Biochemical & protein interaction analysis of calcium-sensing proteins CML 15 and CML 16 in Arabidopsis thaliana

Matt Watson Supervisor: Dr. Wayne Snedden, Committee Members: Dr. Sharon Regan & Dr. Virginia Walker

METHOD: Protein purification & calcium-dependent hydrophobic affinity chromatography

INTRODUCTION

METHOD: Western Blotting

OBJECTIVE 1: Generation of recombinant CMLs for use in affinity chromatography.

v  Recombinant CML 15 & CML16 were successfully purified in a manner consistent with canonical Ca2+ sensors such as CaM

v  CML 16 has detectable expression in 10-day WT seedling tissue from Arabidopsis thaliana Col-0 WT

v  Evidence that CML 16 has putative binding targets within the CD4-30 library, suggesting that a positive target has been isolated from floral tissue. This target will need to be further corroborated with further in vitro and in vivo methods.

This study has set the foundation for future study into the downstream targets of CML 15 & 16. Future work into these CMLs should focus on the development of affinity columns using recombinant CMLs, as well as corroboration of putative targets from the yeast 2 hybrid screen.

CONCLUSIONS METHOD: Yeast 2 hybrid screen- use of a bait CML vector to identify putative binding interactions with a prey vector from a floral cDNA library

.

OBJECTIVE 3: Identify tissue-specific expression patterns of CML 16.

RESULT: Conjugation of both recombinant CMLs to phenyl-Sepharose reconfirmed their characteristics

as putative Ca2+ sensors & produced soluble protein fractions for affinity chromatography.

RESULT: Western Blotting confirmed detection of CML 16 in 10-day old seedling tissue. Evidence of

CML 16 expression provided the rationale for its use in future tissue-specific assays.

RESULT: Putative binding interactions were observed between CML 16 (pGBKT7 vector) and targets in the CD4-30 floral library, suggesting a putative binding

target for CML 16 in floral tissue.

Fig. 2. SDS-PAGE gels showing protein purity of recombinant CMLs purified by calcium-dependent hydrophobic affinity chromatography (CML affinity columns have not yet been generated).

Fig. 5. Western Blot on PVDF membrane showing quantitative expression patterns of CML 16 for 10-day seedling (positive control), root, and recombinant protein samples. Wild-type (WT) Colombia Ecotype (Col-0) tissue was used.

Fig. 3. Original yeast transformation on –LTAH dropout media, Plate C, plated with CML 16 & CD4-30 floral library.

OBJECTIVE 2: Identify putative binding targets for CML 16 in a floral library.

Fig.4. Yeast re-streak of original transformation on –LTAH dropout media, Plate C, plated with CML 16 & CD4-30 floral library.

Within plants such as Arabidopsis, there exists a known superfamily of 50 calmodulin-like proteins (CMLs)4, of which little biochemical or physiological information currently exists. Specifically, the understanding of the downstream binding targets of these CMLs remains unknown, producing a gap in the understanding of their roles within plant responses.

The aim of this study is to develop and employ the use of molecular tools for the

identification of downstream targets of two CMLs, CML 15 and CML 16.

REFERENCES 1. Yang, T., & Poovaiah, B. W. (2003). Calcium/calmodulin-mediated signal network in plants. Trends in plant science, 8(10), 505-512. 2. Snedden, W. & Fromm, H. (2001). Calmodulin as a versatile calcium signal transducer in plants. New Phytologist 151, 35–66. 3. DeFalco, T.A., Bender, K.W. & Snedden, W.A. (2010). Breaking the code: Ca2+ sensors in plant signaling. The Biochemical Journal 425, 27-40. 4. McCormack, E. & Braam, J. (2003). Calmodulins and related potential calcium sensors of Arabidopsis. New Phytologist 159, 585-598.

Acknowledgments I would like to acknowledge Dr. Wayne Snedden for providing me with this project opportunity, as well as Deni Ogunrinde for her guidance and mentorship during the course of this research project. Thank you to Dr. Sharon Regan & Dr. Virginia Walker for their committee support.

Fig. 1. Working model of Ca2+ signalling transduction pathways in eukaryotic organisms.

Plant cells employ the use of calcium ion concentration-specific gradients to illicit a physiological response to external stimuli1. To interpret these calcium signatures that ultimately produce cellular responses, plants make use of calcium-binding proteins, termed ‘calcium sensors’, such as highly conserved protein calmodulin (CaM)2. These Ca2+ sensors are integral to the ability of the plant cell to make calculated responses to environmental changes/cues by transmitting encoded information down a signal cascade to modulate cellular activity. Current working models show reversible binding of Ca2+ ions to these sensors3, producing conformational changes that transduce information down a signal pathway.

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