RESUMEN

Human Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) functions as a dNTPase to maintain dNTP pool balance. In eukaryotes, the limiting step in de novo dNTP biosynthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR). In Arabidopsis, the RNR1 subunit of RNR is encoded by CRINKLED LEAVES 8 (CLS8), and RNR2 by three paralogous genes, including TSO MEANING 'UGLY' IN CHINESE 2 (TSO2). In plants, DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1 (DOV1) catalyzes the first step of the de novo biosynthesis of purines. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4-0 point mutation, whose leaf phenotype was stronger than those of its insertional alleles. Structural predictions suggested that the E249L substitution in the mutated VEN4-0 protein rigidifies its 3D structure. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4-0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis. Our results reveal high functional conservation between VEN4 and SAMHD1 in dNTP metabolism.

Asunto(s)

Arabidopsis , Ribonucleótido Reductasas , Humanos , Proteína 1 que Contiene Dominios SAM y HD/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Fenotipo

RESUMEN

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Smith (Sm) antigen protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of the mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, as for sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and in enhanced resilience to oxidative stress.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Paraquat , Proteómica , Empalme Alternativo , Mutación , ARN Mensajero/metabolismo , Estrés Oxidativo , Regulación de la Expresión Génica de las Plantas , Mamíferos/metabolismo

RESUMEN

Plant breeders have indirectly selected for variation at circadian-associated loci in many of the world's major crops, when breeding to increase yield and improve crop performance. Using an eight-parent Multiparent Advanced Generation Inter-Cross (MAGIC) population, we investigated how variation in circadian clock-associated genes contributes to the regulation of heading date in UK and European winter wheat (Triticum aestivum) varieties. We identified homoeologues of EARLY FLOWERING 3 (ELF3) as candidates for the Earliness per se (Eps) D1 and B1 loci under field conditions. We then confirmed a single-nucleotide polymorphism within the coding region of TaELF3-B1 as a candidate polymorphism underlying the Eps-B1 locus. We found that a reported deletion at the Eps-D1 locus encompassing TaELF3-D1 is, instead, an allele that lies within an introgression region containing an inversion relative to the Chinese Spring D genome. Using Triticum turgidum cv. Kronos carrying loss-of-function alleles of TtELF3, we showed that ELF3 regulates heading, with loss of a single ELF3 homoeologue sufficient to alter heading date. These studies demonstrated that ELF3 forms part of the circadian oscillator; however, the loss of all homoeologues was required to affect circadian rhythms. Similarly, loss of functional LUX ARRHYTHMO (LUX) in T. aestivum, an orthologue of a protein partner of Arabidopsis (Arabidopsis thaliana) ELF3, severely disrupted circadian rhythms. ELF3 and LUX transcripts are not co-expressed at dusk, suggesting that the structure of the wheat circadian oscillator might differ from that of Arabidopsis. Our demonstration that alterations to ELF3 homoeologues can affect heading date separately from effects on the circadian oscillator suggests a role for ELF3 in cereal photoperiodic responses that could be selected for without pleiotropic deleterious alterations to circadian rhythms.

Asunto(s)

Arabidopsis , Relojes Circadianos , Triticum/genética , Arabidopsis/genética , Fitomejoramiento , Ritmo Circadiano/genética , Relojes Circadianos/genética , Regulación de la Expresión Génica de las Plantas

RESUMEN

Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.

Asunto(s)

Pan , Triticum , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Fenotipo , Sitios de Carácter Cuantitativo , Triticum/genética

RESUMEN

Human health is dependent on a plentiful and nutritious supply of food, primarily derived from crop plants. Rhythmic supply of light as a result of the day and night cycle led to the evolution of circadian clocks that modulate most plant physiology, photosynthesis, metabolism, and development. To regulate crop traits and adaptation, breeders have indirectly selected for variation at circadian genes. The pervasive impact of the circadian system on crops suggests that future food production might be improved by modifying circadian rhythms, engineering the timing of transgene expression, and applying agricultural treatments at the most effective time of day. We describe the applied research required to take advantage of circadian biology in agriculture to increase production and reduce inputs.

Asunto(s)

Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Productos Agrícolas/crecimiento & desarrollo , Fitomejoramiento/métodos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Relojes Circadianos/genética , Ritmo Circadiano/genética , Productos Agrícolas/genética , Abastecimiento de Alimentos , Regulación de la Expresión Génica de las Plantas , Sitios Genéticos , Selección Genética

RESUMEN

Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Herbicidas/farmacología , Complejo Mediador/metabolismo , Estrés Oxidativo/fisiología , Amitrol (Herbicida)/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Complejo Mediador/genética , MicroARNs , Estrés Oxidativo/efectos de los fármacos , Paraquat/farmacología , Plantas Modificadas Genéticamente , Dominios Proteicos , Especies Reactivas de Oxígeno/metabolismo , Ácido Salicílico/metabolismo , Factores Generales de Transcripción/genética , Factores Generales de Transcripción/metabolismo

RESUMEN

Nicotinamide-adenine dinucleotide (NAD) is involved in redox homeostasis and acts as a substrate for NADases, including poly(ADP-ribose) polymerases (PARPs) that add poly(ADP-ribose) polymers to proteins and DNA, and sirtuins that deacetylate proteins. Nicotinamide, a by-product of NADases increases circadian period in both plants and animals. In mammals, the effect of nicotinamide on circadian period might be mediated by the PARPs and sirtuins because they directly bind to core circadian oscillator genes. We have investigated whether PARPs and sirtuins contribute to the regulation of the circadian oscillator in Arabidopsis. We found no evidence that PARPs and sirtuins regulate the circadian oscillator of Arabidopsis or are involved in the response to nicotinamide. RNA-seq analysis indicated that PARPs regulate the expression of only a few genes, including FLOWERING LOCUS C. However, we found profound effects of reduced sirtuin 1 expression on gene expression during the day but not at night, and an embryo lethal phenotype in knockouts. Our results demonstrate that PARPs and sirtuins are not associated with NAD regulation of the circadian oscillator and that sirtuin 1 is associated with daytime regulation of gene expression.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiología , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Sirtuinas/metabolismo , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Ritmo Circadiano/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Mutación/genética , NAD+ Nucleosidasa/antagonistas & inhibidores , NAD+ Nucleosidasa/metabolismo , Niacinamida/farmacología , Fenotipo , Poli(ADP-Ribosa) Polimerasas/genética , Semillas/efectos de los fármacos , Semillas/metabolismo

RESUMEN

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca) is a AAA+ enzyme that uses ATP to remove inhibitors from the active site of Rubisco, the central carboxylation enzyme of photosynthesis. Rca α and ß isoforms exist in most higher plant species, with the α isoform being identical to the ß form but having an additional 25-45 amino acids at the Rca C terminus, known as the C-terminal extension (CTE). Rca is inhibited by ADP, and the extent of ADP sensitivity of the Rca complex can be modulated by the CTE of the α isoform, particularly in relation to a disulfide bond structure that is specifically reduced by the redox-regulatory enzyme thioredoxin-f. Here, we introduced single point mutations of Lys-428 in the CTE of Rca-α from wheat (Triticum aestivum) (TaRca2-α). Substitution of Lys-428 with Arg dramatically altered ADP inhibition, independently of thioredoxin-f regulation. We determined that the reduction in ADP inhibition in the K428R variant is not due to a change in ADP affinity, as the apparent constant for ADP binding was not altered by the K428R substitution. Rather, we observed that the K428R substitution strongly increased ATP substrate affinity and ATP-dependent catalytic velocity. These results suggest that the Lys-428 residue is involved in interacting with the γ-phosphate of ATP. Considering that nucleotide-dependent Rca activity regulates Rubisco and thus photosynthesis during fluctuating irradiance, the K428R substitution could potentially provide a mechanism for boosting the performance of wheat grown in the dynamic light environments of the field.

Asunto(s)

Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Mutación Puntual/genética , Triticum/enzimología , Secuencia de Aminoácidos , Estabilidad de Enzimas , Cinética , Especificidad por Sustrato

RESUMEN

The circadian oscillator, an internal time-keeping device found in most organisms, enables timely regulation of daily biological activities by maintaining synchrony with the external environment. The mechanistic basis underlying the adjustment of circadian rhythms to changing external conditions, however, has yet to be clearly elucidated. We explored the mechanism of action of nicotinamide in Arabidopsis thaliana, a metabolite that lengthens the period of circadian rhythms, to understand the regulation of circadian period. To identify the key mechanisms involved in the circadian response to nicotinamide, we developed a systematic and practical modeling framework based on the identification and comparison of gene regulatory dynamics. Our mathematical predictions, confirmed by experimentation, identified key transcriptional regulatory mechanisms of circadian period and uncovered the role of blue light in the response of the circadian oscillator to nicotinamide. We suggest that our methodology could be adapted to predict mechanisms of drug action in complex biological systems.

Asunto(s)

Arabidopsis , Ritmo Circadiano , Regulación de la Expresión Génica de las Plantas , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Modelos Biológicos , Niacinamida/farmacología , Biología de Sistemas , Transcriptoma

RESUMEN

The LATE ELONGATED HYPOCOTYL (LHY) transcription factor functions as part of the oscillatory mechanism of the Arabidopsis circadian clock. This paper reports the genome-wide analysis of its binding targets and reveals a role in the control of abscisic acid (ABA) biosynthesis and downstream responses. LHY directly repressed expression of 9-cis-epoxycarotenoid dioxygenase enzymes, which catalyse the rate-limiting step of ABA biosynthesis. This suggested a mechanism for the circadian control of ABA accumulation in wild-type plants. Consistent with this hypothesis, ABA accumulated rhythmically in wild-type plants, peaking in the evening. LHY-overexpressing plants had reduced levels of ABA under drought stress, whereas loss-of-function mutants exhibited an altered rhythm of ABA accumulation. LHY also bound the promoter of multiple components of ABA signalling pathways, suggesting that it may also act to regulate responses downstream of the hormone. LHY promoted expression of ABA-responsive genes responsible for increased tolerance to drought and osmotic stress but alleviated the inhibitory effect of ABA on seed germination and plant growth. This study reveals a complex interaction between the circadian clock and ABA pathways, which is likely to make an important contribution to plant performance under drought and osmotic stress conditions.

Asunto(s)

Ácido Abscísico/biosíntesis , Arabidopsis/genética , Arabidopsis/metabolismo , Vías Biosintéticas , Ritmo Circadiano , Proteínas de Unión al ADN/metabolismo , Genoma de Planta , Transducción de Señal , Factores de Transcripción/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Vías Biosintéticas/efectos de los fármacos , Relojes Circadianos/efectos de los fármacos , Relojes Circadianos/genética , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ontología de Genes , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos

RESUMEN

Circadian clocks drive rhythms with a period near 24 h, but the molecular basis of the regulation of the period of the circadian clockis poorly understood. We previously demonstrated that metabolites affect the free-running period of the circadian oscillator of Arabidopsis (Arabidopsis thaliana), with endogenous sugars acting as an accelerator and exogenous nicotinamide acting as a brake. Changes in circadian oscillator period are thought to adjust the timing of biological activities through the process of entrainment, in which the circadian oscillator becomes synchronized to rhythmic signals such as light and dark cycles as well as changes in internal metabolism. To identify the molecular components associated with the dynamic adjustment of circadian period, we performed a forward genetic screen. We identified Arabidopsis mutants that were either period insensitive to nicotinamide (sin) or period oversensitive to nicotinamide (son). We mapped son1 to BIG, a gene of unknown molecular function that was shown previously to play a role in light signaling. We found that son1 has an early entrained phase, suggesting that the dynamic alteration of circadian period contributes to the correct timing of biological events. Our data provide insight into how the dynamic period adjustment of circadian oscillators contributes to establishing a correct phase relationship with the environment and show that BIG is involved in this process.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión a Calmodulina/genética , Relojes Circadianos/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Relojes Circadianos/efectos de la radiación , Ritmo Circadiano/genética , Ritmo Circadiano/efectos de la radiación , Luz , Plantas Modificadas Genéticamente

RESUMEN

BACKGROUND: Growth is an important parameter to consider when studying the impact of treatments or mutations on plant physiology. Leaf area and growth rates can be estimated efficiently from images of plants, but the experiment setup, image analysis, and statistical evaluation can be laborious, often requiring substantial manual effort and programming skills. RESULTS: Here we present rosettR, a non-destructive and high-throughput phenotyping protocol for the measurement of total rosette area of seedlings grown in plates in sterile conditions. We demonstrate that our protocol can be used to accurately detect growth differences among different genotypes and in response to light regimes and osmotic stress. rosettR is implemented as a package for the statistical computing software R and provides easy to use functions to design an experiment, analyze the images, and generate reports on quality control as well as a final comparison across genotypes and applied treatments. Experiment procedures are included as part of the package documentation. CONCLUSIONS: Using rosettR it is straight-forward to perform accurate, reproducible measurements of rosette area and relative growth rate with high-throughput using inexpensive equipment. Suitable applications include screening mutant populations for growth phenotypes visible at early growth stages and profiling different genotypes in a wide variety of treatments.

RESUMEN

Cellular signaling pathways are regulated in a highly dynamic fashion in order to quickly adapt to distinct environmental conditions. Acetylation of lysine residues represents a central process that orchestrates cellular metabolism and signaling. In mitochondria, acetylation seems to be the most prevalent post-translational modification, presumably linked to the compartmentation and high turnover of acetyl-CoA in this organelle. Similarly, the elevated pH and the higher concentration of metabolites in mitochondria seem to favor non-enzymatic lysine modifications, as well as other acylations. Hence, elucidating the mechanisms for metabolic control of protein acetylation is crucial for our understanding of cellular processes. Recent advances in mass spectrometry-based proteomics have considerably increased our knowledge of the regulatory scope of acetylation. Here, we review the current knowledge and functional impact of mitochondrial protein acetylation across species. We first cover the experimental approaches to identify and analyze lysine acetylation on a global scale, we then explore both commonalities and specific differences of plant and animal acetylomes and the evolutionary conservation of protein acetylation, as well as its particular impact on metabolism and diseases. Important future directions and technical challenges are discussed, and it is pointed out that the transfer of knowledge between species and diseases, both in technology and biology, is of particular importance for further advancements in this field.

Asunto(s)

Acetilcoenzima A/metabolismo , Lisina/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Biología Computacional , Espectrometría de Masas , Plantas , Proteómica

RESUMEN

Extremophile plants are valuable sources of genes conferring tolerance traits, which can be explored to improve stress tolerance of crops. Lepidium crassifolium is a halophytic relative of the model plant Arabidopsis thaliana, and displays tolerance to salt, osmotic and oxidative stresses. We have employed the modified Conditional cDNA Overexpression System to transfer a cDNA library from L. crassifolium to the glycophyte A. thaliana. By screening for salt, osmotic and oxidative stress tolerance through in vitro growth assays and non-destructive chlorophyll fluorescence imaging, 20 Arabidopsis lines were identified with superior performance under restrictive conditions. Several cDNA inserts were cloned and confirmed to be responsible for the enhanced tolerance by analysing independent transgenic lines. Examples include full-length cDNAs encoding proteins with high homologies to GDSL-lipase/esterase or acyl CoA-binding protein or proteins without known function, which could confer tolerance to one or several stress conditions. Our results confirm that random gene transfer from stress tolerant to sensitive plant species is a valuable tool to discover novel genes with potential for biotechnological applications.

Asunto(s)

Deshidratación , Técnicas Genéticas , Lepidium/genética , Estrés Oxidativo , Plantas Tolerantes a la Sal/genética , Arabidopsis , Biblioteca de Genes , Genes de Plantas , Paraquat

RESUMEN

Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas Portadoras/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interacción de Proteínas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Escherichia coli/genética , Flores/genética , Perfilación de la Expresión Génica , Vectores Genéticos , Germinación , Histona Desacetilasas/genética , Microscopía Confocal , Proteínas Nucleares/genética , Plantas Modificadas Genéticamente , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo

RESUMEN

As challenges to food security increase, the demand for lead genes for improving crop production is growing. However, genetic screens of plant mutants typically yield very low frequencies of desired phenotypes. Here, we present a powerful computational approach for selecting candidate genes for screening insertion mutants. We combined ranking of Arabidopsis thaliana regulatory genes according to their expression in response to multiple abiotic stresses (Multiple Stress [MST] score), with stress-responsive RNA co-expression network analysis to select candidate multiple stress regulatory (MSTR) genes. Screening of 62 T-DNA insertion mutants defective in candidate MSTR genes, for abiotic stress germination phenotypes yielded a remarkable hit rate of up to 62%; this gene discovery rate is 48-fold greater than that of other large-scale insertional mutant screens. Moreover, the MST score of these genes could be used to prioritize them for screening. To evaluate the contribution of the co-expression analysis, we screened 64 additional mutant lines of MST-scored genes that did not appear in the RNA co-expression network. The screening of these MST-scored genes yielded a gene discovery rate of 36%, which is much higher than that of classic mutant screens but not as high as when picking candidate genes from the co-expression network. The MSTR co-expression network that we created, AraSTressRegNet is publicly available at http://netbio.bgu.ac.il/arnet. This systems biology-based screening approach combining gene ranking and network analysis could be generally applicable to enhancing identification of genes regulating additional processes in plants and other organisms provided that suitable transcriptome data are available.

Asunto(s)

Arabidopsis/genética , Expresión Génica , Redes Reguladoras de Genes , Genes de Plantas , Estrés Fisiológico/genética , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos

RESUMEN

Reduced glutathione (GSH) is considered to exert a strong influence on cellular redox homeostasis and to regulate gene expression, but these processes remain poorly characterized. Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities. The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 and -320 mV. Growth in the presence of the GSH-synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately -260 mV. GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECATE), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH-synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO. Inhibition of auxin transport had no effect on organ GSH levels, but exogenous auxin decreased the root GSH pool. We conclude that GSH depletion significantly increases the redox potentials of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses, but not oxidative stress.

Asunto(s)

Arabidopsis/citología , Arabidopsis/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glutatión/farmacología , Ácido Abscísico/genética , Ácido Abscísico/metabolismo , Arabidopsis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Núcleo Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Etilenos/metabolismo , Genes de Plantas , Disulfuro de Glutatión/metabolismo , Ácidos Indolacéticos/farmacología , Meristema/citología , Meristema/efectos de los fármacos , Meristema/genética , Oxidación-Reducción/efectos de los fármacos , Fenotipo , Ftalimidas/farmacología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Tiorredoxinas/metabolismo

RESUMEN

A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production.

Asunto(s)

Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Proteínas de Plantas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Transcriptoma , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Benzamidas/farmacología , Biocombustibles , Biomasa , Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes y Vías Metabólicas/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transcripción Genética

RESUMEN

Reduced glutathione (GSH) is an abundant low molecular weight plant thiol. It fulfills multiple functions in plant biology, many of which remain poorly characterized. A phenomics approach was therefore used to investigate the effects of glutathione homeostasis on growth and stress tolerance in Arabidopsis thaliana. Rosette leaf area was compared in mutants that are either defective in GSH synthesis (cad2, pad2, and rax1) or the export of γ-glutamylcysteine and GSH from the chloroplast (clt) and in wild-type plants under standard growth conditions and following exposure to a range of abiotic stress treatments, including oxidative stress, water stress, and high salt. In the absence of stress, the GSH synthesis mutants had a significantly lower leaf area than the wild type. Conversely, the clt mutant has a greater leaf area and a significantly reduced lateral root density than the wild type. These findings demonstrate that cellular glutathione homeostasis exerts an influence on root architecture and on rosette area. An impaired capacity to synthesize GSH or a specific depletion of the cytosolic GSH pool did not adversely affect leaf area in plants exposed to short-term abiotic stress. However, the negative effects of long-term exposure to oxidative stress and high salt on leaf area were less marked in the GSH synthesis mutants than the wild type. These findings demonstrate the importance of cellular glutathione homeostasis in the regulation of plant growth under optimal and stress conditions.

RESUMEN

Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.

Asunto(s)

Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biomasa , Sequías , Flores/efectos de los fármacos , Flores/enzimología , Flores/genética , Flores/fisiología , Expresión Génica , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Modelos Biológicos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Semillas/efectos de los fármacos , Semillas/enzimología , Semillas/genética , Semillas/fisiología , Estrés Fisiológico