RESUMO
In cereals, C-repeat binding factor genes have been defined as key components of the light quality-dependent regulation of frost tolerance by integrating phytochrome-mediated light and temperature signals. This study elucidates the differences in the lipid composition of barley leaves illuminated with white light or white light supplemented with far-red light at 5 or 15 °C. According to LC-MS analysis, far-red light supplementation increased the amount of monogalactosyldiacylglycerol species 36:6, 36:5, and 36:4 after 1 day at 5 °C, and 10 days at 15 °C resulted in a perturbed content of 38:6 species. Changes were observed in the levels of phosphatidylethanolamine, and phosphatidylserine under white light supplemented with far-red light illumination at 15 °C, whereas robust changes were observed in the amount of several phosphatidylserine species at 5 °C. At 15 °C, the amount of some phosphatidylglycerol species increased as a result of white light supplemented with far-red light illumination after 1 day. The ceramide (42:2)-3 content increased regardless of the temperature. The double-bond index of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine ceramide together with total double-bond index changed when the plant was grown at 15 °C as a function of white light supplemented with far-red light. white light supplemented with far-red light increased the monogalactosyldiacylglycerol/diacylglycerol ratio as well. The gene expression changes are well correlated with the alterations in the lipidome.
Assuntos
Congelamento , Hordeum/metabolismo , Luz , Metabolismo dos Lipídeos , Folhas de Planta/metabolismo , Aclimatação , Resposta ao Choque Frio , Galactolipídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Folhas de Planta/efeitos da radiaçãoRESUMO
NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants' resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs.
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Evolução Molecular , Regulação da Expressão Gênica de Plantas , Proteínas NLR/genética , Imunidade Vegetal , Plantas/metabolismo , Proteínas de Plantas/genética , Plantas/genéticaRESUMO
MicroRNAs are a class of post-transcriptional regulators of plant developmental and physiological processes and responses to environmental stresses. Here, we present the study regarding the annotation and characterization of MIR genes conducted in durum wheat. We characterized the miRNAome of leaf and root tissues at tillering stage under two environmental conditions: irrigated with 100% (control) and 55% of evapotranspiration (early water stress). In total, 90 microRNAs were identified, of which 32 were classified as putative novel and species-specific miRNAs. In addition, seven microRNA homeologous groups were identified in each of the two genomes of the tetraploid durum wheat. Differential expression analysis highlighted a total of 45 microRNAs significantly differentially regulated in the pairwise comparisons leaf versus root. The miRNA families, miR530, miR395, miR393, miR5168, miR396 and miR166, miR171, miR319, and miR167, were the most expressed in leaves in comparison to roots. Putative microRNA targets were predicted for both five and three prime sequences derived from the stem-loop of the MIR gene. Gene ontology analysis showed significant overrepresented gene categories in microRNA targets belonging to transcription factors, phenylpropanoids, oxydases, and lipid binding-protein. This work represents one of the first genome wide characterization of MIR genes in durum wheat, identifying leaf and root tissue-specific microRNAs. This genomic identification of microRNAs together with the analysis of their expression profiles is a well-accepted starting point leading to a better comprehension of the role of MIR genes in the genus Triticum.
Assuntos
Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , RNA de Plantas/genética , Triticum/genética , Secas , Especificidade de Órgãos , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Estresse Fisiológico , Triticum/fisiologiaRESUMO
Plant stress response is a complex molecular process based on transcriptional and posttranscriptional regulation of many stress-related genes. microRNAs are the best-studied class of small RNAs known to play key regulatory roles in plant response to stress, besides being involved in plant development and organogenesis. We analyzed the leaf miRNAome of two durum wheat cultivars (Cappelli and Ofanto) characterized by a contrasting water use efficiency, exposed to heat stress, and mild and severe drought stress. On the whole, we identified 98 miRNA highly similar to previously known miRNAs and grouped in 47 MIR families, as well as 85 novel candidate miRNA, putatively wheat specific. A total of 80 known and novel miRNA precursors were found differentially expressed between the two cultivars or modulated by stress and many of them showed a cultivar-specific expression profile. Interestingly, most in silico predicted targets of the miRNAs coming from the differentially expressed precursors have been experimentally linked in other species to mechanisms controlling stomatal movement, a finding in agreement with previous results showing that Cappelli has a lower stomatal conductance than Ofanto. Selected miRNAs were validated through a standardized and reliable stem-loop qRT-PCR procedure.
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Secas , Temperatura Alta , MicroRNAs/genética , Triticum/genética , Triticum/crescimento & desenvolvimento , Água/metabolismoRESUMO
The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like durum wheat requires the identification and optimization of factors affecting T-DNA delivery and plant regeneration. The purpose of this research was to compare the behavior of diverse durum wheat genotypes during in vitro culture and Agrobacterium tumefaciens-mediated transformation, using immature embryos as explants. Apart from plant genotype, two of the main influencing factors for a successful genetic transformation have been examined here, i.e., auxin source (Dicamba and 2,4-D) and duration of the pre-culture period (one, seven and 21 days). The addition of Dicamba to the media in combination with seven days pre-cultivation resulted in a general enhancement of T-DNA delivery for most of the analyzed cultivars, as revealed by ß-glucuronidase (GUS) histochemical assay. Although all genotypes were able to produce calli, significant differences were detected in regeneration and transformation efficiencies, since only two (Karalis and Neolatino) out of 14 cultivars produced fertile transgenic plants. The estimated transformation efficiencies were 6.25% and 1.66% for Karalis and Neolatino, respectively, and χ² analysis revealed the stable integration and segregation of the gus transgene in T1 and T2 progenies. This research has demonstrated that, among the influencing factors, genotype and auxin type play the most important role in the success of durum wheat transformation.
Assuntos
Agrobacterium tumefaciens/fisiologia , DNA Bacteriano/genética , Ácidos Indolacéticos/farmacologia , Triticum/genética , Ácido 2,4-Diclorofenoxiacético/farmacologia , Dicamba/farmacologia , Genótipo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/microbiologia , Técnicas de Cultura de Tecidos , Transformação Genética , Triticum/crescimento & desenvolvimento , Triticum/microbiologiaRESUMO
BACKGROUND: Alloplasmic lines provide a unique tool to study nuclear-cytoplasmic interactions. Three alloplasmic lines, with nuclear genomes from Triticum aestivum and harboring cytoplasm from Aegilops uniaristata, Aegilops tauschii and Hordeum chilense, were investigated by transcript and metabolite profiling to identify the effects of cytoplasmic substitution on nuclear-cytoplasmic signaling mechanisms. RESULTS: In combining the wheat nuclear genome with a cytoplasm of H. chilense, 540 genes were significantly altered, whereas 11 and 28 genes were significantly changed in the alloplasmic lines carrying the cytoplasm of Ae. uniaristata or Ae. tauschii, respectively. We identified the RNA maturation-related process as one of the most sensitive to a perturbation of the nuclear-cytoplasmic interaction. Several key components of the ROS chloroplast retrograde signaling, together with the up-regulation of the ROS scavenging system, showed that changes in the chloroplast genome have a direct impact on nuclear-cytoplasmic cross-talk. Remarkably, the H. chilense alloplasmic line down-regulated some genes involved in the determination of cytoplasmic male sterility without expressing the male sterility phenotype. Metabolic profiling showed a comparable response of the central metabolism of the alloplasmic and euplasmic lines to light, while exposing larger metabolite alterations in the H. chilense alloplasmic line as compared with the Aegilops lines, in agreement with the transcriptomic data. Several stress-related metabolites, remarkably raffinose, were altered in content in the H. chilense alloplasmic line when exposed to high light, while amino acids, as well as organic acids were significantly decreased. Alterations in the levels of transcript, related to raffinose, and the photorespiration-related metabolisms were associated with changes in the level of related metabolites. CONCLUSION: The replacement of a wheat cytoplasm with the cytoplasm of a related species affects the nuclear-cytoplasmic cross-talk leading to transcript and metabolite alterations. The extent of these modifications was limited in the alloplasmic lines with Aegilops cytoplasm, and more evident in the alloplasmic line with H. chilense cytoplasm. We consider that, this finding might be linked to the phylogenetic distance of the genomes.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Genoma de Planta , Triticum/genética , Triticum/metabolismo , Cloroplastos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Metaboloma , Metabolômica/métodos , Mitocôndrias/metabolismo , Fotossíntese/genética , Infertilidade das Plantas/genética , Transdução de SinaisRESUMO
The chloroplast is the central switch of the plant's response to cold and light stress. The ability of many plant species to develop a cold tolerant phenotype is dependent on the presence of light and photosynthetic activity during low-temperature growth. Light exposure at low temperature stimulates an over-reduction of the plastoquinone pool as well as the accumulation of reactive oxygen species, and both metabolic conditions generate a retrograde signal controlling nuclear gene expression. At the same time the chloroplast is the target of many cold acclimation processes which are the results of the chloroplast-nucleus cross-talk. Often, the extent of cold acclimation of the chloroplast is tightly correlated with the overall plant tolerance to chilling and freezing temperatures, a finding suggesting that the chloroplast cold acclimation could be the rate limiting factor in the adaptation to low temperature.
Assuntos
Aclimatação/fisiologia , Cloroplastos/genética , Cloroplastos/fisiologia , Temperatura Baixa , Proteínas de Plantas/metabolismo , Transdução de Sinais , Regulação da Expressão Gênica de Plantas , Luz , Fotossíntese , Estresse FisiológicoRESUMO
The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles-VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.
RESUMO
Cereal species can be damaged by frost either during winter or at flowering stage. Frost tolerance per se is only a part of the mechanisms that allow plants to survive during winter, while winter-hardiness also considers other biotic or physical stresses that challenge the plants during the winter season, limiting their survival rate. While frost tolerance can also be tested in controlled environments, winter-hardiness can only be determined with field evaluations. Post-heading frost damage occurs from radiation frost events in spring during the reproductive stages. A reliable evaluation of winter-hardiness or of post heading frost damage should be carried out with field trials replicated across years and locations to overcome the irregular occurrence of natural conditions which satisfactorily differentiate genotypes. The evaluation of post-heading frost damage requires a specific attention to plant phenology. The extent of frost damage is traditionally determined with a visual score at the end of the winter, although, recently an image-based phenotyping coupled with unmanned aerial vehicles (UAVs) has been proposed.
Assuntos
Adaptação Biológica , Grão Comestível/fisiologia , Congelamento , Melhoramento Vegetal , Estações do Ano , FenótipoRESUMO
We investigated the pigment composition and the transcriptome of albina (alb-e ( 16 ) and alb-f ( 17 )) and xantha (xan-s ( 46 ) and xan-b ( 12 )) barley mutants to provide an overall transcriptional picture of genes whose expression is interconnected with chloroplast activities and to search for candidate genes associated with the mutations. Beside those encoding plastid-localized proteins, more than 3,000 genes involved in non-chloroplast localized metabolism were up-/down-regulated in the mutants revealing the network of chloroplast-dependent metabolic pathways. The alb-e ( 16 ) mutant was characterized by overaccumulation of protoporphyrin IX upon ALA (5-amino levulinic acid) feeding and down-regulation of the gene encoding one subunit of Mg-chelatase, suggesting a block of the chlorophyll biosynthetic pathway before Mg-protoporphyrin IX biosynthesis, while alb-f ( 17 ) overaccumulated Mg-protoporphyrin IX and repressed PorA expression, without alterations in Mg-chelatase mRNA level. The alb-f ( 17 )mutant also showed overexpression of several genes involved in phytochrome and in phytochrome-dependent pathways. The results indicate that the down-regulation of Lhcb genes in alb-e ( 16 ) cannot be mediated by the accumulation of Mg-protoporphyrin IX. After ALA treatment, xan-s ( 46 ) showed overaccumulation of Mg-protoporphyrin IX, while the relative porphyrin composition of xan-b ( 12 ) was similar to wild type. The transcripts encoding the components of several mitochondrial metabolic pathways were up-regulated in albina/xantha leaves to compensate for the absence of active chloroplasts. The mRNAs encoding gun3, gun4, and gun5 barley homologous genes showed significant expression variations and were used to search for co-expressed genes across all samples. These analyses provide additional evidences on a chloroplast-dependent covariation of large sets of nuclear genes.
Assuntos
Cloroplastos/genética , Perfilação da Expressão Gênica , Hordeum/genética , Liases/genética , Ácido Aminolevulínico/farmacologia , Cloroplastos/efeitos dos fármacos , Cloroplastos/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hordeum/efeitos dos fármacos , Hordeum/metabolismo , Liases/metabolismo , Proteínas de Plantas/genética , Subunidades Proteicas/genética , Protoporfirinas/farmacologia , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.
Assuntos
Triticum/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Cromossomos de Plantas/genética , Domesticação , Variação Genética , Genoma de Planta , Filogenia , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Seleção Genética , Sintenia , Tetraploidia , Triticum/classificação , Triticum/metabolismoRESUMO
Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 isoform in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter::GUS transient expression was examined in barley developing caryopses at 30-day postfertilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and comparable Km values for both substrates, i.e. 0.89x10(-5) M and 0.5x10(-5) M for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activity significantly differs in substrate specificity and pH optimum when compared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles.
Assuntos
Duplicação Gênica , Hordeum/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Proteínas de Plantas/genética , Sequência de Bases , Evolução Molecular , Genes Duplicados , Hordeum/anatomia & histologia , Hordeum/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Distribuição Tecidual/genética , Poliamina OxidaseRESUMO
Drought and heat tolerance are complex quantitative traits. Moreover, the adaptive significance of some stress-related traits is more related to plant survival than to agronomic performance. A web of regulatory mechanisms fine-tunes the expression of stress-related traits and integrates both environmental and developmental signals. Both post-transcriptional and post-translational modifications contribute substantially to this network with a pivotal regulatory function of the transcriptional changes related to cellular and plant stress response. Alternative splicing and RNA-mediated silencing control the amount of specific transcripts, while ubiquitin and SUMO modify activity, sub-cellular localization and half-life of proteins. Interactions across these modification mechanisms ensure temporally and spatially appropriate patterns of downstream-gene expression. For key molecular components of these regulatory mechanisms, natural genetic diversity exists among genotypes with different behavior in terms of stress tolerance, with effects upon the expression of adaptive morphological and/or physiological target traits.
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MicroRNAs (miRNAs) are short non-coding RNA molecules produced from hairpin structures and involved in gene expression regulation with major roles in plant development and stress response. Although each annotated miRNA in miRBase (www.mirbase.org) is a single defined sequence with no further details on possible variable sequence length, isomiRs - namely the population of variants of miRNAs coming from the same precursors - have been identified in several species and could represent a way of broadening the regulatory network of the cell. Next-gen-based sequencing makes it possible to comprehensively and accurately assess the entire miRNA repertoire including isomiRs. The aim of this work was to survey the complexity of the peach miRNome by carrying out Illumina high-throughput sequencing of miRNAs in three replicates of five biological samples arising from a set of different peach organs and/or phenological stages. Three hundred-ninety-two isomiRs (miRNA and miRNA*-related) corresponding to 26 putative miRNA coding loci, have been highlighted by mirDeep-P and analyzed. The presence of the same isomiRs in different biological replicates of a sample and in different tissues demonstrates that the generation of most of the detected isomiRs is not random. The degree of mature sequence heterogeneity is very different for each individual locus. Results obtained in the present work can thus contribute to a deeper view of the miRNome complexity and to better explore the mechanism of action of these tiny regulators.
RESUMO
The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at -11°C and -12°C. Frost tolerance was assessed measured the F(v)/F(m) parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Oryza/genética , Fatores de Transcrição/genética , Adaptação Fisiológica/genética , Anaerobiose/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Temperatura Baixa , Congelamento , Germinação , Hordeum/enzimologia , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Oryza/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20-24 nt, known to play key roles in post-transcriptional gene regulation, that can be coded either by intergenic or intragenic loci. Intragenic (exonic and intronic) miRNAs can exert a role in the transcriptional regulation and RNA processing of their host gene. Moreover, the possibility that the biogenesis of exonic miRNAs could destabilize the corresponding protein-coding transcript and reduce protein synthesis makes their characterization very intriguing and suggests a possible novel mechanism of post-transcriptional regulation of gene expression. This work was designed to carry out the computational identification of putative exonic miRNAs in 30 plant species and the analysis of possible mechanisms involved in their regulation. The results obtained represent a useful starting point for future studies on the complex networks involved in microRNA-mediated gene regulation in plants.
Assuntos
Regulação da Expressão Gênica de Plantas , Genoma de Planta , MicroRNAs/genética , Plantas/genética , Biologia Computacional , Éxons , Etiquetas de Sequências Expressas , MicroRNAs/metabolismo , Plantas/metabolismoRESUMO
We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level.
Assuntos
Hordeum/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Oxirredução , Plastoquinona/química , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Cloroplastos/metabolismo , Eletroforese em Gel Bidimensional , Focalização Isoelétrica , Luz , Fotossíntese , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteômica/métodos , RNA Mensageiro/metabolismo , Tilacoides/metabolismoRESUMO
Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; > or = 2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold.
Assuntos
Aclimatação/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Hordeum/genética , RNA Mensageiro/metabolismo , Cloroplastos/metabolismo , Cloroplastos/fisiologia , Análise por Conglomerados , Perfilação da Expressão Gênica , Hordeum/crescimento & desenvolvimento , Hordeum/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fotossíntese , Transdução de SinaisRESUMO
WRKY proteins constitute a large family of plant specific transcription factors implicated in many different processes. Here we describe Hv-WRKY38, a barley gene coding for a WRKY protein, whose expression is involved in cold and drought stress response. Hv-WRKY38 was early and transiently expressed during exposure to low non-freezing temperature, in ABA-independent manner. Furthermore, it showed a continuous induction during dehydration and freezing treatments. A WRKY38:YFP fusion protein was found to localise into the nucleus upon introduction into epidermal onion cells. Bacterially expressed Hv-WRKY38 was able to bind in vitro to the W-box element (T)TGAC(C/T) also recognisable by other WRKY proteins. Hv-WRKY38 genomic DNA was sequenced and mapped onto the centromeric region of the barley chromosome 6H. Arabidopsis and rice sequences homologous to Hv-WRKY38 were also identified. Our results indicate that Hv-WRKY38 transcription factor may play a regulatory role in abiotic stress response.
Assuntos
Hordeum/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Núcleo Celular/metabolismo , Temperatura Baixa , DNA de Plantas/química , DNA de Plantas/genética , Ensaio de Desvio de Mobilidade Eletroforética , Éxons , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hordeum/efeitos dos fármacos , Íntrons , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Água/farmacologiaRESUMO
The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutant a(n), implying a chloroplast control on mRNAs accumulation. By using albino and xantha mutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation of cor14b, tmc-ap3, and blt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant a(n) leaf explants using transient beta-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides -274 and -247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14b promoter was equally active in green and in albino a(n) leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of the vir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b.