- American Society of Plant Biologists
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). A MAPK cascade consists of a core module of three kinases that act in sequence: a MAPK kinase kinase (MAPKKK) that activates, via phosphorylation, a MAPK kinase (MAPKK), which activates a MAPK. Activated MAPKs phosphorylate a number of different target proteins; the majority of targets appear to be transcription factors, but other targets include various protein kinases, phospholipases, and cytoskeletal proteins, all of which effect changes in gene expression and/or physiological responses appropriate to the stimulus in question (Widmann et al., 1999). In this issue of The Plant Cell, Krysan et al. (pages 1109–1120) report on the isolation and analysis of knockout mutants of the ANP family of MAPKKK genes in Arabidopsis and show that the ANP kinases likely are involved in the control of cell division.
MAPKKKs AND THE REGULATION OF CELL DIVISION
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.
Cell Division Is Impaired in anp2 anp3 Double-Mutant Embryos.
The incomplete cell walls and binucleate cells seen in the anp2 anp3 double mutant are indicative of failed cell division. (Figure courtesy of Peter Jester and Patrick Krysan.)
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.
A ROLE FOR ANP MAPKKKs IN STRESS RESPONSES?
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) conducted microarray gene expression analysis using an Arabidopsis oligonucleotide microarray containing sequences corresponding to 8200 genes and found that a group of pathogen- and stress-related genes were upregulated in the anp2 anp3 mutants compared with wild-type seedlings. However, it is not known if this response is related to a role for ANP proteins in stress responses in wild-type plants or if it is merely the result of the “stress” of the mutant phenotype caused by the loss of normal ANP2 and ANP3 function. Further experiments, including overexpression of ANP genes, analyses of patterns of gene expression and localization, and analyses of the responses of various mutant and transgenic plants to different stresses, are necessary to determine the role of ANP proteins in regulating stress responses.
SIGNALING NETWORKS
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) found that the H2O2-mediated MAPK cascade initiated by ANP1 in Arabidopsis protoplasts likely involves the activation of MPK3 and MPK6. Asai et al. (2001) identified a complete MAPK cascade that consists of MPK3 and MPK6 together with the MAPKKs MKK4 and MKK5 activated by the MAPKKK MEKK1 that functions downstream of the flagellin receptor FLS2 in the Arabidopsis protoplast system. This cascade was found to activate the transcription factors WRKY22 and WRKY29 and to be associated with the development of resistance to bacterial and fungal pathogens. Thus, MPK3 and MPK6 appear to be involved in more than one type of MAPK cascade, because H2O2-mediated activation of these proteins was associated with different downstream gene expression responses than MKK4- and MKK5-mediated responses associated with the FLS2 stimulus (Asai et al., 2001).
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). In Saccharomyces, the mating response to pheromone involves the activation of a MAPK cascade consisting of the MAPKKKSTE11, MAPKKSTE7, and MAPKFUS3, and FUS3 activates a number of other factors that control the mating response and the formation of the mating projection (an outgrowth of the cell that facilitates mating). In contrast, a signal produced when cells are grown under nutrient deprivation activates a MAPK module consisting of MAPKKKSTE11, MAPKKSTE7, and MAPKKSS1, which leads to cell cycle arrest and a change of growth habit to a pseudohyphal form.
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) provides an important foothold on one set of MAPK cascade components.
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.
PHYLOGENY-BASED GENE KNOCKOUTS
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.
