EIRPP: Early Intracellular Response Profiling of Plants Using Targeted Fluorescent Protein Probes

The Laboratory of Plant Development & Interaction Lab works in three major areas of plant biology shown below.
The major research program in the lab is aimed at generating an Early Intracellular Response Profile for higher plants.

As consumers, we realize that our future survival on earth relies on our capability to harness, carefully manage and optimize the productivity of plants. And yet, we do not truly understand plants very well. For example, we know that like all other living organisms, plants too suffer from stress; but we do not know the first indications of stress shown by a plant cell. We know that plants age, but we do not understand the earliest signs of aging shown by a plant cell; we know by experience that plants are susceptible to pests and diseases but we have only a hazy idea about the earliest response of a plant cell to invasion of its surface. In general, our understanding of plant response to a stimulus comes from our observations of plants usually taken long after the causal event has taken place. Consequently, most plant management practices are not preventative, they are designed to help in reducing losses after damage has been initiated. Our limited understanding of plants as living organisms cohabiting the world with us is partly because until now we have not been able to look inside living plants and see things as they were happening.

Our approach towards understanding plants and their inner workings is undergoing a rapid transformation as we rely increasingly on “live imaging” technology for visualizing, tracking and quantifying gene products in living plant cells. Fluorescent proteins such as the Aequorea victoria derived Green Fluorescent Protein and its color variants have played a major role in this revolutionary approach. Today a large number of transgenic plants expressing fluorescent probes targeted to different sub-cellular structures and compartments are available to plant scientists.

Our lab uses a judicious combination of Molecular-Genetic and Cell biological tools including numerous fluorescent transgenic marker lines and cytoskeletal mutants to understand organelle dynamics and subcellular interactions during plant development.

Our 'live-cell' visualization approach is aimed at generating an exhaustive Cell biological profile, backed by a dissection of Molecular-Genetic mechanisms underlying early subcellular responses of plants to diverse biotic and abiotic stimuli.

Sub-cellular 'living colour' Probes in <em>Arabidopsis</em>
Experimental Approach
Step 1
A. Creating transgenic lines in Arabidopsis and tobacco, carrying multiple targeted fluorescent markers for simultaneous multicolor live visualization of sub-cellular components. See multiple markers above and in Sinclair et. al 2006.

Step 2
B. Charting single / multiple organelle localization and behaviour using 4D-imaging and tracking techniques on normally developing, non- stimulated model cells to create base line parameters.

Step 3
C. Exposing wild-type plants to different abiotic (eg. temperature, salt, osmotic) and biotic (eg. wounding, bacterial / fungal infestation) stimuli. Observing conditional responses of different organelles and comparing them against the typical responses noted in non-stimulated model cells earlier.

Step 4
D. Treating wild-type plants to different cytoskeletal drugs to dampen cytoskeletal dynamics and then exposing them to abiotic and biotic stimuli specified in 'C'. Observing alterations in organelle response and sub-cellular response and compare against the typical wild-type responses noted in 'A', & 'B'.

E. Exposing mutant plants (eg. for ARP2/3-complex / ROP-GTPases/ MAPs) to abiotic and biotic stimuli. Observing sub-cellular response and comparing against the typical wild-type responses noted in 'A, B & C'.

Expected outcome
Documentation of early sub-cellular response of plant cells to diverse stimuli. Identifying common molecular mechanisms that might be triggered in response to completely different environmental stimuli. Laying down the ground-work for understanding the basic laws that must be involved in the creation of order from apparent chaos: a phrase that sums up and links sub-cellular behaviour to the development of a precise cellular response.

  1. NSERC funded project 046947.