Study of Alfalfa Leaf Proteome Response Under Biological Stress Conditions Caused by Alfalfa Leaf Weevil (Hypera Postica Gell.) Feeding Using Polyacrylamide Gel Electrophoresis, Isoelectric Focusing and Two-dimensional Electrophoresis

Abstract

Abstract Alfalfa is an important forage plant. Alfalfa leaf Weevil (Hypera postica Gell.) is considered a first-class pest of this plant, which causes a lot of damage every year, especially to the first layer of this plant. Knowledge about initial molecular signaling and proteins associated with sensing the damage of pests, especially the weevil pest in the alfalfa plant in among crop plants is limited. In this study, an attempt has been made to investigate the overall protein expression pattern of the leaf of this plant in response to the stress caused by the alfalfa leaf weevil (Hypera postica Gell.) using the proteomics technique, to take a step in investigating the resistance mechanisms of this plant to the aforementioned pest. For this purpose, a sample of stress (under pest feeding) and non-stress stage (Control) was obtained under the same growth conditions. In order to determine the significant difference in protein expression in control and stress conditions caused by alfalfa leaf weevil pest, t-test method was used. The extracted proteins were separated in two dimensions by IPG gels with a gradient pH of 4–7 and with length 18 cm and 12.5% acrylamide gels. The results of the statistical evaluation using Image Master 2D platinum of Melani 6 software showed that out of a total of 241 repeatable protein points, 28 protein points showed changes in expression in stress levels caused by alfalfa leaf weevil. These changes included increased and decreased expression. Mass spectrometry results led to the identification of proteins involved in stress response mechanisms, energy production, metabolism, synthesis and photosynthesis. The evaluation of different protein classes showed that the proteome responding to biological stress in this plant follows two distinct trends in terms of co-expression. The results showed that among the 28 protein spots with significant expression changes in the Yazdi genotype, most of them i.e. 17.85% were expressed for energy production and the same amount was expressed in response to stress in the plant. In general, the results showed that studying the amount of changes in the expression of individual proteins alone will not be the solution, but knowing the set of co-expressed proteins and studying the pattern of their collective changes in response to different levels of biological stress caused by alfalfa leaf weevil. It is very important and gives a better understanding. It is obvious that conducting more studies on other alfalfa genotypes can provide a suitable molecular model for modifying alfalfa leaf weevil resistance in other alfalfa genotypes. These results clarify our understanding of the underlying mechanisms in alfalfa plant tolerance to alfalfa leaf weevil.