Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought-and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electro- kinases, also known as extracellular signal-regulated kinases (ERKs), were originally found to be involved in differentiation and reentry into the cell cycle. Later, mammalian osmoregulated and stress-activated MAP kinases were discovered. The family of SAPK (stress-activated protein kinase)/JNK (Jun N-terminal kinase)/p38 is activated by various types of stress (13-15). JNK and p38 can functionally replace the HOG1 yeast MAP kinase that is necessary for adaptation to high extracellular osmolarity. Genetic and biochemical studies in yeast revealed several distinct MAP kinase cascades in signaling different extracellular stimuli. These kinase cascades are important regulators in pheromone response, pseudohyphal differentiation, and osmolarity responses (16,17).Activation of MAP kinases requires tyrosine and threonine phosphorylation (4). The highly conserved threonine and tyrosine residues are located close to kinase domain VIII and seem to be also important for activation of plant MAP kinases (7). Phosphorylation of these crucial residues is performed by a dual specific MAP kinase kinase that in turn has to be activated by a serine/threonine MAP kinase kinase kinase (18,19). These kinase cascades seem to be conserved in modular form throughout evolution, mediating distinct signal transduction pathways. Isolation of homologous upstream kinases from plants (20-23) indicate the presence of similar biochemical modules for extracellular signal transmission.Despite the fact that all of the components of MAP kinase modules have been identified in plants, little is known about their functions. An Arabidopsis MAP kinase was proposed to be involved in auxin signal transduction (24). Genetic studies and the isolation of the CTR1 gene suggest that a MAP kinase cascade may also be involved in mediating ethylene responses (21). Recently, MAP kinases have been demonstrated to be activated upon cutting of leaves (22,28) and exposure of cells to fungal elicitor (25). Increased transcript levels of genes encoding a MAP kinase module have been taken as evidence for the involvement of a MAP kinase pathway in signaling touch, cold, salt, and water stress (26).In this article, we present evidence that environmental stresses are mediated by posttranslational activation of a specific MAP kinase in alfalfa. The MAP kinase pathway appears to mediate only specific forms of stress, because cold and drought, but not high temperature and osmotic stress, induce the activation of this pathway. MATERIALS AND METHODSIsolation, Sequence Analysis, ...

In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses. MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif. In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues. Recently, we showed that salt stress-induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions. Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase). SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system. Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK. SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases. Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding. In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs. Moreover, SIMKK enhances the salt-induced activation of SIMK. These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK.

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