Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Area of Science:

• Plant molecular genetics
• Calcium signaling
• Forward genetics

Background:

• Forward genetic screens are crucial for unbiasedly identifying genes in biological pathways.
• Cytosolic calcium (Ca2+) elevation is a vital early signaling response to stress.
• The molecular players (receptors, channels, pumps, transporters) in Ca2+ signaling remain largely unknown in many systems.

Purpose of the Study:

• To develop a forward genetic screen for identifying novel components of calcium (Ca2+) signaling in Arabidopsis.
• To utilize a Ca2+ reporter system for high-throughput screening of mutants with altered Ca2+ responses.

Main Methods:

• Generated random mutations in Arabidopsis using ethyl methane sulfonate (EMS) in a transgenic aequorin (Ca2+ reporter) background.
• Screened the M2 generation for altered Ca2+ responses using a 96-well high-throughput Ca2+ measurement protocol.
• Rescued and propagated mutants exhibiting the desired phenotype to obtain homozygous lines.

Main Results:

• Successfully established a forward genetic screening protocol in a Ca2+ reporter background.
• Identified several novel mutants with varying real-time Ca2+ responses.
• Demonstrated the capability to discover new Ca2+-regulated targets.

Conclusions:

• This protocol facilitates efficient forward genetic screens for Ca2+ signaling components in plants.
• The approach enables the discovery of previously unknown genes involved in Ca2+ regulation.
• The identified mutants provide valuable resources for understanding plant stress signaling pathways.