Yüksek sıcaklık stresinin çilek bitkisinde enzimatik ve enzimatik olmayan antioksidanlar ile protein metabolizmasına etkileri
Loading...
Files
Date
2012
Authors
Ergin, Sergül
Journal Title
Journal ISSN
Volume Title
Publisher
Uludağ Üniversitesi
Abstract
Çilek bitkisinde (Fragaria x ananassa Duch.) yüksek sıcaklıklara tolerans mekanizmasının araştırıldığı bu çalışmada yüksek sıcaklığa tolerant (R. Hope) ve hassas (CG3) iki çilek çeşidi kullanılmıştır. Çeşitlere ait frigo fideler perlit, torf ve elenmiş bahçe toprağı karışımı (1:1:1) içeren 14 12 cm çapındaki saksılarda, 30/15°C sıcaklıkta ve ortalama % 65 oransal nemde 2 ay boyunca yetiştirilmişlerdir. Daha sonra çeşitlere ait yaprak örneklerine yapay olarak kademeli ve şok yüksek sıcaklık testleri (35, 40, 43, 46, 49, 52, 55 ve 60°C) uygulanmıştır. Yüksek sıcaklık uygulamalarını takiben hücrede meydana gelen zararlanmaları belirlemek için iyon sızıntısı testleri yapılmış ve hidrojen peroksit (H2O2) birikimi ölçülmüştür. Yüksek sıcaklıklara ve uygulama şekline göre yaprak dokularında oluşan enzimatik olmayan antioksidanlardan askorbik asit (AsA) ve glutatiyon miktarı (GSH) ile enzimatik antioksidanlardan askorbat peroksidaz (APX), katalaz (CAT), glutatiyon redüktaz (GR) ve peroksidaz (PRX) enzim aktiviteleri saptanmıştır. Ayrıca, yüksek sıcaklığa toleransta protein metabolizmasının açıklanması ve bu konuda kullanılabilecek bir protein markırının araştırılması amacıyla toplam çözünebilir protein değişimleri ölçülerek SDS-PAGE yöntemiyle toplam çözünebilir protein profilleri çıkarılmıştır. Ardından immunoblot tekniği ile örneklerde HSP 60 ve HSP 70 proteinleri araştırılmıştır.Yüksek sıcaklık uygulamaları sonucu hücresel zararlanmanın sıcaklık artışına paralel olarak arttığı saptanmıştır. AsA ve GSH miktarının uygulama şekline ve sıcaklıklara göre değiştiği belirlenmiştir. Enzimatik antioksidanlardan APX, CAT ve PRX aktivitesinin sıcaklık artışıyla birlikte arttığı, GR aktivitesinin ise değişmediği tespit edilmiştir. SDS-PAGE'de uygulamalara göre değişen pek çok protein bandı görülmüş ancak HSP60 için yapılan immunoblot çalışmalarında sadece 23 kDa proteinin sıcaklığa bağlı olduğu tespit edilmiştir. 23 kDa HSP'nin her iki çeşitte de yüksek sıcaklıkta biriktiği ve yoğunluğunun da R.Hope çeşidinde daha fazla olduğu belirlenmiştir. Dolayısıyla 23 kDa HSP, çilek yaprak dokularının yüksek sıcaklık toleransı ile ilişkilendirilmiştir.Sonuç olarak, R.Hope çilek çeşidinin yaprak dokularının yüksek sıcaklık koşullarında CAT, APX ve PRX gibi enzim aktivitelerini arttırarak antioksidatif savunma sistemini harekete geçirmek ve 23 kDa HSP sentezlemek suretiyle hücre zarı yapısını koruyabildiği saptanmıştır.
In this study, the mechanism of tolerance to high temperatures was investigated in strawberry plants (Fragaria x ananassa Duch) using two strawberry cultivars which were high temperature tolerant (R. Hope) and sensitive (CG3). Seedlings were planted in 14x12 cm pots using perlite, peat and garden mold mix (1:1:1) and grown for 2 months at 30/15°C and 65% relative humidity. Then, gradually and shock high temperature tests (35, 40, 43, 46, 49, 52, 55 and 60°C) were applied to the leaf samples. To determine the cell damages that occurred after high-temperature applications, ion leakage tests were conducted and the accumulation of hydrogenperoxide (H2O2) was determined. The level of non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH), and some enzymatic antioxidants such as ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and peroxidase (PRX) enzyme activities were measured. In addition, to investigate protein metabolism and identify a protein marker in tolerance to high temperature of strawberry, total soluble protein profiles were determined using SDS-PAGE method and levels of HSP 60 proteins were analysed using immunoblotting method.As a result of high temperature applications, cellular injury was increase in parallel to the temperatures. AsA and GSH contents were varied according to the applications and temperatures. APX, CAT and PRX activities were increased by high temperature, but GR activity was not changed. On SDS-PAGE many protein bands were detected depending on the treatments. However anti-HSP60 immunoblots revealed that the 23 kDa polypeptides were detectable during heat-acclimation of strawberry cultivars. The 23 kDa HSP accumulated significantly in high temperature treatments in both cultivars. Indeed the intensities of the HSP in R. Hope were more than CG3. Therefore accumulation of 23 kDa HSP was correlated with heat-tolerance of the cultivars.In conclusion, strawberry leaf tissues of cultivar R.Hope were found to enhance the structural stability of cellular membranes under high temperature by increasing both the activity of such enzymes as CAT, APX and PRX to activate the antioxtidative systems and the expression of 23 kDa HSP.
In this study, the mechanism of tolerance to high temperatures was investigated in strawberry plants (Fragaria x ananassa Duch) using two strawberry cultivars which were high temperature tolerant (R. Hope) and sensitive (CG3). Seedlings were planted in 14x12 cm pots using perlite, peat and garden mold mix (1:1:1) and grown for 2 months at 30/15°C and 65% relative humidity. Then, gradually and shock high temperature tests (35, 40, 43, 46, 49, 52, 55 and 60°C) were applied to the leaf samples. To determine the cell damages that occurred after high-temperature applications, ion leakage tests were conducted and the accumulation of hydrogenperoxide (H2O2) was determined. The level of non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH), and some enzymatic antioxidants such as ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and peroxidase (PRX) enzyme activities were measured. In addition, to investigate protein metabolism and identify a protein marker in tolerance to high temperature of strawberry, total soluble protein profiles were determined using SDS-PAGE method and levels of HSP 60 proteins were analysed using immunoblotting method.As a result of high temperature applications, cellular injury was increase in parallel to the temperatures. AsA and GSH contents were varied according to the applications and temperatures. APX, CAT and PRX activities were increased by high temperature, but GR activity was not changed. On SDS-PAGE many protein bands were detected depending on the treatments. However anti-HSP60 immunoblots revealed that the 23 kDa polypeptides were detectable during heat-acclimation of strawberry cultivars. The 23 kDa HSP accumulated significantly in high temperature treatments in both cultivars. Indeed the intensities of the HSP in R. Hope were more than CG3. Therefore accumulation of 23 kDa HSP was correlated with heat-tolerance of the cultivars.In conclusion, strawberry leaf tissues of cultivar R.Hope were found to enhance the structural stability of cellular membranes under high temperature by increasing both the activity of such enzymes as CAT, APX and PRX to activate the antioxtidative systems and the expression of 23 kDa HSP.
Description
Keywords
Çilek, Fragaria x ananassa, Sıcaklık stresi, Antioksidan enzim, Protein, Strawberry, Heat stress, Antioxidative enzyme
Citation
Ergin, S. (2012). Yüksek sıcaklık stresinin çilek bitkisinde enzimatik ve enzimatik olmayan antioksidanlar ile protein metabolizmasına etkileri. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.