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.

Abcission, the natural shedding of leaves, flowers and fruits, is a fundamental component of plant development. Abscission is a highly regulated process that occurs at distinct zones of cells that undergo enlargement and subsequent separation. Although some components of abscission, including accumulation of the hormone ethylene and cell wall-degrading enzymes, have been described, the regulatory pathways remain largely unknown. In this paper we describe a critical component required for floral organ abscission in Arabidopsis thaliana, the receptor-like protein kinase HAESA. Histochemical analysis of transgenic plants harboring a HAESA promoter:: β-glucuronidase reporter gene and in situ RNA hybridization experiments show HAESA expression in the abscission zones where the sepals, petals, and stamens attach to the receptacle, at the base of pedicels, and at the base of petioles where leaves attach to the stem. Immunodetection, immunoprecipitation, and protein kinase activity assays reveal HAESA is a plasma membrane serine/threonine protein kinase. The reduction of function of HAESA in transgenic plants harboring an antisense construct results in delayed abscission of floral organs, and the severity of the phenotype is directly correlated with the level of HAESA protein. These results demonstrate that HAESA functions in developmentally regulated floral organ abscission.

Enzymes of the eukaryotic protein kinase superfamily catalyze the reversible transfer of the [gamma]-phosphate from ATP to amino acid side chains of proteins. Protein kinase function can be counteracted by the action of phosphoprotein phosphatases. Phosphorylation status of a protein can have profound effects on its activity and interaction with other proteins. An estimated 1 to 3% of functional eukaryotic genes encode protein kinases, suggesting that they are involved in many aspects of cellular regulation and metabolism. In plants, protein phosphorylation has been implicated in responses to many signals, including light, pathogen invasion, hormones, temperature stress, and nutrient deprivation. Activities of several plant metabolic and regulatory enzymes are also controlled by reversible phosphorylation. As might be expected from this diversity of function, there is a large array of different protein kinases. Purification of protein kinases and their subsequent cloning, facilitated by the PCR and advances in homology-based cloning techniques, as well as functional analyses, including complementation of conditional yeast mutants and positional cloning of mutant plant genes, has already led to identification of more than 70 plant protein kinase genes. However, the precise functional roles of specific protein kinases and phosphatases during plant growth and development have been elucidated for only a few.

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.