Calcium Signaling in Arabidopsis Thaliana

My lab was sent Arabidopsis thaliana seeds by another lab and asked to bulk the lines and analyze their phenotypes. In practice, “bulking the lines” entails a lot of plant care: planting seeds on plates, transplanting them to soil, watering, tying up unruly stems, and harvesting tissue. As the plants develop, we try to observe potential phenotypes that change their growth or appearance. Once we harvest tissue, we can genotype the plants. We do this by extracting DNA from leaves, running it through PCR, and then screening them for their tDNA insert. Essentially, we want to double-check that the genotype is actually present for any phenotypes we suspect. The lines we’ve been given that we are most interested in are knockouts such as calcium-dependent protein kinase 3 (CPK3) and CPK6. We screen our PCR products on a gel to see if they return a band for the tDNA insert, and then we’ll move on to imaging tissue.

Calcium is an important signaling molecule in plants, especially in stimulus-response pathways. Many unknowns exist about how calcium works, especially its transduction pathways’ specific, mechanistic details. However, it is suspected that when the plant is stressed or initiating an immune response, CPKs mediate phosphorylation events that increase the rates of stomatal closure, likely through the upregulation of slow anion channels.  Calcium is part of a concert of biomolecules that work toward stomatal closure. Whether calcium signals come before or after other important players in the plant immune response, such as abscisic acid (ABA), has not been determined. Other calcium-independent processes could also be more responsible for the closure of stomata. My project aims to discover as much as possible about the calcium-related phenotype for the mutant lines that we were sent and, in doing so, come to a better understanding of the role calcium plays in the plant immune system and cytoskeletal response (such as stomatal closure).

To accomplish this, we can compare the calcium signaling in wild-type Arabidopsis to the mutant lines we were sent. The plant samples have a  genetically-coded indicator, R-GECO1. This molecule functions as a biosensor, allowing us to pick up on calcium signaling because the R-GECO1 is adapted from red fluorescent proteins (RFPs) that fluoresce when excited by calcium. While imaging under the confocal, as I spoke about in my last post, we can induce an immune response by treating our tissue sample with the pathogen flagellum protein flg22. The result from the microscope is a video where you can see a wave of calcium cross through the plant cells, which we call calcium’s spatiotemporal dynamics. Image processing techniques can be used to gather statistics about the response to quantitatively compare between samples. After we’ve genotyped and screened all samples, we’ll select the most representative ones from each line to subject to further experimentation and see how each mutant line’s calcium signaling behavior differs, if at all, from the wild-type. For example, if the CPK3 knockout line has significantly suppressed calcium signals and resulted in reduced stomata closure, it could be a clue that CPK3 is an important part of the pathway. If the CPK3 mutant and the wildtype have very similar calcium responses under the confocal, it could be that CPK3 is downstream completely of the calcium response.