generations. While drought-associated epi-alleles in the methylome were detected within a generation, they did not correlate with drought-responsive gene expression. Six traits were analyzed for transgenerational stress memory, and the descendants of drought-stressed lineages showed one case of memory in the form of increased seed dormancy, and that persisted one generation removed from stress. With respect to transgenerational drought stress, there were negligible conserved differentially methylated regions in drought-exposed lineages compared with unstressed lineages. Instead, the majority of observed variation was tied to stochastic or preexisting differences in the epigenome occurring at repetitive regions of the Arabidopsis genome. Furthermore, the experience of repeated drought stress was not observed to influence transgenerational epi-allele accumulation. Our findings demonstrate that, while transgenerational memory is observed in one of six traits examined, they are not associated with causative changes in the DNA methylome, which appears relatively impervious to drought stress.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Metilação de DNA , Regulação da Expressão Gênica de Plantas/fisiologia , Água , Alelos , Proteínas de Arabidopsis/genética , DNA de Plantas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Estresse Fisiológico
17.
Plant Cell Environ ; 41(7): 1657-1672, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29707792

RESUMO

The capacity for plant stress priming and memory and the notion of this being underpinned by DNA methylation-mediated memory is an appealing hypothesis for which there is mixed evidence. We previously established a lack of drought-induced methylome variation in Arabidopsis thaliana (Arabidopsis); however, this was tied to only minor observations of physiological memory. There are numerous independent observations demonstrating that photoprotective mechanisms, induced by excess-light stress, can lead to robust programmable changes in newly developing leaf tissues. Although key signalling molecules and transcription factors are known to promote this priming signal, an untested question is the potential involvement of chromatin marks towards the maintenance of light stress acclimation, or memory. Thus, we systematically tested our previous hypothesis of a stress-resistant methylome using a recurring excess-light stress, then analysing new, emerging, and existing tissues. The DNA methylome showed negligible stress-associated variation, with the vast majority attributable to stochastic differences. Yet, photoacclimation was evident through enhanced photosystem II performance in exposed tissues, and nonphotochemical quenching and fluorescence decline ratio showed evidence of mitotic transmission. Thus, we have observed physiological acclimation in new and emerging tissues in the absence of substantive DNA methylome changes.


Assuntos
Metilação de DNA/efeitos da radiação , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Clorofila/metabolismo , Genoma de Planta/genética , Luz , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/efeitos da radiação , Análise de Sequência de DNA , Estresse Fisiológico , Xantofilas/metabolismo
18.
Plant Physiol ; 171(3): 1734-49, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27288360

RESUMO

Distinct ROS signaling pathways initiated by singlet oxygen ((1)O2) or superoxide and hydrogen peroxide have been attributed to either cell death or acclimation, respectively. Recent studies have revealed that more complex antagonistic and synergistic relationships exist within and between these pathways. As specific chloroplastic ROS signals are difficult to study, rapid systemic signaling experiments using localized high light (HL) stress or ROS treatments were used in this study to uncouple signals required for direct HL and ROS perception and distal systemic acquired acclimation (SAA). A qPCR approach was chosen to determine local perception and distal signal reception. Analysis of a thylakoidal ascorbate peroxidase mutant (tapx), the (1)O2-retrograde signaling double mutant (ex1/ex2), and an apoplastic signaling double mutant (rbohD/F) revealed that tAPX and EXECUTER 1 are required for both HL and systemic acclimation stress perception. Apoplastic membrane-localized RBOHs were required for systemic spread of the signal but not for local signal induction in directly stressed tissues. Endogenous ROS treatments revealed a very strong systemic response induced by a localized 1 h induction of (1)O2 using the conditional flu mutant. A qPCR time course of (1)O2 induced systemic marker genes in directly and indirectly connected leaves revealed a direct vascular connection component of both immediate and longer term SAA signaling responses. These results reveal the importance of an EXECUTER-dependent (1)O2 retrograde signal for both local and long distance RBOH-dependent acclimation signaling that is distinct from other HL signaling pathways, and that direct vascular connections have a role in spatial-temporal SAA induction.


Assuntos
Aclimatação/fisiologia , Arabidopsis/fisiologia , Oxigênio Singlete/metabolismo , Aldeídos/metabolismo , Antocianinas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Diterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Mutação , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Proc Natl Acad Sci U S A ; 110(35): 14474-9, 2013 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-23918368

RESUMO

Sunlight provides energy for photosynthesis and is essential for nearly all life on earth. However, too much or too little light or rapidly fluctuating light conditions cause stress to plants. Rapid changes in the amount of light are perceived as a change in the reduced/oxidized (redox) state of photosynthetic electron transport components in chloroplasts. However, how this generates a signal that is relayed to changes in nuclear gene expression is not well understood. We modified redox state in the reference plant, Arabidopsis thaliana, using either excess light or low light plus the herbicide DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), a well-known inhibitor of photosynthetic electron transport. Modification of redox state caused a change in expression of a common set of about 750 genes, many of which are known stress-responsive genes. Among the most highly enriched promoter elements in the induced gene set were heat-shock elements (HSEs), known motifs that change gene expression in response to high temperature in many systems. We show that HSEs from the promoter of the ASCORBATE PEROXIDASE 2 (APX2) gene were necessary and sufficient for APX2 expression in conditions of excess light, or under low light plus the herbicide. We tested APX2 expression phenotypes in overexpression and loss-of-function mutants of 15 Arabidopsis A-type heat-shock transcription factors (HSFs), and identified HSFA1D, HSFA2, and HSFA3 as key factors regulating APX2 expression in diverse stress conditions. Excess light regulates both the subcellular location of HSFA1D and its biochemical properties, making it a key early component of the excess light stress network of plants.


Assuntos
Arabidopsis/fisiologia , Proteínas de Choque Térmico/fisiologia , Luz , Fatores de Transcrição/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/genética , Dibromotimoquinona/farmacologia , Regulação da Expressão Gênica de Plantas , Fotossíntese
20.
Plant Cell ; 23(11): 3992-4012, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22128124

RESUMO

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Nucleotidases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Núcleo Celular/genética , Secas , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Mitocôndrias/metabolismo , Mutação , Nucleotidases/genética , Monoéster Fosfórico Hidrolases , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
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