Signal Transduction
Background Information:
Our laboratory is interested in understanding the fundamental principles that underlie signal transduction. Signal transduction in the process by which information flows through a living system. We have chosen to explore signal transduction using the plant species Arabidposis thaliana. Arabidopsis is an excellent genetic model system that offers many unique benefits as an experimental subject: it has a small, fully sequenced genome; knockout mutants are readily available; the generation time is short; genetic crosses are easy and quick; genome-wide expression analysis chips are commercially available; e.t.c.
Most of the experimental strategies that we use in our lab fall under the category of "Functional Genomics". This buzz-word is used to refer to a new way of approaching biological questions in which the whole-genome sequence of an organism serves as the starting point for planning and implementing experiments. One of the exciting aspects of Functional Genomics is that it allows one to imagine and perform experiments that were unthinkable in the pre-genomic era. Because it is such a new field of study, the opportunities for exciting research within Functional Genomics are abundant.
Reverse-genetics is the area of Functional Genomics in which our laboratory specializes. The term "Reverse-genetics" refers to an experimental strategy where you are given the sequence of a gene and you work towards describing that gene's function. The first step in the process is to isolate an individual organism in which the gene of interest has been "knocked-out". These individuals are then grown under a variety of environmental conditions and carefully screened for the presence of abnormal phenotypes. The characteristics of any mutant phenotypes that do arise allow one to build an understanding of the gene's function.
We have chosen to focus our reverse-genetic efforts on a group of Arabidopsis genes that encodes MAP kinase cascade components. MAP kinase cascades are a found in all eucaryotic cells and play a central role in many signal transduction pathways. Although MAP kinase cascades have been well studied in yeast, we have only just begun to understand how these signaling modules operate within multi-cellular organisms. Some of the processes thought to involve MAP kinase signaling in plant cells include oxidative stress responses, pathogen responses, the regulation of cell division, ethylene responses, and numerous other unknown signaling pathways.
The diagram below presents a simplified view of a generic signaling pathway that includes a MAP kinase cascade. The MAP kinase cascade itself is composed of three interacting protein kinases: a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). Signals from various upstream receptor molecules lead to the activation of the MAP3K, causing it to phosphorylate the MAP2K, which in turn phosphorylates the MAPK. This activated MAPK then phosphorylates a number of downstream targets.
The genome of Arabidopsis contains twenty-three MAPK, nine MAP2K, and sixty MAP3K genes (1,2). Because individual MAPK cascade components can participate in more than one signaling pathway, it is expected that there will be a large number of distinct MAPK cascades operating within Arabidopsis plants. Characterizing the function and identity of each of these MAPK pathways is an exciting challenge that will encompass a variety of interesting areas of plant biology.
Our current research is focused on the MAP3K's. Although there are sixty MAP3K genes in Arabidopsis, only two of these loci have emerged from forward-geneticscreens (3,4). This situation suggested that functional redundancy might be masking the appearance of mutant phenotypes for most of the MAP3K genes. In order to test this hypothesis, we used reverse-genetics to directly investigate the function of the MAP3K genes ANP1, ANP2, and ANP3 (5). The phylogenetic tree of a subset of the 60 MAP3K genes shown in Figure 2 demonstrates that the ANPs form a distinct sub-family of MAP3K's in Arabidopsis.
We isolated knockout mutations for each of the ANP genes by screening large populations of T-DNA transformed Arabidopsis plants. The procedure used to screen for knockouts was originally developed by myself, Jeff Young, and Frans Tax while working as post-docs in the laboratory of Mike Sussman (6,7). This PCR-based method has proven to be extremely valuable and forms the basis of the Arabidopsis Knockout Facility at the University of Wisconsin. This NSF-funded service facility provides scientists world-wide with access to Arabidopsis knockout mutations, and has been used by over 500 principal investigators.
We determined that single-mutant plants for each of the ANP loci were phenotypically normal. This result was consistent with our hypothesis that functional redundancy could be masking the appearance of mutant phenotypes for these genes. To test this possibility we attempted to create the various double- and triple-mutant combinations of the three ANP loci by performing genetic crosses. All of the double-mutant combinations were created; however, no triple mutants could be made. We later determined that the triple-mutant is lethal in both gametes, thereby preventing its isolation in adult plants.
Analysis of the phenotypes displayed by the three ANP double-mutant combinations revealed that each double-mutant gave a different phenotype. A summary of the phenotypes is provided in the table below:
| Genotype | Phenotype | Organs affected |
| anp1 / anp2 | no mutant phenotype | - - - |
| anp1 / anp3 | cell division defects | flowers |
| anp2 / anp3 | cell division defects; reduced plant size | whole plant |
The main cellular defect seen in the double-mutant plants was a disruption of cell division. The scanning electron microscope images below demonstrate the effect that disrupted cell division has on the epidermal cell layers in flowers and hypocotyls:
In addition to the structural defects shown above, we also documented molecular differences between wild-type plants and the double-mutants. Using DNA microarray technology we performed genome-wide gene expression analysis to search for differences in RNA levels between wild-type plants and anp2/anp3 double-mutants. This analysis revealed that a large number of genes related to stress response and pathogen response are up-regulated in the anp2/anp3 double-mutants when compared to wild-type. It is not clear at this point if this up-regulation is a direct consequence of the anp2/anp3 mutations, or if it reflects a more indirect consequence that stems from the structural changes caused by the ANP mutations. A more detailed description of our work with the ANP gene family has been published (Krysan et al, 2002). In addition, a feature article by the Plant Cell editorial staff provides an outside perspective on our work with this gene family (In This Issue, Plant Cell).
Literature Cited
1. Tena G, Asai T, Chiu WL, Sheen J. 2001. Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol. 4(5):392-400..
2. MAPK Group (Kazuya Ichimura et al.). 2002. Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci. 7(7):301-8.
3. Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR. 1993. CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell. 72(3):427-41.
4. Frye CA, Tang D, Innes RW. 2001. Negative regulation of defense responses in plants by a conserved MAPKK kinase. Proc Natl Acad Sci U S A. 98(1):373-8.
5. Krysan, P.J., P.J. Jester, J.R. Gottwald, and M.R. Sussman. 2002. An Arabidopsis MAPKK kinase gene family encodes essential positive regulators of cytokinesis. Plant Cell 14:1109-20.
6. Krysan, P. J., J. C. Young, F. Tax, and M. R. Sussman. 1996. Identification of T-DNA insertions within Arabidopsis genes involved in signal transduction and ion transport. Proceedings of the National Academy of Sciences (USA) 93:8145-8150.
7. Krysan, P. J., J. C. Young, and M. R. Sussman. 1999. T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283-2290.