PhD Exit Seminar – Thomas DeFalco (Yoshioka lab)

PhD Exit Seminar

Friday December 2^nd, 10:10 am – Ramsay Wright Building, Rm. 432

Thomas DeFalco (Yoshioka lab)

“Regulation of Cyclic Nucleotide-Gated Channels and Plant Calcium Signaling”

Abstract

Plants must coordinate developmental processes and responses to environmental stimuli via signaling at the cellular level. Ca²⁺ serves a common second messenger in many signal transduction pathways, wherein increases in cellular Ca²⁺ levels are interpreted by a suite of downstream Ca²⁺-binding sensor proteins, such as calmodulin (CaM). While Ca²⁺ is known to mediate responses to diverse stimuli in plants, including biotic stress, comparably little is known regarding the channels involved in Ca²⁺ signaling or their regulation. Cyclic nucleotide-gated channels (CNGCs) represent one of the major classes of Ca²⁺-permeable channels thought to mediate Ca²⁺ flux in plants, and several isoforms of the 20-member Arabidopsis CNGC family, including CNGC12 and CNGC2, have been implicated in immune responses. In this thesis, I present a thorough analysis of the regulation of Arabidopsis CNGCs by CaM, and demonstrate that CNGC12 function is both positively and negatively regulated by CaM-binding to multiple, distinct sites at the cytosolic termini of the channel. My results with CNGC12 indicate that plant CNGCs are likely subject to complex regulation by Ca²⁺, suggesting that CaM is required for both channel function and feedback inhibition.

My findings with CNGC12 are expanded to the entire 20-member CNGC family, and I have shown that Arabidopsis CNGCs possess diverse CaM-binding sites, both in terms of number and mode of CaM-binding. In particular, the IQ motif appears to be broadly conserved across the CNGC family, and in most members, mediates permanent association with CaM via Ca²⁺-independent interaction with the CaM C-lobe. These data indicate that CaM functions as a Ca²⁺-sensing subunit of CNGC complexes in plants.

Finally, I developed transgenic Nicotiana benthamiana and Nicotiana tabacum (tobacco) lines expressing the fluorescent Ca²⁺ indicator, GCaMP3, which can be used to visualize and measure Ca²⁺ signals in response to diverse abiotic or biotic stimuli. I examined the dynamics of Ca²⁺ signaling in response to stimuli including cold-shock, mechanical wounding, and pathogen-associated molecular patterns (PAMPs). These plants represent an excellent tool to dissect the molecular components of Ca²⁺ signaling via gain- or loss-of-function studies.

Ramsay Wright is a wheelchair accessible building.