Diagonal two-dimensional electrophoresis (D-2DE): a new approach to study the effect of osmotic stress induced by polyethylene glycol in durum wheat (Triticum durum Desf.).
Acclimatization to stress is associated with profound changes in proteome composition. The use of plant cell and tissue culture offers a means to investigate the physiological and biochemical processes involved in the adaptation to osmotic stress. We employed a new proteomic approach to further understand the response of calli to dehydration induced by polyethylene glycol (PEG6000). Calli of three durum wheat genotypes Djenah Khetifa, Oued Zenati and Waha were treated with two concentrations of polyethylene glycol to mimic osmotic stress. Changes in protein relative abundance were analyzed using a new electrophoretic approach named diagonal two-dimensional electrophoresis (D-2DE), combined with mass spectrometry. Total proteins were extracted from 30-day-old calli from three durum wheat genotypes that showed contrasting levels of drought stress tolerance in the field. The combination of one-dimensional electrophoresis and D-2DE gave a specific imprint of the protein extracts under osmotic stress, as well as characterizing and identifying individual target proteins. Of the variously expressed proteins, three were selected (globulin, GAPDH and peroxidase) and further analyzed using qRT-PCR at the transcriptome level in order to compare the results with the proteomic data. Western blot analysis was used to further validate the differences in relative abundance pattern. The proteins identified through this technique provide new insights as to how calli respond to osmotic stress. Our method of study provides an original and relevant approach of analyzing the osmotic-responsive mechanisms at the cellular level of durum wheat with agronomic perspectives.
Department of Life Sciences, Walloon Agricultural Research Centre, Chaussée de Charleroi, 234, 5030, Gembloux, Belgium. kacem.nadia@yahoo.fr.|Laboratory of Genetic Biochemistry and Plant Biotechnology, Faculty of Nature and Life Sciences, University Frères Mentouri Constantine, 25017, Constantine, Algeria. kacem.nadia@yahoo.fr.
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Kacem
Department of Life Sciences, Walloon Agricultural Research Centre, Chaussée de Charleroi, 234, 5030, Gembloux, Belgium.
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Mauro
Department of Life Sciences, Walloon Agricultural Research Centre, Chaussée de Charleroi, 234, 5030, Gembloux, Belgium.
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Muhovski
Department of Life Sciences, Walloon Agricultural Research Centre, Chaussée de Charleroi, 234, 5030, Gembloux, Belgium.
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Delporte
Environmental Research and Innovation Department (ERIN), Integrative Biology Facility, Luxembourg Institute of Science and Technology, 41, Rue Du Brill, 4422, Belvaux, Luxembourg.
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Renaut
Laboratory of Genetic Biochemistry and Plant Biotechnology, Faculty of Nature and Life Sciences, University Frères Mentouri Constantine, 25017, Constantine, Algeria.
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Djekoun
Department of Life Sciences, Walloon Agricultural Research Centre, Chaussée de Charleroi, 234, 5030, Gembloux, Belgium.
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Watillon
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D005916:Globulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D009997:Osmotic Pressure; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology*; D013312:Stress, Physiological; D014908:Triticum / Q000378:metabolism*
D005916:Globulins; D010940:Plant Proteins; D011092:Polyethylene Glycols; D009195:Peroxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Diagonal two-dimensional electrophoresis; Mature embryo; Osmotic stress; PEG; Triticum durum