Irini Nianiou-Obeidat

Overexpression of a specific soybean GmGSTU4 isoenzyme improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants

Plant glutathione transferases (GSTs) superfamily consists of multifunctional enzymes and forms a major part of the plants herbicide detoxification enzyme network. The tau class GST isoenzyme GmGSTU4 from soybean, exhibits catalytic activity towards the diphenyl ether herbicide fluorodifen and is active as glutathione-dependent peroxidase (GPOX). Transgenic tobacco plants of Basmas cultivar were generated via Agrobacterium transformation. The aim was to evaluate in planta, GmGSTU4s role in detoxifying the diphenyl ether herbicides fluorodifen and oxyfluorfen and the chloroacetanilides alachlor and metolachlor. Transgenic tobacco plants were verified by PCR and Southern blot hybridization and expression of GmGSTU4 was determined by RT-PCR. Leaf extracts from transgenic plants showed moderate increase in GST activity towards CDNB and a significant increase towards fluorodifen and alachlor, and at the same time an increased GPOX activity towards cumene hydroperoxide. GmGSTU4 overexpressing plants when treated with 200 μM fluorodifen or oxyfluorfen exhibited reduced relative electrolyte leakage compared to wild type plants. Moreover all GmGSTU4 overexpressing lines exhibited significantly increased tolerance towards alachlor when grown in vitro at 7.5 mg/L alachlor compared to wild type plants. No significant increased tolerance was observed to metolachlor. These results confirm the contribution of this particular GmGSTU4 isoenzyme from soybean in the detoxification of fluorodifen and alachlor, and provide the basis towards the development of transgenic plants with improved phytoremediation capabilities for future use in environmental cleanup of herbicides.

Summer Squash Identification by High-Resolution-Melting (HRM) Analysis Using Gene-Based EST–SSR Molecular Markers

Cucurbita pepo (squash, pumpkin, gourd), a worldwide cultivated vegetable of American origin, is extremely variable in fruit characteristics. Most of its widely grown commercial types are known as summer squashes and belong to the elongated forms of C. pepo ssp. pepo (Cocozelle, Vegetable marrow and Zucchini groups). Here, we have integrated the high-resolution-melting (HRM) analysis method with expressed sequence tags–simple sequence repeat (EST–SSR) marker genotyping, in order to facilitate the identification of 36 summer squash landraces originated from Greece. The six EST–SSR loci used were informative and generated a unique melting curve profile of EST-derived microsatellites for each accession allowing their comparison and classification. Moreover, HRM was highly informative, as by using only four microsatellite markers we were able to discriminate 36 summer squash landraces and by using six EST–SSRs. We were able to construct a highly informative and discriminative dendrogram where the 36 genotypes were classified in six distinct clusters. Furthermore, we acquired information about the genes containing the EST–SSRs using bioinformatics tools. We found that the EST–SSRs used in this study were hybridizing to genes involved in stress response to heavy metals and biotic stresses or the production of flavonoids or symporters of important nitrogen sources, like xanthine and uric acid amongst others. The results presented here suggest that the panel of EST–SSR markers used in combination with HRM analysis could be useful in a variety of applications, like squash biodiversity assessment but most importantly in managing squash germplasm to improve breeding programs.

Plant glutathione transferase-mediated stress tolerance: functions and biotechnological applications

Plant glutathione transferases (EC 2.5.1.18, GSTs) are an ancient, multimember and diverse enzyme class. Plant GSTs have diverse roles in plant development, endogenous metabolism, stress tolerance, and xenobiotic detoxification. Their study embodies both fundamental aspects and agricultural interest, because of their ability to confer tolerance against biotic and abiotic stresses and to detoxify herbicides. Here we review the biotechnological applications of GSTs towards developing plants that are resistant to biotic and abiotic stresses. We integrate recent discoveries, highlight, and critically discuss the underlying biochemical and molecular pathways involved. We elaborate that the functions of GSTs in abiotic and biotic stress adaptation are potentially a result of both catalytic and non-catalytic functions. These include conjugation of reactive electrophile species with glutathione and the modulation of cellular redox status, biosynthesis, binding, and transport of secondary metabolites and hormones. Their major universal functions under stress underline the potential in developing climate-resilient cultivars through a combination of molecular and conventional breeding programs. We propose that future GST engineering efforts through rational and combinatorial approaches, would lead to the design of improved isoenzymes with purpose-designed catalytic activities and novel functional properties. Concurrent GST–GSH metabolic engineering can incrementally increase the effectiveness of GST biotechnological deployment.

Shoot regeneration and micrografting of micropropagated hybridtomatoes

Summary The use of grafted tomato plants is expanding. An in vitro propagation system was established for three commercial F1 cultivars (‘Garnet 622’, ‘Jumbo’ and ‘Marvel’) to combine micropropagation with micrografting. Nodal tissue explants were excised from the central stem of plantlets and micropropagated on MS medium. A micropropagation rate of 6 shoots per explant was obtained for all three hybrids. Micropropagated shoots were then micrografted onto 3 week-old ‘RT-79’ rootstocks with a union rate of over 80%. After acclimatisation, micropropagated plants were established ex vivo, flowered and matured normally. In addition, regeneration protocols were tested using cotyledon explants from 10 d-old seedlings. Adventitious shoot organogenesis from three parts of the cotyledon explants (proximal, middle, distal) was monitored on MS media supplemented with different concentrations of zeatin (Z) and indole-3-acetic acid (IAA). The highest percentage of shoot formation was observed on MS medium supplemented with 0.5 mg l–1 Z and 0.1 mg l–1 IAA acid with cotyledon explants that originated from the proximal part. Up to 80% of explants formed adventitious shoots, with an average of three shoots per explant. Three week-old regenerated shoots were placed on MS medium without growth regulators for elongation, rooting and further development of plantlets.

De novo transcriptome assembly of two contrasting pumpkin cultivars

Cucurbita pepo (squash, pumpkin, gourd), a worldwide-cultivated vegetable of American origin, is extremely variable in fruit characteristics. However, the information associated with genes and genetic markers for pumpkin is very limited. In order to identify new genes and to develop genetic markers, we performed a transcriptome analysis (RNA-Seq) of two contrasting pumpkin cultivars. Leaves and female flowers of cultivars, ‘Big Moose’ with large round fruits and ‘Munchkin’ with small round fruits, were harvested for total RNA extraction. We obtained a total of 6 GB (Big Moose; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056882) and 5 GB (Munchkin; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056883) sequence data (NCBI SRA database SRX1502732 and SRX1502735, respectively), which correspond to 18,055,786 and 14,824,292 150-base reads. After quality assessment, the clean sequences where 17,995,932 and 14,774,486 respectively. The numbers of total transcripts for ‘Big Moose’ and ‘Munchkin’ were 84,727 and 68,051, respectively. TransDecoder identified possible coding regions in assembled transcripts. This study provides transcriptome data for two contrasting pumpkin cultivars, which might be useful for genetic marker development and comparative transcriptome analyses.

Maintenance of metabolic homeostasis and induction of cytoprotectants and secondary metabolites in alachlor-treated GmGSTU4-overexpressing tobacco plants, as resolved by metabolomics

Herbicides are an invaluable tool for agricultural production scaling up. However, their continuous and intensive use has led to an increased incidence of herbicide resistant weeds and environmental pollution. Plant glutathione transferases (GSTs) are tightly connected with crop and weed herbicide tolerance capacitating their efficient metabolic detoxification, thus GSTs can be biotechnologically exploited towards addressing those issues. However, information on their effects at a “systems” level in response to herbicides is lacking. Here, we aimed to study the effects of the chloroacetanilide herbicide alachlor on the metabolome of wild-type and tobacco plants overexpressing the soybean tau class glutathione transferase GmGSTU4. Alachlor-treated wild-type plants This system, naturally serving the detoxification of endogenous exhibited an abiotic stress-like response with increased abundance of compatible solutes, decrease in TCA cycle intermediates and decreased sugar and amino acid content. Transgenic plants responded distinctly, exhibiting an increased induction of abiotic stress responsive metabolites, accumulation of secondary metabolites and its precursors, and metabolic detoxification by-products compared to wild-type plants. These results suggest that the increased metabolic capacity of GmGSTU4 overexpressing plants is accompanied by pleiotropic metabolic alterations, which could be the target for further manipulation in order to develop herbicide resistant crops, plants with increased phytoremediation potential, as well as efficient management of non-target site, GST induced, herbicide resistance in weeds.

Plant Glutathione Transferases: Structure, Antioxidant Catalytic Function and in planta Protective Role in Biotic and Abiotic Stress

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Structure and Antioxidant Catalytic Function of Plant Glutathione Transferases

Plant cytosolic glutathione transferases (GSTs) are an ancient enzyme superfamily with multiple and diverse functions which are important in counteracting biotic and abiotic stress. GSTs play an important role in catalyzing the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), such as pesticides, chemical carcinogens, environmental pollutants, which leads to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroper- oxides formed during oxidative stress, biosynthesis of sulfur-containing secondary metabolites, and exhibit thioltrans- ferase and dehydroascorbate reductase activity. This review focuses on plant GSTs, and attempts to give an overview of the new insights into the catalytic function and structural biology of these enzymes.

Plant Glutathione Transferases in Abiotic Stress Response and Herbicide Resistance

Plant responses and adaptations to stress conditions are of great interest for both basic and applied science, and represent the key factors for the improvement of economically important crops worldwide. Glutathione S-transferases (GSTs, EC. 2.5.1.18) are multifunctional enzymes encoded by a highly divergent ancient gene family. GSTs catalyze the conjugation of tripeptide glutathione (GSH) with endogenous electrophilic compounds (secondary metabolites, hydroperoxides) and xenobiotics, such as herbicides, leading to their cellular detoxification. Therefore, GSTs are implicated in metabolism-based herbicide resistance in crop weeds. This chapter discusses the involvement of plant GSTs in abiotic stress response with focus on metabolism-based herbicide resistance and attempts to give an overview of their catalytic roles and in planta function.

Plant Adaptation to Stress Conditions: The Case of Glutathione S-Transferases (GSTs)

Plants, unlike animals, are anchored to one place and, therefore, forced to sustain any environmental condition present. Unfavourable environmental conditions include abiotic (extreme temperatures, water deficits, floods, salinity, light intensities) and biotic (pests, viral, bacterial and fungal diseases) stress factors. Both types of stresses induce the production of reactive oxygen species (ROS), which damage macromolecules such as proteins, lipids, nucleic acids and cell structures like membranes. The effect of each stress factor depends on its intensity. When the stress is severe and the production of ROS is high, it might result to plant death. To avoid such event, plants have developed advanced physiological and chemical defence mechanisms of stress avoidance and/or tolerance, which allow growth only when the environmental conditions are optimum for each species, like in the case of seed dormancy. Plants have also evolved specific enzymatic defence mechanisms, including enzymes like catalase, peroxidase, super oxide dismutase and glutathione transferases. These defence mechanisms help plants either to avoid adverse environmental conditions or to combat their negative effects. A major defence mechanism involves the action of antioxidant enzymes. Glutathione transferases (GSTs) are antioxidant enzymes of great importance for the detoxification of plants from toxic compounds. GSTs have also important involvement in plant stress tolerance against biotic and abiotic stress tolerance like extreme heat, cold, salt and herbicides.