MSc Exit Seminar – Van Phan -Friday, May 11th, 2018

MSc Exit Seminar

Friday, May 11^th, 2018 at 10:10am Earth Sciences Building, Room 3087

Van Phan (Yoshioka Lab)

Abstract:

Evaluation of GCaMP3 Protoplasts for the Analysis of Ca²⁺ Signaling in Plant Immunity

Cytoplasmic calcium [Ca²⁺]_cyt elevation is an early event that occurs after recognition of various environmental stimuli. Utilization of Ca²⁺visualization tools such as genetically-encoded Ca²⁺ indicators has advanced our knowledge of the temporal and spatial nature of Ca²⁺ signals. GCaMP3 is a green fluorescence protein (GFP)-based genetically-encoded indicator that can be detected by a conventional fluorescence microscope and a plate reader system. In this study, the use of GCaMP3 to study plant Ca²⁺ signals in protoplasts was evaluated.

Firstly, Arabidopsis thaliana leaf mesophyll protoplasts derived from stable transgenic lines expressing GCaMP3 were evaluated in comparison to leaf discs. Using fluorescence microscopy and a plate reader, Ca²⁺ signals upon abiotic and biotic stimuli in protoplasts were assessed in a quantitative and qualitative manner. Ca²⁺signals upon various stimuli were detected by both microscope and a plate reader. Ca²⁺ signals detected in protoplasts were generally quicker and stronger than those in leaf discs. However, the signal patterns were fundamentally similar between protoplasts and leaf discs. Thus, it was concluded that protoplasts expressing GCaMP3 can be utilized to study Ca²⁺ signaling.

Next, the usage of the transient expression of GCaMP3 in protoplasts (by transfection) was tested. This can be a powerful tool to analyze Ca²⁺signals in various mutants. Several methods to detect Ca²⁺signals from GCaMP3-transfected protoplasts were evaluated utilizing the bacterial elicitor, flg22. However, the detection by a plate reader was not successful, likely due to the insufficient number of the cells expression GCaMP3 by transfection, while some signals could be observed in individual protoplasts under the microscope.

Taken together, protoplasts can be used for investigating Ca²⁺signals upon various stimuli using GCaMP3. However, further optimization of the protocol for transfection is required.