László Bögre

Complexity, cross talk and integration of plant MAP kinase signalling

Mitogen-activated protein kinases (MAPKs) link information transfer from external stimuli-activated sensors to cellular responses. The completed Arabidopsis genome sequence revealed an extraordinary complexity in MAPK-signalling components in plants. Information obtained from Arabidopsis provides a framework for a unified nomenclature and the assembly and function of MAPK-signalling pathways. Strategies and tools are evolving to connect MAPK pathways and to determine their function. As a result, MAPK signalling modules emerged, one of which appears to antagonistically regulate stress- and growth-responses and another that regulates cytokinesis.

Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease

We have found that a major target for effectors secreted by Pseudomonas syringae is the abscisic acid (ABA) signalling pathway. Microarray data identified a prominent group of effector‐induced genes that were associated with ABA biosynthesis and also responses to this plant hormone. Genes upregulated by effector delivery share a 42% overlap with ABA‐responsive genes and are also components of networks induced by osmotic stress and drought. Strongly induced were NCED3, encoding a key enzyme of ABA biosynthesis, and the abscisic acid insensitive 1 (ABI1) clade of genes encoding protein phosphatases type 2C (PP2Cs) involved in the regulation of ABA signalling. Modification of PP2C expression resulting in ABA insensitivity or hypersensitivity led to restriction or enhanced multiplication of bacteria, respectively. Levels of ABA increased rapidly during bacterial colonisation. Exogenous ABA application enhanced susceptibility, whereas colonisation was reduced in an ABA biosynthetic mutant. Expression of the bacterial effector AvrPtoB in planta modified host ABA signalling. Our data suggest that a major virulence strategy is effector‐mediated manipulation of plant hormone homeostasis, which leads to the suppression of defence responses.

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid‐signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2‐1, regulated by the 3′‐phosphoinositide‐dependent kinase‐1 (AtPDK1). AGC2‐1 specifically interacts with AtPDK1 through a conserved C‐terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2‐1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2‐1 in an AtPDK1‐dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2‐1 is abundant in fast‐growing organs and dividing cells, and activated during re‐entry of cells into the cell cycle after sugar starvation‐induced G1‐phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1‐regulated AGC2‐1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP‐AGC2‐1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2‐1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2‐1 in root hair growth and development.

Wounding Induces the Rapid and Transient Activation of a Specific MAP Kinase Pathway.

Mechanical injury in plants induces responses that are involved not only in healing but also in defense against a potential pathogen. To understand the intracellular signaling mechanism of wounding, we have investigated the involvement of protein kinases. Using specific antibodies, we showed that wounding alfalfa leaves specifically induces the transient activation of the p44MMK4 kinase, which belongs to the family of mitogen-activated protein kinases. Whereas activation of the MMK4 pathway is a post-translational process and was not blocked by [alpha]-amanitin and cycloheximide, inactivation depends on de novo transcription and translation of a protein factor(s). After wound-induced activation, the MMK4 pathway was subject to a refractory period of 25 min, during which time restimulation was not possible, indicating that the inactivation mechanism is only transiently active. After activation of the p44MMK4 kinase by wounding, transcript levels of the MMK4 gene increased, suggesting that the MMK4 gene may be a direct target of the MMK4 pathway. In contrast, transcripts of the wound-inducible MsWIP gene, encoding a putative proteinase inhibitor, were detected only several hours after wounding. Abscisic acid, methyl jasmonic acid, and electrical activity are known to mediate wound signaling in plants. However, none of these factors was able to activate the p44MMK4 kinase in the absence of wounding, suggesting that the MMK4 pathway acts independently of these signals.

SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK.

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.

A MAP Kinase Is Activated Late in Plant Mitosis and Becomes Localized to the Plane of Cell Division

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells. The MMK3 protein could be found during all stages of the cell cycle, but its protein kinase activity was transient in mitosis and correlated with the timing of phragmoplast formation. Depolymerization of microtubules by short treatments with the drug amiprophosmethyl during anaphase and telophase abolished MMK3 activity, indicating that intact microtubules are required for MMK3 activation. During anaphase, MMK3 was found to be concentrated in between the segregating chromosomes; later, it localized at the midplane of cell division in the phragmoplast. As the phragmoplast microtubules were redistributed from the center to the periphery during telophase, MMK3 still localized to the whole plane of division; thus, phragmoplast microtubules are not required to keep MMK3 at this location. Together, these data strongly support a role for MMK3 in the regulation of plant cytokinesis.

Growth signalling pathways in Arabidopsis and the AGC protein kinases

Lipid-derived signals are central to regulating a multitude of cellular processes but, in plants, little is known of the downstream signalling pathways. The Arabidopsis 3-phosphoinositide-dependent protein kinase (PDK1) could couple lipid signals to the activation of several protein kinases of the so-called AGC kinase family. The Arabidopsis AGC kinases contain sequence motives required for the docking of PDK1 and phosphorylation of their activation loop in the kinase catalytic domain. It is becoming evident that specific members of the AGC kinases are implicated in key growth signalling pathways. For example, Arabidopsis p70(S6K) might be a nodal point able to integrate hormonal and developmental signals with nutritional inputs, together with the Arabidopsis Target of Rapamycin (TOR) protein.

The Role of the Arabidopsis E2FB Transcription Factor in Regulating Auxin-Dependent Cell Division

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA.

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen‐activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress‐activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F‐actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co‐localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root‐derived cell culture. Loss of tip‐focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain‐of‐function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.

Molecular Biology of Somatic Embryogenesis

Similar to other higher eukaryotic organisms, in flowering plants, embryo development is the consequence of fertilization events. Union of gametes as the male sperm nucleus and the female egg results in the zygote which later develops into an embryo within the ovule. During the sexual reproductive cycle, the egg cell is prepared for initiation of the embryogenic development that is triggered by signals after sperm-egg contact. In vivo the gametophytic and sporophytic cell differentiation is separated and the haploid gametes are specialized for sexual fusion and the fertilized egg has the potential to develop into a new organism. In most higher eukaryotes, the differentiation of totipotent embryogenic cells is controlled by a pre-set developmental program and terminally differentiated cells are formed. In early embryos, the cells have rapid division cycles and the chromatin becomes transcriptionally active after variable number of division cycles during embryo development.