Glow and behold: using transient expression of fluorescent proteins to study target plant proteins
This article was originally published as part of the ASPB Conviron Scholars program
When most people hear the term “GMO”, they think of the genetically engineered crops that are used for food, fuel and fiber. While these types of GMOs are beneficial to consumers and farmers, there is a largely undiscussed category of genetically engineered plants that are of immense value to plant biologists. As a plant molecular biologist, I use genetically modified versions of model organism Arabidopsis thaliana discover the location and function of proteins.
The same principles and methods used for making genetically engineered crops also apply to plant research. We can remove, edit and add genes to plants in precise ways that let us study the effects of the modification. In other words, if we change nothing else in a plant’s genome except one gene, any changes to how the plant grows can be attributed to that one gene. This approach is referred to as reverse genetics, where scientists start with the gene and discover the biological trait, or phenotype, associated with it.
The germline can be engineered such that the organism’s genome is permanently altered. The seeds from the engineered plant will also have the modification. Conversely, special lab techniques allow for temporary expression of specific genes. We can create a temporary “GMO” that gives us an otherwise impossible glimpse into plant biology. Engineering a plant’s genome can take months to complete, but transiently expressing genes can be done in a matter of hours.
Since DNA has the same structure in all living organisms, we can in theory use a gene from any species for use in plants. Plant scientists must carefully design gene “constructs” that can be inserted for the desired effect. These constructs are usually made in a bacterial plasmid. Bacterial plasmids are circular strands of DNA that contain several useful genes. First, they include genes necessary for growth in its host species. Second, plasmids have antibiotic resistance markers that allow biologists to isolate only cells that successfully carry the plasmid. Together, these components comprise of the plasmid’s “backbone.” Biologists can add other DNA sequences to the plasmid backbone to fit their experiment.
You might be surprised how simple it is to transiently express plasmid DNA in living Arabidopsis thaliana leaf cells. To get the DNA into the cell, the outer layer of the leaf is removed to expose the mesophyll cells inside. Next, the cell wall is digested using a special enzyme solution. After a few hours, you can see tiny living cells floating in solution. Finally, the plasma membrane is made permeable with another special mixture that allows the plasmid DNA to be incorporated. The cells are left to incubate overnight and can be studied the next day (Wu et al. 2009).
Fluorescent proteins have the fascinating ability to emit a strong signal when excited by light of certain wavelength. The fluorescent protein “reports” its location when excited by a laser at a specific wavelength. The first fluorescent protein was discovered in the 1960’s while studying a species of bioluminescent jellyfish. It was isolated and named Green Fluorescent Protein (GFP). Today, there are several engineered versions of GFP that have a spectrum of excitation and emission values. We can exploit the differences in excitation/emission properties of fluorescent proteins to express more than one protein-reporter plasmids in a single cell (Kremers et al. 2011; Nelson et al. 2007).
The temporary expression of protein-protein fusions from plasmid DNA can be visualized by fluorescent microscopy. Specifically, scanning laser confocal microscopy allows us to visualize the protein of interest fused to the fluorescent protein. Essentially, a laser hits a very thin layer of cells on a microscope slide and excites the fluorescent proteins. Using more than one laser at a time excites more than one fluorescent protein. The cells “glow” and reveal the location of the protein of interest!
The figure below shows how fluorescent reporter proteins help determine the subcellular location of a protein. My colleague Chris Yuen transformed Arabidopsis thaliana mesophyll cells with two different plasmid constructs. The protein of interest is Protein Disulfide Isomerase-7 (PDI7), a protein-folding enzyme whose subcellular location was previously unknown (Panel 1). The PDI7 protein was fused to GFP and the control protein was fused to mCherry, a modified version of Red Fluorescent Protein. The control protein is designed to only be found in the endoplasmic reticulum (ER) (Panel 2).
When we merge the two images created by the different fluorescent proteins, we see the overlap of PDI7 with the ER. Complete overlap is an orange-yellow color. The third panel shows that some, but not all of the PDI7 proteins found in the cell, overlap with the ER. As the paper discusses in detail, PDI7 likely cycles between the ER and cis-Golgi bodies (Yuen et al. 2017) .
Fluorescent reporter proteins are a popular approach to finding the location of a previously unstudied protein, as well as seeing if two proteins reside in the same part of the cell. In certain applications we can even quantify the amount of one protein relative to another. These “GMO” Arabidopsis cells will never make it to the supermarket, but they and other genetically engineered plants used only for research are invaluable to our understanding of plants.
References and further reading:
Kremers, G.-J., Gilbert, S. G., Cranfill, P. J., Davidson, M. W., Piston, D. W. 2011. Fluorescent proteins at a glance. J Cell Sci 124: 157-160.
Nelson, B. K., Cai, X., Nebenführ, A. 2007. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. The Plant Journal 51(6): 1126–1136.
Wu, F. H., Shen, S. C., Lee, L. Y., Lee, S. H., Chan, M. T., & Lin, C. S. 2009. Tape-Arabidopsis sandwich - A simpler Arabidopsis protoplast isolation method. Plant Methods, 5(1): 1–10.
Yuen, C. Y. L., Wang, P., Kang, B.-H., Matsumoto, K., Christopher, D. A. 2017. A Non-Classical Member of the Protein Disulfide Isomerase Family, PDI7 of Arabidopsis thaliana, Localizes to the cis-Golgi and Endoplasmic Reticulum Membranes. Plant and Cell Physiology 58(6): 1103-1117.