Huachun Wang

Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis

Stomata are specialized epidermal structures that regulate gas (CO2 and O2) and water vapor exchange between plants and their environment. In Arabidopsis thaliana, stomatal development is preceded by asymmetric cell divisions, and stomatal distribution follows the one-cell spacing rule, reflecting the coordination of cell fate specification. Stomatal development and patterning are regulated by both genetic and environmental signals. Here, we report that Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, two environmentally responsive mitogen-activated protein kinases (MAPKs), and their upstream MAPK kinases, MKK4 and MKK5, are key regulators of stomatal development and patterning. Loss of function of MKK4/MKK5 or MPK3/MPK6 disrupts the coordinated cell fate specification of stomata versus pavement cells, resulting in the formation of clustered stomata. Conversely, activation of MKK4/MKK5-MPK3/MPK6 causes the suppression of asymmetric cell divisions and stomatal cell fate specification, resulting in a lack of stomatal differentiation. We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. The establishment of a complete MAPK signaling cascade as a key regulator of stomatal development and patterning advances our understanding of the regulatory mechanisms of intercellular signaling events that coordinate cell fate specification during stomatal development.

MEKK1 Is Required for flg22-Induced MPK4 Activation in Arabidopsis Plants

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1s full kinase activity.

Regulation of floral organ abscission in Arabidopsis thaliana

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone.

Haplo-Insufficiency of MPK3 in MPK6 Mutant Background Uncovers a Novel Function of These Two MAPKs in Arabidopsis Ovule Development

The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage–dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3−/− mpk6+/− plants are fertile. Genetic analysis revealed that the female sterility of mpk3+/− mpk6−/− plants is a sporophytic effect. In mpk3+/− mpk6−/− mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3+/− mpk6−/− ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development.

Regulation of Arabidopsis Early Anther Development by the Mitogen-Activated Protein Kinases, MPK3 and MPK6, and the ERECTA and Related Receptor-Like Kinases

Mitogen-activated protein kinase (MAPK) and leucine-rich repeat receptor-like kinase (LRR-RLK) signaling pathways have been shown to regulate diverse aspects of plant growth and development. In Arabidopsis, proper anther development relies on intercellular communication to coordinate cell proliferation and differentiation. Two closely related genes encoding MAPKs, MPK3 and MPK6, function redundantly in regulating stomatal patterning. Although the mpk6 mutant has reduced fertility, the function of MPK3 and MPK6 in anther development has not been characterized. Similarly, the ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 genes encoding LRR-RLKs function together to direct stomatal cell fate specification and the er-105 erl1-2 erl2-1 triple mutant is sterile. Because the mpk3 mpk6 double null mutant is embryo lethal, anther development was characterized in the viable mpk3/+ mpk6/- and er-105 erl1-2 erl2-1 mutants. We found that both mutant anthers usually fail to form one or more of the four anther lobes, with the er-105 erl1-2 erl2-1 triple mutant exhibiting more severe phenotypes than those of the mpk3/+ mpk6/- mutant. The somatic cell layers of the differentiated mutant lobes appeared larger and more disorganized than that of wild-type. In addition, the er-105 erl1-2 erl2-1 triple mutant has a reduced number of stamens, the majority of which possess completely undifferentiated or under-differentiated anthers. Furthermore, sometimes, the mpk3/+ mpk6/- mutant anthers do not dehisce, and the er-105 erl1-2 erl2-1 anthers were not observed to dehisce. Therefore, our results indicate that both ER/ERL1/ERL2 and MPK3/MPK6 play important roles in normal anther lobe formation and anther cell differentiation. The close functional relationship between these genes in other developmental processes and the similarities in anther developmental phenotypes of the two types of mutants reported here further suggest the possibility that these genes might also function in the same pathway to regulate anther cell division and differentiation.

A MAPK Cascade Downstream of ERECTA Receptor-Like Protein Kinase Regulates Arabidopsis Inflorescence Architecture by Promoting Localized Cell Proliferation

Coordinated spatiotemporal-specific cell proliferation is critical to plant growth and development. This study demonstrates the function of a mitogen-activated protein kinase cascade downstream of the ERECTA receptor-like kinase in regulating localized cell proliferation, which determines the inflorescence architecture and size in Arabidopsis thaliana. Spatiotemporal-specific cell proliferation and cell differentiation are critical to the formation of normal tissues, organs, and organisms. The highly coordinated cell differentiation and proliferation events illustrate the importance of cell–cell communication during growth and development. In Arabidopsis thaliana, ERECTA (ER), a receptor-like protein kinase, plays important roles in promoting localized cell proliferation, which determines inflorescence architecture, organ shape, and size. However, the downstream signaling components remain unidentified. Here, we report a mitogen-activated protein kinase (MAPK; or MPK) cascade that functions downstream of ER in regulating localized cell proliferation. Similar to an er mutant, loss of function of MPK3/MPK6 or their upstream MAPK kinases (MAPKKs; or MKKs), MKK4/MKK5, resulted in shortened pedicels and clustered inflorescences. Epistasis analysis demonstrated that the gain of function of MKK4 and MKK5 transgenes could rescue the loss-of-function er mutant phenotype at both morphological and cellular levels, suggesting that the MPK3/MPK6 cascade functions downstream of the ER receptor. Furthermore, YODA (YDA), a MAPKK kinase, was shown to be upstream of MKK4/MKK5 and downstream of ER in regulating inflorescence architecture based on both gain- and loss-of-function data. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the ER receptor in regulating localized cell proliferation, which further shapes the morphology of plant organs.

The Protein Phosphatases and Protein Kinases of Arabidopsis thaliana

Protein kinases and protein phosphatases are major post-translational regulators of numerous cellular processes. These enzymes regulate metabolic pathways and are intimately involved in cellular signaling networks. There are over 1000 genes (Wang et al., 2003) in Arabidopsis that encode protein kinases and another 112 genes (Kerk et al., 2002) that encode protein phosphatase catalytic subunits (Table 1)​1).. While Arabidopsis contains orthologs of many of the protein kinases found in other eukaryotes, Arabidopsis, and most likely plants in general, also has an unique set of protein kinases. These include the receptor-like protein kinases and related cytoplasmic protein kinases, the calcium-dependent protein kinases and several members of the putative mitogen-activated protein kinase kinase kinases (Wang et al., 2003). The Arabidopsis protein phos-phatase catalytic subunits encompass orthologs of the majority of the protein phosphatases found in other eukaryotes. However, the type 2C protein phosphatase family is notably large in number in Arabidopsis (Kerk et al., 2002). The distinct representation of genes encoding protein kinases and phosphatases in the Arabidopsis genome, relative to other eukaryotes, is a reflection of the evolutionary history of plants. The understanding that plants have developed cellular communication systems and basic developmental mechanisms independently from other multicellular eukaryotes (Meyerowitz, 2002) explains why plants have evolved a unique collection of enzymes that regulate protein phopshorylation. Indeed, we pointed out over a decade ago (Stone and Walker, 1995), before the exceptionality of the Arabidopsis kinome was fully appreciated, that plants have an unique repertoire of protein kinases that control the early steps in signaling pathways which is reflective of the unique developmental and environmental responses that govern plant growth and development. Table 1. Overview of protein kinases and phosphatases in Arabidopsis. Table 1 (continued). Overview of protein kinases and phosphatases in Arabidopsis. Progress in understanding the role of protein phosphorylation in plant development and environmental responses has made some significant steps in the past few years. While much of the research is focused on Arabidopsis, important insights are also being made in other plant species. Indeed, as genomic and functional data becomes more complete for other plant species, we should be better equipped to answer questions about the fundamental mechanisms plants employ to control their growth, development and responses to environmental stimuli and the role that protein phosphorylation plays in these processes. This chapter on the protein phosphatases and protein kinases of Arabidopsis takes a gene-centric approach to summarize our current understanding of the functional roles of these important mediators of cellular processes. We have tried to focus on the unique aspects of protein kinases and phosphatases.