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Fusarium oxysporum f. sp. lentis (Fol) is considered the most destructive disease for lentil (Lens culinaris Medik.) worldwide. Despite the extensive studies elucidating plants' metabolic response to fungal agents, there is a knowledge gap in the biochemical mechanisms governing Fol-resistance in lentil. Τhis study aimed at comparatively evaluating the metabolic response of two lentil genotypes, with contrasting phenotypes for Fol-resistance, to Fol-inoculation. Apart from gaining insights into the metabolic reprogramming in response to Fol-inoculation, the study focused on discovering novel biomarkers to improve early selection for Fol-resistance. GC-MS-mediated metabolic profiling of leaves and roots was employed to monitor changes across genotypes and treatments as well as their interaction. In total, the analysis yielded 178 quantifiable compounds, of which the vast majority belonged to the groups of carbohydrates, amino acids, polyols and organic acids. Despite the magnitude of metabolic fluctuations in response to Fol-inoculation in both genotypes under study, significant alterations were noted in the content of 18 compounds, of which 10 and 8 compounds referred to roots and shoots, respectively. Overall data underline the crucial contribution of palatinitol and L-proline in the metabolic response of roots and shoots, respectively, thus offering possibilities for their exploitation as metabolic biomarkers for Fol-resistance in lentil. To the best of our knowledge, this is the first metabolomics-based approach to unraveling the effects of Fol-inoculation on lentil's metabolome, thus providing crucial information related to key aspects of lentil-Fol interaction. Future investigations in metabolic aspects of lentil-Fol interactions will undoubtedly revolutionize the search for metabolites underlying Fol-resistance, thus paving the way towards upgrading breeding efforts to combat fusarium wilt in lentil.
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Introduction: Drought stress is one of the most devastating environmental stressors, especially in the arid and semi-arid regions of the world. Considering the major constraints that drought stress poses to crop production and the consequent yield losses in food crops, breeding for climate-resilient crops is an efficient means to mitigate stress conditions. Materials and methods: This study aimed at evaluating the response of four squash (Cucurbita maxima Duchesne) landraces to drought stress at germination and at plant stage. Drought stress was induced by different concentrations of D-mannitol (-0.24, -0.47 and -0.73 MPa). The tested parameters at germination stage included germination percentage, seedling vigor index, seed water absorbance and seedling growth potential. At the plant stage, leaf chlorophyll and carotenoids content, chlorophyll fluorescence, evapotranspiration, photosynthesis activity and several biomarkers, namely malondialdehyde, proline, total phenols content, total flavonoids content and DPPH radical scavenging activity were evaluated in both roots and leaves. Results and discussion: Our results indicate a magnitude of drought stress effects reflected via repression of germination and seedling growth as well as adjustments in physiological functions at later growth stages, in a genotype depended manner. Among landraces, "751" and "746" showed better performance, as evidenced by higher seed germination and seedling growth potential even at high stress levels (-0.47 and - 0.73 MPa), whereas "747" was the most sensitive landrace to drought stress at both tested stages. In conclusion, our findings highlight the importance of squash landraces selection for the identification of elite genotypes with increased tolerance to drought stress.
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Salt stress is considered as one of the most common abiotic stresses reducing the productivity and fruit quality of crop plants. The present study was carried out to assess the salt tolerance among 15 local squash (Cucurbita maxima Duchesne) landraces. Different salt (NaCl) concentrations of 0, 100, 200 and 300 mM were selected in order to evaluate the response of the study germplasm to salt stress based on 12 agronomic parameters and 3 biochemical traits, proline, malondialdehyde (MDA) and chlorophylls. A varied effect of the salt stress level was observed among the studied landraces based on germination potential, as well as on growth and biochemical parameters at seedling stage. Results showed that all landraces were drastically affected at high stress level with a significant variation in their stress response, indicating the existence of considerable genetic variability. Landraces "746" and "747" were the best performing cultivars across stress levels, whereas "1007", "1008" and "1009" were the most negatively affected. Based on the tested landrace performance, four landraceswere selected and further evaluated at biochemical level, focusing on the determination of compounds that play a key role in the ability to withstand salt stress. The mean MDA content across landraces was generally increased in stressed plants, as compared to the control treatment; the increase was attributed to a peak in MDA content at specific stress levels. In particular, "746" and "1007" showed the maximum content at 100 mM NaCl, while in landrace "751", MDA content reached its peak at 300 mM NaCl. In addition, the response of most landraces to salt stress involved an increase in free proline content, with the exception of "746", with the maximum content being observed either at 200 mM ("748" and "751" landraces) or at 300 mM NaCl, where only "747" expressed the highest content. These findings can be extrapolated into efforts to develop more salt-tolerant squash landraces and exhaust the possibilities of using saline water or soils under changing climate conditions.
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The hrpZPsph gene from Pseudomonas syringae pv. phaseolicola, in its secretable form (SP/hrpZPsph), has previously proven capable of conferring resistance against rhizomania disease as well as abiotic stresses in Nicotiana benthamiana plants, while enhancing plant growth. This study aimed at investigating the response of SP/hrpZPsph-expressing plants under cadmium stress. Transgenic N. benthamiana lines, homozygous for the SP/hrpZPsph gene, and wild-type plants were exposed to Cd at different stress levels (0, 50, 100, 150 µΜ CdCl2). Plants' response to stress was assessed at germination and at the whole plant level on the basis of physiological and growth parameters, including seed germination percentage, shoot and root length, total chlorophyll content, fresh and dry root weight, as well as overall symptomatology, and Cd content in leaves and roots. At germination phase, significant differences were noted in germination rates and post-germination growth among stress levels, with Cd effects being in most cases analogous to the level applied but also among plant categories. Although seedling growth was adversely affected in all plant categories, especially at high stress level, lines #6 and #9 showed the lowest decrease in root and shoot length over control. The superiority of these lines was further manifested at the whole plant level by the absence of stress-attributed symptoms and the low or zero reduction in chlorophyll content. Interestingly, a differential tissue-specific Cd accumulation pattern was observed in wt- and hrpZPsph-plants, with the former showing an increased Cd content in leaves and the latter retaining Cd in the roots. These data are discussed in the context of possible mechanisms underlying the hrpZPsph-based Cd stress resistance.
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Cádmio , Germinação , Raízes de Plantas , Plantas Geneticamente Modificadas , Plântula , Estresse Fisiológico , Nicotiana/genéticaRESUMO
The plant tau class glutathione transferases (GSTs) perform diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. In the present work, the tau class isoenzyme GSTU2-2 from Glycine max (GmGSTU2-2) was characterized. Gene expression analysis of GmGSTU2 suggested a highly specific and selective induction pattern to osmotic stresses, indicating that gene expression is controlled by a specific mechanism. Purified, recombinant GmGSTU2-2 was shown to exhibit wide-range specificity towards xenobiotic compounds and ligand-binding properties, suggesting that the isoenzyme could provide catalytic flexibility in numerous metabolic conditions. Homology modeling and phylogenetic analysis suggested that the catalytic and ligand binding sites of GmGSTU2-2 are well conserved compared to other tau class GSTs. Structural analysis identified key amino acid residues in the hydrophobic binding site and provided insights into the substrate specificity of this enzyme. The results established that GmGSTU2-2 participates in a broad network of catalytic and regulatory functions involved in the plant stress response.
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Glutationa Transferase/química , Glutationa/química , Glycine max/enzimologia , Proteínas de Plantas/química , Plântula/enzimologia , Proteínas tau/química , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Cinética , Simulação de Acoplamento Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Plântula/química , Plântula/genética , Alinhamento de Sequência , Glycine max/química , Glycine max/genética , Estresse Fisiológico , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Glutathione transferases (GSTs) represent a major group of detoxification enzymes. All plants possess multiple cytosolic GSTs, each of which displays distinct catalytic as well as non-catalytic binding properties. The progress made in recent years in the fields of genomics, proteomics and protein crystallography of GSTs, coupled with studies on their molecular evolution, diversity and substrate specificity has provided new insights into the function of these enzymes. In plants, GSTs appear to be implicated in an array of different functions, including detoxification of xenobiotics and endobiotics, primary and secondary metabolism, stress tolerance, and cell signalling. This review focuses on plant GSTome and attempts to give an overview of its catalytic and functional role in xenome and plant stress regulatory networks.
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Glutationa Transferase/metabolismo , Plantas/enzimologia , Plantas/genética , Estresse Fisiológico , Animais , Biocatálise , Genômica , Glutationa Transferase/genética , Humanos , Especificidade por SubstratoRESUMO
With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employment of different sources of resistance to Beet necrotic yellow vein virus was pursued. To this purpose, Nicotiana benthamiana transgenic plants that simultaneously produce dsRNA originating from a conserved region of the BNYVV replicase gene and the HrpZ(Psph) protein in a secreted form (SP/HrpZ(Psph)) were produced. The integration and expression of both transgenes as well as proper production of the harpin protein were verified in all primary transformants and selfed progeny (T1, T2). Transgenic resistance was assessed by BNYVV-challenge inoculation on T2 progeny by scoring disease symptoms and DAS-ELISA at 20 and 30 dpi. Transgenic lines possessing single transformation events for both transgenes as well as wild type plants were included in inoculation experiments. Transgenic plants were highly resistant to virus infection, whereas in some cases immunity was achieved. In all cases, the resistant phenotype of transgenic plants carrying both transgenes was superior in comparison with the ones carrying a single transgene. Collectively, our findings demonstrate, for a first time, that the combination of two entirely different resistance mechanisms provide high level resistance or even immunity against the virus. Such a novel approach is anticipated to prevent a rapid virus adaptation that could potentially lead to the emergence of isolates with resistance breaking properties.
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Beta vulgaris/imunologia , Beta vulgaris/virologia , Resistência à Doença/genética , Engenharia Genética/métodos , Doenças das Plantas/imunologia , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Beta vulgaris/genética , Vírus de Plantas/enzimologia , Plantas Geneticamente Modificadas , RNA de Cadeia Dupla/genética , RNA Viral/genética , Fatores de Tempo , Nicotiana/genética , Transgenes/genética , Proteínas Virais/genéticaRESUMO
In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.
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Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Glutationa Transferase/genética , Estresse Oxidativo , Agrobacterium tumefaciens/enzimologia , Agrobacterium tumefaciens/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Estresse Fisiológico/efeitos dos fármacos , Homologia Estrutural de Proteína , Especificidade por Substrato , Transcrição Gênica , Xenobióticos/farmacologiaRESUMO
To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.
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Proteínas da Membrana Bacteriana Externa/genética , Beta vulgaris/genética , Genes Bacterianos/genética , Imunidade Inata/genética , Nicotiana/genética , Doenças das Plantas/imunologia , Pseudomonas syringae/genética , Beta vulgaris/crescimento & desenvolvimento , Beta vulgaris/virologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica de Plantas , Necrose , Doenças das Plantas/virologia , Folhas de Planta/virologia , Raízes de Plantas/virologia , Vírus de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Nicotiana/crescimento & desenvolvimento , Nicotiana/virologia , TransgenesRESUMO
Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.
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Lotus/genética , Simportadores/genética , Xilitol/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Lotus/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismoRESUMO
Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species.