Summer Research Opportunity in Genetics, Molecular Biology, & Plant Physiology
Chan Laboratory

What do we study

My research interest is in the area of calcium signal transduction. Calcium is arguably one of the most important signaling molecules in eukaryotes. In plants, similar to other eukaryotes, its cellular concentrations are carefully maintained at homeostatic levels by the coordinated activities of calcium uptake, extrusion and sequestration systems. Specific temporal and spatial changes in calcium concentrations are correlated with various changes in environmental and physiological conditions and their subsequent biological responses. My research focuses on the identification and characterization of various components of calcium transduction pathways. It is important for achieving a better understanding of many aspects of plant physiology as well as basic mechanisms of eukaryotic signal transduction. In particular, I concentrate on studying proteins that sense and transmit changes in cellular calcium concentrations using the model plant Arabidopsis thaliana as the experimental system. The two proteins that are the recent foci of my research are a calcium channel and a calcium-activated protein kinase.

What will student researchers do?

There are several on-going projects in my laboratory and the ones that are most suitable for summer projects are listed below.

(1) Identification and screening of plants which are hypersensitive to calcium in the growth medium

I have previously identified a calcium channel protein, CNGC2, that when rendered non-functional by mutations, causes a plant to become hypersensitive to external calcium ions. Such mutant plants are stunted in growth when calcium concentrations in growth media are nominally increased. To the best of my knowledge, this is the first calcium hypersensitivity phenotype reported in plants. My working hypothesis is that this calcium channel is responsible for sensing and subsequently initiating appropriate adaptive responses to high external calcium concentrations. To further to elucidate the mechanism of calcium-sensing and adaptation in plants, I would like to identify other proteins that participate in the same or parallel calcium signaling pathway as CNGC2. Preliminary data suggest that transcriptional changes of a number of genes accompany the mutant calcium hypersensitivity phenotype, and are candidate that for further investigation. If these genes are involved in calcium-sensing and adaptation, plants that are mutated in these genes may display calcium-dependent growth defects similar to the phenotype of CNGC2 mutants. I, along with students in my laboratory, am in the process of identifying and isolating individual mutant plants through the use of a molecular technique - polymerase chain reaction (PCR). I anticipate that the isolation of mutants will continue through the summer. Mutant plants that have been identified then be grown in agar- or soil-based medium supplemented with excess calcium salt, and their growth will be compared to wild-type plants grown in parallel. In this manner, we can screen for additional calcium-hypersensitive mutants. These mutants will then be targets for further investigation, including genetics and molecular characterization. Depending on our progress, we may be able to initiate some of these other studies towards the end of the summer project. .

(2) Rescuing a mutant phenotype through the introduction of an exogenous copy of the wild-type gene.

Another focus of my research involves the characterization of a calcium-activated protein kinase. Protein kinases are enzymes that add phosphate moieties to other proteins. This modification has been shown to affect the activities of the phosphorylated proteins and is a commonly utilized mechanism for transducing cellular signals in eukaryotes. My current data strongly suggest that a calcium-dependent kinase, CPK28, is essential for proper embryo development in plants. CPK28 may also be important for other aspects of plant growth, such as initiation and maintenance of root growth and pollen dispersal (for pollination). During the summer, I would like to expand my current studies on CPK28 in two areas, as briefly described below. Embryos that lack CPK28 are lethal. I plan to test if this lethality can be rescued, i.e. whether embryo viability can be restored, by providing a wild-type copy of CPK28 in trans. This can be achieved through the process of molecular transformation: The coding sequence of CPK28 is inserted into the plant genome through a naturally-occurring, bacteria-mediated transformation process. Progeny (seeds) of the transformed plants will be collected, grown and analyzed with a PCR-based technique to ascertain whether the rescue is successful. If the rescue is successful, it will serve as a very strong piece of evidence to support the hypothesis that CPK28 and therefore, proper calcium signaling, is required for embryo development in a higher plant. Preliminary data on the localization of CPK28 suggests that it may be important for several aspects of vegetative and reproductive growth. My students and I are in the process of generating 'CPK28 knock-down' plants - plants in which the expression level of CPK28 is reduced to different levels. During the summer, I plan to analyze the phenotype of these plants, focusing on potential defects on root growth and pollen dispersal. The frequency of root initiation, the rate of root growth and the percentage of pollen dispersed are assayed by growing the appropriate tissues on various agar media. Responses of the knock-down plants and the wild type will be compared and if systematic differences are found, we will try to correlate the difference with the expression level of CPK28. If CPK28 is indeed important for the physiological processes described above, plants with the least amount of CPK28 are predicted to display the most severe defects.

What kind of skills and knowledge will I take from this experience?

My laboratory uses a combination of molecular, genetics and physiological techniques to study fundamental and crucial signaling mechanisms in plants. Students in my laboratory typically participate in multiple, concurrent projects. I believe that students can benefit greatly by being exposed to a wide variety of techniques and biological problems at the same time. All students will be involved in library and on-line research, and are expected to participate in and contribute to discussion of current scientific literature that is relevant to our research projects. Teamwork is highly encouraged in my laboratory for it promotes learning and increases productivity.

Research Papers