ABSTRACT
Protein metabolism and expression play important role in plant adaptation to water stress. The objectives of this study were to examine proteomic responses to water stress induced by polyethylene glycol (PEG) in creeping bentgrass (Agrostis stolonifera L.) leaves and to identify proteins associated with stress tolerance. Plants of two cultivars ('Penncross' and 'Penn-A4') differing in water stress tolerance were grown in sand irrigated daily with water (control) or PEG solution (osmotic potential of -0.66MPa) to induce water stress, for 28d in growth chambers. Shoot extension rate, relative water content and cell membrane stability were measured to compare drought tolerance between the two cultivars. All parameters maintained at a significantly higher level in 'Penn-A4' than in 'Penncross' under PEG treatment. After 28d of water stress, proteins were extracted from leaves and separated by difference gel electrophoresis. Among 56 stress-responsive protein spots, 46 were identified using mass spectrometry. Some proteins involved in primary nitrogen and carbon metabolism were down-regulated by PEG-induced water stress in both cultivars. The abundance of antioxidant enzyme proteins (ascorbate peroxidase, catalase and glutathione-S-transferase) increased under water stress, particularly ascorbate peroxidase in 'Penn-A4'. The abundance levels of actins, UDP-sulfoquinovose synthase and glucan exohydrolase were greater in 'Penn-A4' than in 'Penncross' under PEG treatment. Our results suggest that proteins involved in membrane synthesis, cell wall loosening, cell turgor maintenance, and antioxidant defense may play roles in perennial grass adaptation to PEG-induced water stress.
Subject(s)
Adaptation, Physiological/drug effects , Agrostis/drug effects , Agrostis/physiology , Polyethylene Glycols/pharmacology , Proteome/metabolism , Proteomics , Stress, Physiological/drug effects , Agrostis/cytology , Amino Acids/metabolism , Antioxidants/metabolism , Cell Proliferation/drug effects , Cell Respiration/drug effects , Cell Wall/drug effects , Cell Wall/metabolism , Dehydration , Electrophoresis, Gel, Two-Dimensional , Heat-Shock Proteins/metabolism , Photosynthesis/drug effects , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Proteins/classification , Plant Proteins/metabolism , Plant Shoots/drug effects , Plant Shoots/growth & developmentABSTRACT
Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant.
