Arabidopsis Thaliana’da Tuz Ön Uygulaması ile Bor Tolerans Kazanımında Antosiyaninin Çoklu Fonksiyonel Rollerinin Araştırılması

Abstract

In the scope of this thesis, it is aimed to elucidate the acquisition of boron tolerance by salt pretreatment in the model plant Arabidopsis thaliana by investigating 1) some physiological, photochemical and antioxidant enzyme activities and 2) the changes in some genes expression involved in the synthesis pathway of anthocyanin which has multiple functions.

After Arabidopsis seeds were subjected to stratification in petri dishes containing MS medium for 3 days at +4°C in dark conditions, the germinated plants were grown in a controlled growth chamber (22± 1°C temperature, 16/8 photoperiod, µmol.m-2s-1 light intensity and 50-60% humidity) for 12 days. Then, the pre-treatment groups were transferred to media containing NaCl-type salt for 3 days, and at the end of 3 days, they were transferred to petri dishes containing different boron concentrations (3 and 5 mM H3BO3) together with the groups without pre-treatment and exposed to boron toxicity for 7 days.

Due to boron accumulation in Arabidopsis plant, shoot biomass and leaf area decreased. In addition to this, boron toxicity caused an increase in Cl- uptake and a decrease in K+ uptake with boron accumulation in shoots of Arabidopsis plants. Toxic levels of boron transported to leaves adversely affected photosynthetic apparatus and photosynthetic activity. Boron toxicity led to changes in fluorescence transitions (Kaustky curve), specific (ABS/RC, TRO/RC, ETO/RC, DIO/RC, RE/RC) and phenomenological (ABS/CS, TRO/CSO, ETO/CSO, DIO/CSO) energy flows in thylakoid membranes, quantum yield (ϕEO), the efficiencies of the donor side(VK/VJ) of PSII and the acceptor (ΔVIP) side of PSI. These changes caused significant reductions in photosynthetic performance (PIABS / PITOP) and components. It has been revealed that the decrease in the photochemical efficiency of plants under boron toxicity is associated with the decrease in the active reaction center and the increase in the loss of energy in the form of heat. In addition, it was determined that B toxicity affects both photosystems negatively and PSI is affected more than PSII. Although this negative effect caused by boron toxicity in the model plant affected the membrane integrity and the amounts of pigments in the antenna and active reaction centers, it has been determined that this effect was not at a level to cease the functionality of the photosystems and could be alleviated with salt pre-treatment. These findings are also consistent with the MDA results and the change in biomass.

Arabidopsis plant gained tolerance against boron toxicity by regulating its protective defense systems with the increases in antioxidant enzymes activities and the content of phenolic compounds (anthocyanin and flavonoid) with salt pretreatment. The significant and coherent increase in the activity of antioxidant enzymes, especially POD, at toxic boron levels has showed that it plays an active role in the detoxification of H2O2 resulting from oxidative damage. Besides, increases in the activities of APX, GR and GST, which are involved in the ascorbate-glutathione pathway, have been involved in protecting the plant against damages. The activities of all these enzymes were higher in salt pre-treated groups.

Multiple functions of anthocyanin in defense, the changes in expression of some genes and transcription factors involved in anthocyanin synthesis were investigated. Expressions of MYB75, MYB114 and MYBD, which are transcription factors, increased under toxicity. Expression of PAL2, 4CL3, C4H genes involved in the synthesis pathway increased, but no difference was observed in the ANS gene. The increase of expression of GSH1 and GST26 genes might have played a role in protection against oxidative damage by forming anthocyanin-GSH and/or GST-anthocyanin complexes and binding the boron element.

It has been exhibited that salt pre-treatment has been inducing the defense system of plants before they encounter destructive toxicity and, it provides tolerance to this situation when it encounters high concentrations of boron. In addition, the increased expression amounts in anthocyanin-related genes which are involved in the anthocyanin synthesis pathway show that this system plays an important role in defense.