Cam-binding property and roles in stress response of myosin heavy chain in rice oryza sativa L.

Abstract

When plants encounter to environmental stimuli both biotic and abiotic stresses, plants will response using the signal transduction pathway by rapid increasing the concentration of calcium ion in the cytosol of plant cell. Calcium signals are then conveyed by the action of calcium sensors. Calmodulin (CaM), which is one of the calcium sensors, will bind to calcium ions and transmit the calcium signal by binding to and activating the target proteins within the cell. The activities of target proteins affect physiological responses to specific stimuli received by plant cells. To identify CaM function, it is necessary to find its target proteins. In previous study, the putative OsCaM1 target proteins were identified from rice (Oryza sativa L.) by cDNA expression library screening. One of the putative OsCaM1 target proteins is Myosin heavy chain. In this research, the yeast two-hybrid system was performed to examine the interaction of Myosin heavy chain with CaM and CML from rice. The result showed that Myosin heavy chain can interact only with OsCML4, OsCML5 and OsCML8 both in the presence and in the absence of calcium but not with OsCaM1. In the addition, in this research, the CaM Pull-down assay was performed to confirm the interaction of CaM with Myosin heavy chain. In the result, Myosin heavy chain was found to bind CaM in the presence of calcium. Moreover, the expression pattern of a Myosin heavy chain gene in rice under salt and drought stresses was investigated using real-time RT-PCR. In the two-week-old rice seedlings, this gene did not clearly exhibited lower expression level in the leaf tissues when rice plants exposed to salt stress. In the case of drought stress, the lower expression level in the leaf tissues of rice was detected (p<0.05). In addition, in this research, we have successfully generated four transgenic Arabidopsis lines overexpressing Myosin heavy chain gene including line C2, C6, E1 and E3. In order to characterize the role of Myosin heavy chain protein, the seed germination rate and percentage was observed. In the result, no significant change of the germination rate and percentage between wild-type and transgenic lines under both salt and drought stresses was detected. Next, fresh and dry weight of wild-type and transgenic lines were compared under both salt and drought stress conditions. We found that both fresh and dry weight measurement did not show significant difference in each stress treatment. When comparing the reduction percentage of fresh and dry weight, there was a decrease in the fresh weight reduction percentage in line E1 under both salt and drought stress conditions and only drought stress of line C6 was detected but, the dry weight reduction percentage was decrease only in drought stress condition of line C6 and E3.