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American Society of Plant Biologists
Good Things Come in ThreesA Trio of Triple Kinases Essential for Cell Division in Arabidopsis
The mitogen-activated protein kinase (MAPK) phosphorylation cascade is a highly conserved signal transduction mechanism that plays a key role in regulating many aspects of growth and development in eukaryotes. In plants, MAPK cascades have been associated with hormonal, abiotic stress, and disease defense responses and with the regulation of the cell cycle (Tena et al., 2001
Krysan et al. (2002)  isolated single, double, and triple knockout mutants of the three genes ANP1, ANP2, and ANP3, which constitute a distinct branch of MAPKKKs in Arabidopsis. The three genes were found to have overlapping or partially redundant functions, because no obvious phenotypes were observed in homozygous single-mutant plants of any of the genes. The anp2 anp3 double mutant, which showed the strongest phenotype of the various double-mutant combinations, suggested a positive role for the kinases in the control of cell division. The anp2 anp3 double mutants showed an overall reduction in plant size compared with the wild type, and defective cell growth was observed in the epidermis of hypocotyls and floral organs using scanning electron microscopy. Transmission electron microscopy showed characteristics of a disruption in cytokinesis in anp2 anp3 hypocotyls and embryos, such as the presence of cell wall stubs and binucleate cells (Figure 1) . Interestingly, anp1 anp3 double mutants did not show an obvious macroscopic phenotype, but scanning electron microscopy revealed defective cell growth in floral organs that was similar to or more severe than that of the anp2 anp3 double mutant.
The ANP genes were isolated by Nishihama et al. (1997) on the basis of their high degree of similarity to the tobacco MAPKKK protein NPK1, which also appears to function in the regulation of cell division. Nishihama et al. (2001) found that NPK1 was required for cell plate formation during cytokinesis, because expression of a kinase-negative mutant of NPK1 resulted in the production of multinucleate cells with incomplete cell plates. These results are similar to the phenotype of the anp2 anp3 double mutant in Arabidopsis, suggesting that ANP and NPK1 genes may share the same functions.
There also is evidence linking ANP/NPK1 gene function to various stress responses. Kovtun et al. (2000) found that ANP1 initiated an H2O2-mediated MAPK cascade in an Arabidopsis protoplast system that was linked to activation of the stress response genes encoding a glutathione S-transferase (GST6) and a heat shock protein (HSP18.2) and to repression of the auxin-responsive GH3 promoter. Furthermore, expression of constitutively active NPK1 in transgenic tobacco resulted in enhanced tolerance to freezing, heat, and salt stress (Kovtun et al., 2000).
Krysan et al. (2002)
It is clear that signals are perceived and transduced within complex networks of interacting pathways in higher plants and other eukaryotes. MAPK cascade components represent prime candidates for signaling components that participate in more than one signal transduction pathway. MAPK cascades are defined by the three kinases that make up the core module. Eukaryotic genomes typically contain relatively large families of the three types of kinases, so the specificity of a module may lie with specific combinations of module components. MAPKKs are restricted to interaction with specific subsets of MAPKs, but MAPKKKs, which receive inputs from the primary signal, evidently are capable of mixing and matching with many different MAPKK-MAPK combinations (Widmann et al., 1999 ). It is not surprising to find that MAPKKK gene families are frequently the largest of the three, which may allow for a diversity of incoming signals from different stimuli to feed into specific MAPK cascade modules. The Arabidopsis genome, for example, contains some 23 MAPKs, 10 MAPKKs, and >25 MAPKKKs (Tena et al., 2001).
In Arabidopsis, the MAPKs MPK3 and MPK6 may be involved in more than one pathway. Kovtun et al. (2000)
A well-characterized example of the specificity of the MAPK core module, but individual components playing a role in more than one pathway, can be found in the yeast Saccharomyces cerevisiae (for review, see Widmann et al., 1999 Although the activities of FUS3 and KSS1 are at least partially redundant (e.g., expression of KSS1 can complement the mating defect induced by the loss of FUS3 in genetic experiments), the in vivo activities of these proteins appear to be restricted mainly to the separate mating response and nutrient deprivation pathways, based on patterns of gene expression and protein activity in wild-type cells. In addition, MAPKKKSTE11 has been found to be part of a MAPK module that includes MAPKKPBS2 and MAPKHOG1 and is induced by high osmolarity. Thus, specificity for a response does not lie solely with the MAPKKK in a module, and clearly, other factors must be involved in regulating the interaction of a MAPKKK with different MAPKK-MAPK combinations.
This example also may serve as a caveat for understanding ANP gene function in Arabidopsis. Although the three ANP genes have at least partially overlapping functions that can compensate for the loss of any one of the genes, the primary function of each of these genes may turn out to be quite different in wild-type plants in vivo. Furthermore, a single ANP protein could be found to participate in more than one response, as is the case for STE11 in Saccharomyces. Although we have just begun to understand how MAPKs operate in plant cell signaling networks, the work of Krysan et al. (2002) The observation that different double-mutant combinations displayed developmentally distinct patterns of the mutant phenotype provides an opportunity to explore how cellular context affects the activity of different members of this gene family. For example, it will be of interest to determine why the anp1 anp3 double mutant shows obvious defects in cell division only in flowers, whereas the anp2 anp3 double mutant is affected throughout development. It also will be important to determine the nature of ANP involvement in plant stress responses. This information may help us understand how effective signaling networks may arise from functional redundancy within gene families.
The work of Krysan et al. (2002) provides a valuable example of how systematic phylogeny-based gene knockouts can be used to understand gene function. The ANP genes were chosen for systematic knockout investigation because analysis of the complete Arabidopsis genome showed that these three genes make up a distinct branch of the MAPKKK phylogenetic tree (Jouannic et al., 1999). Krysan et al. (2002) show that although they have partially redundant functions, collectively, the three genes in this family appear to be essential for viability, because no triple mutants were obtained. Interestingly, every homozygous double mutant isolated at any two of the three loci was found to be homozygous wild type at the third locus (i.e., no plant homozygous for two mutant loci and heterozygous for the third was identified), despite the fact that the three loci are not linked genetically. This finding suggested that triple-mutant haploid gametes are inviable. Obviously, this approach is not limited to genes involved in MAPK signaling. Rather, it represents a generic approach to understanding gene function in the face of functional redundancy, and it further demonstrates the value of complete genome sequencing.
Asai, T., Tena, G., Plotnikova, J., Willmann, M.R., Chiu, W.-L., Gomez-Gomez, L., Boller, T., Ausubel, F.M., and Sheen, J. (2001). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977–983. Jouannic, S., Hamal, A., Leprince, A.S., Tregear, J.W., Kreis, M., and Henry, Y. (1999). Plant MAP kinase kinase kinases structure, classification and evolution. Gene 233, 1–11.[Medline]
Kovtun, Y., Chium, W.L., Tena, G., and Sheen, J. (2000). Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. USA 97, 2940–2945.
Krysan, P.J., Jester, P.J., Gottwald, J.R., and Sussman, M.R. (2002). An Arabidopsis mitogen-activated protein kinase ki-nase kinase gene family encodes essential positive regulators of cytokinesis. Plant Cell 14, 1109–1120. Nishihama, R., Banno, H., Kawahara, E., Irie, K., and Machida, Y. (1997). Possible involvement of differential splicing in regulation of the activity of Arabidopsis ANP1 that is related to mitogen-activated protein kinase kinase kinases (MAPKKKs). Plant J. 12, 39–48.[CrossRef][ISI][Medline]
Nishihama, R., Ishikawa, M., Araki, S., Soyano, T., Asada, T., and Machida, Y. (2001). The NPK1 mitogen-activated protein kinase kinase kinase is a regulator of cell-plate formation in plant cytokinesis. Genes Dev. 15, 352–363. Tena, G., Asai, T., Chiu, W.-L., and Sheen, J. (2001). Plant mitogen-activated protein kinase signaling cascades. Curr. Opin. Plant Biol. 4, 392–400.[CrossRef][ISI][Medline]
Widmann, C., Gibson, S., Jarpe, M., and Johnson, G.L. (1999). Mitogen-activated protein kinase: Conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143–180. Related articles in Plant Cell:
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