A Proteomic approach to the comparison of two pine species: proteomes of pinus brutia and pinus sylvestris under environmental stress

Yılmaz, Can
Drought and cold are environmental factors that affect the development and dispersion of plant species. In this study, two pine species adapted to different environmental conditions: Pinus brutia Ten. (Turkish red pine) and Pinus sylvestris L. (Scots pine) were subjected to a proteome research under those stresses. 20 trees from each species, located in the same region and elevation at Yalıncak area in METU Campus, were marked and sampled for their needles, three times in a year: at the end of dry and hot summer, cold winter and, mild and rainy spring. The changes in the photosynthetic pigments, biochemical stress markers such as proline, Malodialdehyde (MDA), Glutathione (GSH) contents and Glutathione S-transferase (GST) enzyme activities were recorded to provide supportive data for the presence and magnitude of stress for both conifer species. The changes in air temperature, humidity and total rainfall provided by Turkish State Meteorological Service were also used to strengthen the hypothesis. A new and relatively more efficient protein extraction method was introduced and by using this method two dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) images of samples were analysed and compared with each other. The analysis of LC-MS/MS data were performed by two different search machines (OMMSA and X!Tandem) having different algorithms and the results of these applications and supportive data provided by western blotting experiments revealed the proteomic changes in P. brutia and P. sylvestris needles with respect to season. As stated in many studies, proline content of both pine species were relatively higher in winter season to protect osmotic balance against increased water stress created by sub-freezing temperatures. The fate of this molecule in summer season might be shaped by some other metabolic necessities. MDA levels didn’t exhibited expected changes, however the changes in the GSH pool were purposeful with respect to the specific activity values of the total GST in both species: when the enzymatic activity rose, the pool was waned because of increased demand. Moreover, this relationship strengthens the proposition of higher oxidative stress in summer for P. sylvestris and in winter for P. brutia. P. sylvestris decreased photosynthetic machinery, exalted chlorophyll-(a+b)/carotenoid ratio and significantly increased HSPs and chaperonin levels as a part of cold-hardening. On the other hand, as expected, P. brutia trees were not as successful as scots pine in management of sub-freezing temperatures. The meteorological data showed the presence of frost days in spring season and, an early increase in the photosynthetic machinery in the P. brutia needles in this period might be a reason for elevated stress levels. Under cold stress conditions both of the species tend to increase the rate or the dimension of energy harvesting mechanism to meet the ATP need. Up-regulated respiratory system enzymes such as UDP-Glucose Pyrophosphate, Phosphoglycerate kinase, ATP-citrate synthase beta chain protein 1-like, citrate synthase, citrate synthase 3, NAD-dependent malic enzyme 1 pointed an increased ATP production. Moreover, the increase in the amount of Alcohol Dehydrogenase, which protects the sustainable proceeding of glycolysis, might be an indicator of this exalted need for energy because it seemed that it forced needle cells to gather energy even from anaerobic respiration.
Citation Formats
C. Yılmaz, “A Proteomic approach to the comparison of two pine species: proteomes of pinus brutia and pinus sylvestris under environmental stress,” Ph.D. - Doctoral Program, Middle East Technical University, 2013.