Protein and carbohydrate analyses of abiotic stress underlying cryopreservation in potato

Abstract

Cryopreservation complements classical conservation methods, which are carried out in the field or in vitro. It involves the storage of biological material in liquid nitrogen (- 196°C). At this temperature all chemical and physical processes are stopped, allowing a safe storage over an unlimited period of time. The aim of our work is to understand the effects of pretreatments, by increasing the osmotic pressure of the medium, on the cryopreservation ability of potato shoot tips and more specifically to try to evaluate the effects of such pretreatments on the metabolism of potato. Indeed, drought acclimation is known to improve recovery after cryopreservation in potato and other species In vitro Désirée potato shoots were precultured for 21 days on regular MS and MS complemented with 0.055M, 0.11M, and 0.22M sorbitol. Directly after pre-treatment, leaf and shoot tip samples were taken and stored at 80°C for proteomic and carbohydrate analyses. In addition, cryopreservation was carried on the precultured shoot-tips. For the cryopreservation, potato shoot tips were cut from pre-treated plants and incubated in a high osmotic Loading Solution. Afterwards, shoot tips were placed on an aluminum foil strip in droplets of Plant Vitrification Solution 2 and plunged into liquid nitrogen (Agrawal, 2004). Thawing was done in a highly osmotic Recovery Solution at room temperature, to prevent osmotic shock. After cryopreservation, shoot tips were transferred into the dark for 1 week (Panis, 2005). During the initial days of post-culture, shoot tips were maintained on MS media containing 0.3M sucrose. Afterwards, regular MS media were used. After 30 days, recovery was calculated as the percentage of shoot-tips forming new shoot. Proteins from shoot tip and leaf samples were extracted, using a TCA/Acetone ex-traction method. After quantification, 40 mg protein was labelled using three different fluorescent dyes (Cy2, Cy3 and Cy5). In this way, 2 samples and an internal standard - containing a mix of all the samples - was loaded on the same IEF strip (pI 4-7). Isoelectric focusing was carried out using the GE Healthcare Ettan IPGphor. The second dimension was run in a GE Healthcare Ettan Dalt Six electrophoresis system. Gels were scanned using the Typhoon 9400 scanner and subsequently analysed with the GE Healthcare DeCyder program and EDA module. As such, differences in protein expression could be quantified and differentially expressed spots picked by the GE Healthcare Ettan Spot Handling Workstation (Renaut, 2006). In a following step, spots were digested, using Trypsin and spotted on a MALDI plate. Protein identification was done, using the Applied Biosystems MALDI 4800 TOF/TOF analyzer. For the analysis of carbohydrates and polyols, roughly 100 mg leaf samples was used for carbohydrate and polyol extractions. The following carbohydrates and polyos were measured: sucrose, glucose, fructose, galactose, stachyose, arabinose, melibiose, maltose, trehalose, inositol, mannitol, galactinol and sorbitol. Carbohydrates were analyzed on a Dionex HPLC ICS2500-Bio LC, using a Carbopac PA-20 column. HPAEC-PAD analyses for polyols was conducted on a Dionex DX-500 chromatograph, using a Dionex Car-bopac MA1 column. Recovery rates without sorbitol were around 50% but increased with increasing concentration of sorbitol pre-treatment up to a recovery rate up to 80%. For 2D-DIGE proteomics, preliminary analysis of the gels showed differences in protein patterns, when plants were precultured on different sorbitol media. Fifteen up- or downregulated proteins were isolated and identified. Interestingly, these preliminary results indicate strong alteration of the primary metabolism and more precisely in carbon fixation. These results are sustained by the carbohydrate and polyol analyses. Indeed, sucrose, glucose, fructose, mannitol, arabinose, galactinol, meli-biose and stachyose increased with increasing concentrations of exogenousely supplied sorbitol up to 0.11M sorbitol. At 0.22M sorbitol their concentrations decreased. Trehalose and sorbitol concentrations increased exponentially with an increasing molarity of sorbitol pretreatment. Maltose was only observed when plantlets were treated with 0.22M sorbitol. When plants were treated with up to 0.11M sorbitol during 21 days, carbohydrate and polyol concentrations increased. When 0.22M sorbitol was applied as pre-treatment, most sugar concentrations decreased. Since high intracellular osmolyte concentrations are needed to allow successful recovery after cryopreservation, the results from carbohydrate and polyol analysis (Bhandal, 1985) may explain the higher recovery rate after cryopreservation observed after sorbitol treatment. Extra cryopreservation experiments are needed to confirm these results. Beside the sugar analysis, these changes affecting the primary metabolism have been observed at the protein level by using differential in gel electrophoresis. Further experiments including also chilling pretreatments will lead to a better understanding of the physiological status of the tissue and will hopefully explain the reason of the better results of cryopreservation.vokMyyynti MTT, Tietopalvelut 31600 Jokioine