Investigation of epigenetic changes in chickpea plants upon alkali stress by arbuscular mycorrhizal fungus (AMF) application

Alkaline stress negatively affects growth, yield, and development, particularly in chickpea (Cicer arietinum L.), a legume. While the effects of alkaline stress on chickpea and the roles of arbuscular mycorrhizal fungi (AMF) in chickpea plants have been discussed separately in the literature, studies investigating the combination of alkaline stress and AMF within the framework of epigenetic approaches are limited. In this study, epigenetic changes and their biological implications were investigated in Damla-89 and Aydoğan chickpea genotypes grown under control (0 mM) and 100 mM Na₂CO₃ alkaline stress concentrations, following AMF application. The effects of AMF application under alkaline stress conditions on morphological, biochemical, elemental content, and DNA methylation level were evaluated. The results showed that in both varieties, the AMF+100 mM alkali stress combination increased AMF spore density and shoot and root lengths. Biochemical analyses revealed an increase in proline content in AMF+100 mM alkali stress application, while SOD, POD, and APX activities varied depending on the applications and genotypes. Elemental analyses showed an increase in Na, Cu, Fe, Ni, and P content. Within the scope of CRED–iPBS analyses, 11 different iPBS primers were evaluated and their polymorphism levels were determined. According to the analysis results, polymorphic band profiles were detected in almost all primers. The primers with the most pronounced polymorphism were found to be iPBS-2083, iPBS-2377 and iPBS-2400. Furthermore, it revealed that changes related to DNA methylation occurred in all applications; there was an increase in the total number of bands obtained with the MspI and HpaII enzymes compared to the control group. Furthermore, an increase was observed in leaf and root tissues, particularly in Class IV band types, while a general increase was determined in HMO, FMO, and TMO (%) values; however, a decrease was detected in HMO values in both genotypes.The findings indicate that AMF application can contribute to stress tolerance in chickpea plants under alkali stress conditions through epigenetic modifications.