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1.
New Phytol ; 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597191

RESUMO

Salinity stress limits plant growth and has a major impact on agricultural productivity. Here, we identify NAC transcription factor SlTAF1 as a regulator of salt tolerance in cultivated tomato (Solanum lycopersicum). While overexpressing SlTAF1 improves salinity tolerance compared to wild type, lowering SlTAF1 expression causes stronger salinity-induced damage. Under salt stress, shoots of SlTAF1 knockdown plants accumulate more toxic Na+ ions, while SlTAF1 overexpressors accumulate less, in accordance with an altered expression of the Na+ transporter genes SlHKT1;1 and SlHKT1;2. Furthermore, stomatal conductance and pore area are increased in SlTAF1 knockdown plants during salinity stress, but decreased in SlTAF1 overexpressors. We identified stress-related transcription factor, ABA metabolism, and defense-related genes as potential direct targets of SlTAF1, correlating it with reactive oxygen species (ROS) scavenging capacity and changes in hormonal response. Salinity-induced changes in tricarboxylic acid cycle intermediates and amino acids are more pronounced in SlTAF1 knockdown than wild-type plants, but less so in SlTAF1 overexpressors. The osmoprotectant proline accumulates more in SlTAF1 overexpressors than knockdown plants. In summary, SlTAF1 controls tomato's response to salinity stress by combating both, osmotic and ion toxicity, highlighting it as a promising candidate for the future breeding of stress-tolerant crops.

2.
Curr Biol ; 29(15): 2465-2476.e5, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31327714

RESUMO

In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thaliana mRNAs harboring the modified base 5-methylcytosine (m5C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m5C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.

3.
Int J Mol Sci ; 20(12)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242611

RESUMO

Abiotic stress is one of the major threats to plant crop yield and productivity. When plants are exposed to stress, production of reactive oxygen species (ROS) increases, which could lead to extensive cellular damage and hence crop loss. During evolution, plants have acquired antioxidant defense systems which can not only detoxify ROS but also adjust ROS levels required for proper cell signaling. Ascorbate peroxidase (APX), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) are crucial enzymes involved in ROS detoxification. In this study, 40 putative APX, 28 GPX, 16 CAT, and 41 SOD genes were identified from genomes of the resurrection species Boea hygrometrica, Selaginella lepidophylla, Xerophyta viscosa, and Oropetium thomaeum, and the mesophile Selaginella moellendorffii. Phylogenetic analyses classified the APX, GPX, and SOD proteins into five clades each, and CAT proteins into three clades. Using co-expression network analysis, various regulatory modules were discovered, mainly involving glutathione, that likely work together to maintain ROS homeostasis upon desiccation stress in resurrection species. These regulatory modules also support the existence of species-specific ROS detoxification systems. The results suggest molecular pathways that regulate ROS in resurrection species and the role of APX, GPX, CAT and SOD genes in resurrection species during stress.

4.
Cell Mol Life Sci ; 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31250033

RESUMO

Oxidative stress can lead to plant growth retardation, yield loss, and death. The atr7 mutant of Arabidopsis thaliana exhibits pronounced tolerance to oxidative stress. Using positional cloning, confirmed by knockout and RNA interference (RNAi) lines, we identified the atr7 mutation and revealed that ATR7 is a previously uncharacterized gene with orthologs in other seed plants but with no homology to genes in lower plants, fungi or animals. Expression of ATR7-GFP fusion shows that ATR7 is a nuclear-localized protein. RNA-seq analysis reveals that transcript levels of genes encoding abiotic- and oxidative stress-related transcription factors (DREB19, HSFA2, ZAT10), chromatin remodelers (CHR34), and unknown or uncharacterized proteins (AT5G59390, AT1G30170, AT1G21520) are elevated in atr7. This indicates that atr7 is primed for an upcoming oxidative stress via pathways involving genes of unknown functions. Collectively, the data reveal ATR7 as a novel seed plants-specific nuclear regulator of oxidative stress response.

5.
Nat Commun ; 10(1): 2615, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197154

RESUMO

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing ß-carotene and co-producing ß-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.


Assuntos
Vias Biossintéticas/genética , Engenharia Metabólica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Flavanonas/biossíntese , Recombinação Homóloga/genética , Norisoprenoides/biossíntese , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Transcrição/genética , beta Caroteno/biossíntese
6.
J Exp Bot ; 70(10): 2727-2740, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31002305

RESUMO

NAC transcription factors (TFs) are important regulators of expressional reprogramming during plant development, stress responses, and leaf senescence. NAC TFs also play important roles in fruit ripening. In tomato (Solanum lycopersicum), one of the best characterized NACs involved in fruit ripening is NON-RIPENING (NOR), and the non-ripening (nor) mutation has been widely used to extend fruit shelf life in elite varieties. Here, we show that NOR additionally controls leaf senescence. Expression of NOR increases with leaf age, and developmental as well as dark-induced senescence are delayed in the nor mutant, while overexpression of NOR promotes leaf senescence. Genes associated with chlorophyll degradation as well as senescence-associated genes (SAGs) show reduced and elevated expression, respectively, in nor mutants and NOR overexpressors. Overexpression of NOR also stimulates leaf senescence in Arabidopsis thaliana. In tomato, NOR supports senescence by directly and positively regulating the expression of several senescence-associated genes including, besides others, SlSAG15 and SlSAG113, SlSGR1, and SlYLS4. Finally, we find that another senescence control NAC TF, namely SlNAP2, acts upstream of NOR to regulate its expression. Our data support a model whereby NAC TFs have often been recruited by higher plants for both the control of leaf senescence and fruit ripening.

7.
Nat Plants ; 4(11): 863-864, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30374092
8.
PLoS Genet ; 14(7): e1007484, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29985961

RESUMO

Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the Arabidopsis thaliana TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF OBP3-RESPONSIVE GENE 3 (ORG3). While grf9 knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing GRF9 (GRF9ox). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that GRF9 restricts cell proliferation in the early developing leaf. Performing in vitro binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified ORG3 as a direct downstream, and positively regulated target of GRF9. Genetic analysis of grf9 org3 and GRF9ox org3 double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf.

9.
Plant Physiol ; 177(3): 1286-1302, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29760199

RESUMO

Leaf senescence is an essential physiological process in plants that supports the recycling of nitrogen and other nutrients to support the growth of developing organs, including young leaves, seeds, and fruits. Thus, the regulation of senescence is crucial for evolutionary success in wild populations and for increasing yield in crops. Here, we describe the influence of a NAC transcription factor, SlNAP2 (Solanum lycopersicum NAC-like, activated by Apetala3/Pistillata), that controls both leaf senescence and fruit yield in tomato (S. lycopersicum). SlNAP2 expression increases during age-dependent and dark-induced leaf senescence. We demonstrate that SlNAP2 activates SlSAG113 (S. lycopersicum SENESCENCE-ASSOCIATED GENE113), a homolog of Arabidopsis (Arabidopsis thaliana) SAG113, chlorophyll degradation genes such as SlSGR1 (S. lycopersicum senescence-inducible chloroplast stay-green protein 1) and SlPAO (S. lycopersicum pheide a oxygenase), and other downstream targets by directly binding to their promoters, thereby promoting leaf senescence. Furthermore, SlNAP2 directly controls the expression of genes important for abscisic acid (ABA) biosynthesis, S. lycopersicum 9-cis-epoxycarotenoid dioxygenase 1 (SlNCED1); transport, S. lycopersicum ABC transporter G family member 40 (SlABCG40); and degradation, S. lycopersicum ABA 8'-hydroxylase (SlCYP707A2), indicating that SlNAP2 has a complex role in establishing ABA homeostasis during leaf senescence. Inhibiting SlNAP2 expression in transgenic tomato plants impedes leaf senescence but enhances fruit yield and sugar content likely due to prolonged leaf photosynthesis in aging tomato plants. Our data indicate that SlNAP2 has a central role in controlling leaf senescence and fruit yield in tomato.

10.
Plant Physiol ; 177(3): 1319-1338, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29789435

RESUMO

The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any visible senescence. Here, we investigated the molecular mechanisms of this adaptation to prolonged (30 d) darkness and subsequent return to light. H. rhodopensis plants remained green and viable throughout the dark treatment. Transcriptomic analysis revealed that darkness regulated several transcription factor (TF) genes. Stress- and autophagy-related TFs such as ERF8, HSFA2b, RD26, TGA1, and WRKY33 were up-regulated, while chloroplast- and flowering-related TFs such as ATH1, COL2, COL4, RL1, and PTAC7 were repressed. PHYTOCHROME INTERACTING FACTOR4, a negative regulator of photomorphogenesis and promoter of senescence, also was down-regulated. In response to darkness, most of the photosynthesis- and photorespiratory-related genes were strongly down-regulated, while genes related to autophagy were up-regulated. This occurred concomitant with the induction of SUCROSE NON-FERMENTING1-RELATED PROTEIN KINASES (SnRK1) signaling pathway genes, which regulate responses to stress-induced starvation and autophagy. Most of the genes associated with chlorophyll catabolism, which are induced by darkness in dark-senescing species, were either unregulated (PHEOPHORBIDE A OXYGENASE, PAO; RED CHLOROPHYLL CATABOLITE REDUCTASE, RCCR) or repressed (STAY GREEN-LIKE, PHEOPHYTINASE, and NON-YELLOW COLORING1). Metabolite profiling revealed increases in the levels of many amino acids in darkness, suggesting increased protein degradation. In darkness, levels of the chloroplastic lipids digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylglycerol, and sulfoquinovosyldiacylglycerol decreased, while those of storage triacylglycerols increased, suggesting degradation of chloroplast membrane lipids and their conversion to triacylglycerols for use as energy and carbon sources. Collectively, these data show a coordinated response to darkness, including repression of photosynthetic, photorespiratory, flowering, and chlorophyll catabolic genes, induction of autophagy and SnRK1 pathways, and metabolic reconfigurations that enable survival under prolonged darkness.

11.
Nat Commun ; 9(1): 1931, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29789561

RESUMO

The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.

12.
New Phytol ; 218(4): 1543-1557, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29659022

RESUMO

Leaf senescence is a key process in plants that culminates in the degradation of cellular constituents and massive reprogramming of metabolism for the recovery of nutrients from aged leaves for their reuse in newly developing sinks. We used molecular-biological and metabolomics approaches to identify NAC transcription factor (TF) RD26 as an important regulator of metabolic reprogramming in Arabidopsis thaliana. RD26 directly activates CHLOROPLAST VESICULATION (CV), encoding a protein crucial for chloroplast protein degradation, concomitant with an enhanced protein loss in RD26 overexpressors during senescence, but a reduced decline of protein in rd26 knockout mutants. RD26 also directly activates LKR/SDH involved in lysine catabolism, and PES1 important for phytol degradation. Metabolic profiling revealed reduced γ-aminobutyric acid (GABA) in RD26 overexpressors, accompanied by the induction of respective catabolic genes. Degradation of lysine, phytol and GABA is instrumental for maintaining mitochondrial respiration in carbon-limiting conditions during senescence. RD26 also supports the degradation of starch and the accumulation of mono- and disaccharides during senescence by directly enhancing the expression of AMY1, SFP1 and SWEET15 involved in carbohydrate metabolism and transport. Collectively, during senescence RD26 acts by controlling the expression of genes across the entire spectrum of the cellular degradation hierarchy.

13.
Methods Mol Biol ; 1744: 339-358, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29392679

RESUMO

Leaf senescence is an essential developmental process that involves diverse metabolic changes associated with degradation of macromolecules allowing nutrient recycling and remobilization. In contrast to the significant progress in transcriptomic analysis of leaf senescence, metabolomics analyses have been relatively limited. A broad overview of metabolic changes during leaf senescence including the interactions between various metabolic pathways is required to gain a better understanding of the leaf senescence allowing to link transcriptomics with metabolomics and physiology. In this chapter, we describe how to obtain comprehensive metabolite profiles and how to dissect metabolic shifts during leaf senescence in the model plant Arabidopsis thaliana. Unlike nucleic acid analysis for transcriptomics, a comprehensive metabolite profile can only be achieved by combining a suite of analytic tools. Here, information is provided for measurements of the contents of chlorophyll, soluble proteins, and starch by spectrophotometric methods, ions by ion chromatography, thiols and amino acids by HPLC, primary metabolites by GC/TOF-MS, and secondary metabolites and lipophilic metabolites by LC/ESI-MS. These metabolite profiles provide a rich catalogue of metabolic changes during leaf senescence, which is a helpful database and blueprint to be correlated to future studies such as transcriptome and proteome analyses, forward and reverse genetic studies, or stress-induced senescence studies.

14.
Rice (N Y) ; 11(1): 2, 2018 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-29313187

RESUMO

BACKGROUND: Flooding during seasonal monsoons affects millions of hectares of rice-cultivated areas across Asia. Submerged rice plants die within a week due to lack of oxygen, light and excessive elongation growth to escape the water. Submergence tolerance was first reported in an aus-type rice landrace, FR13A, and the ethylene-responsive transcription factor (TF) gene SUB1A-1 was identified as the major tolerance gene. Intolerant rice varieties generally lack the SUB1A gene but some intermediate tolerant varieties, such as IR64, carry the allelic variant SUB1A-2. Differential effects of the two alleles have so far not been addressed. As a first step, we have therefore quantified and compared the expression of nearly 2500 rice TF genes between IR64 and its derived tolerant near isogenic line IR64-Sub1, which carries the SUB1A-1 allele. Gene expression was studied in internodes, where the main difference in expression between the two alleles was previously shown. RESULTS: Nineteen and twenty-six TF genes were identified that responded to submergence in IR64 and IR64-Sub1, respectively. Only one gene was found to be submergence-responsive in both, suggesting different regulatory pathways under submergence in the two genotypes. These differentially expressed genes (DEGs) mainly included MYB, NAC, TIFY and Zn-finger TFs, and most genes were downregulated upon submergence. In IR64, but not in IR64-Sub1, SUB1B and SUB1C, which are also present in the Sub1 locus, were identified as submergence responsive. Four TFs were not submergence responsive but exhibited constitutive, genotype-specific differential expression. Most of the identified submergence responsive DEGs are associated with regulatory hormonal pathways, i.e. gibberellins (GA), abscisic acid (ABA), and jasmonic acid (JA), apart from ethylene. An in-silico promoter analysis of the two genotypes revealed the presence of allele-specific single nucleotide polymorphisms, giving rise to ABRE, DRE/CRT, CARE and Site II cis-elements, which can partly explain the observed differential TF gene expression. CONCLUSION: This study identified new gene targets with the potential to further enhance submergence tolerance in rice and provides insights into novel aspects of SUB1A-mediated tolerance.

15.
Plant Biotechnol J ; 16(2): 354-366, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28640975

RESUMO

Water deficit (drought stress) massively restricts plant growth and the yield of crops; reducing the deleterious effects of drought is therefore of high agricultural relevance. Drought triggers diverse cellular processes including the inhibition of photosynthesis, the accumulation of cell-damaging reactive oxygen species and gene expression reprogramming, besides others. Transcription factors (TF) are central regulators of transcriptional reprogramming and expression of many TF genes is affected by drought, including members of the NAC family. Here, we identify the NAC factor JUNGBRUNNEN1 (JUB1) as a regulator of drought tolerance in tomato (Solanum lycopersicum). Expression of tomato JUB1 (SlJUB1) is enhanced by various abiotic stresses, including drought. Inhibiting SlJUB1 by virus-induced gene silencing drastically lowers drought tolerance concomitant with an increase in ion leakage, an elevation of hydrogen peroxide (H2 O2 ) levels and a decrease in the expression of various drought-responsive genes. In contrast, overexpression of AtJUB1 from Arabidopsis thaliana increases drought tolerance in tomato, alongside with a higher relative leaf water content during drought and reduced H2 O2 levels. AtJUB1 was previously shown to stimulate expression of DREB2A, a TF involved in drought responses, and of the DELLA genes GAI and RGL1. We show here that SlJUB1 similarly controls the expression of the tomato orthologs SlDREB1, SlDREB2 and SlDELLA. Furthermore, AtJUB1 directly binds to the promoters of SlDREB1, SlDREB2 and SlDELLA in tomato. Our study highlights JUB1 as a transcriptional regulator of drought tolerance and suggests considerable conservation of the abiotic stress-related gene regulatory networks controlled by this NAC factor between Arabidopsis and tomato.

16.
Artigo em Inglês | MEDLINE | ID: mdl-29098147

RESUMO

Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host's endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

17.
Nucleic Acids Res ; 45(15): 9193-9205, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28911120

RESUMO

Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h.


Assuntos
Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Engenharia Genética/métodos , Proteínas de Homeodomínio/genética , Fitocromo B/genética , Saccharomyces cerevisiae/genética , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Luz , Transdução de Sinal Luminoso , Ficobilinas/biossíntese , Ficobilinas/genética , Ficocianina/biossíntese , Ficocianina/genética , Fitocromo B/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação
18.
Plant Cell Environ ; 40(11): 2644-2662, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28555890

RESUMO

Since its discovery over two decades ago as an important cell death regulator in Arabidopsis thaliana, the role of LESION SIMULATING DISEASE 1 (LSD1) has been studied intensively within both biotic and abiotic stress responses as well as with respect to plant fitness regulation. However, its molecular mode of action remains enigmatic. Here, we demonstrate that nucleo-cytoplasmic LSD1 interacts with a broad range of other proteins that are engaged in various molecular pathways such as ubiquitination, methylation, cell cycle control, gametogenesis, embryo development and cell wall formation. The interaction of LSD1 with these partners is dependent on redox status, as oxidative stress significantly changes the quantity and types of LSD1-formed complexes. Furthermore, we show that LSD1 regulates the number and size of leaf mesophyll cells and affects plant vegetative growth. Importantly, we also reveal that in addition to its function as a scaffold protein, LSD1 acts as a transcriptional regulator. Taken together, our results demonstrate that LSD1 plays a dual role within the cell by acting as a condition-dependent scaffold protein and as a transcription regulator.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Oxirredução , Estresse Oxidativo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Mapas de Interação de Proteínas , Multimerização Proteica
19.
ACS Synth Biol ; 6(9): 1742-1756, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28531348

RESUMO

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação Fúngica da Expressão Gênica/genética , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Melhoramento Genético/métodos , Proteínas de Saccharomyces cerevisiae/genética , Transfecção/métodos
20.
Front Plant Sci ; 8: 214, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28326087

RESUMO

The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripening-related genes, and leads to an increase in the levels of various amino acids (mostly proline, ß-alanine, and phenylalanine), γ-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.

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