Johannes W. Stratmann

RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development.

A 5-kDa polypeptide was isolated from tobacco leaves that induced a rapid alkalinization of the culture medium of tobacco suspension-cultured cells and a concomitant activation of an intracellular mitogen-activated protein kinase. An N-terminal sequence was obtained, and a cDNA coding for the 49-aa polypeptide was isolated from a tobacco cDNA library. The cDNA encoded a preproprotein of 115 amino acids that contained the polypeptide at its C terminus. A search among known expressed sequence tags revealed that genes encoding Rapid ALkalinization Factor (RALF) preproproteins were present in various tissues and organs from 16 species of plants representing 9 families. A tomato homolog of the polypeptide was synthesized and, when supplied to germinating tomato and Arabidopsis seeds, it caused an arrest of root growth and development. Although its specific role in growth has not been established, the polypeptide joins the ranks of the increasing number of polypeptide hormones that are known to regulate plant stress, growth, and development.

Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum.

Plant respiratory burst oxidase homologs (Rboh) are homologs of the human neutrophil pathogen-related gp91phox. Antisense technology was employed to ascertain the biological function of Lycopersicon esculentum (tomato) Rboh. Lines with diminished Rboh activity showed a reduced level of reactive oxygen species (ROS) in the leaf, implying a role for Rboh in establishing the cellular redox milieu. Surprisingly, the antisense plants acquired a highly branched phenotype, switched from indeterminate to determinate growth habit, and had fasciated reproductive organs. Wound-induced systemic expression of proteinase inhibitor II was compromised in the antisense lines, indicating that ROS intermediates supplied by Rboh are required for this wound response. Extending these observations by transcriptome analysis revealed ectopic leaf expression of homeotic MADS box genes that are normally expressed only in reproductive organs. In addition, both Rboh-dependent and -independent wound-induced gene induction was detected as well as transcript changes related to redox maintenance. The results provide novel insights into how the steady state cellular level of ROS is controlled and portrays the role of Rboh as a signal transducer of stress and developmental responses.

Production of multiple plant hormones from a single polyprotein precursor.

Some animal and yeast hormone genes produce prohormone polypeptides that are proteolytically processed to produce multiple copies of hormones with the same or different functions. In plants, four polypeptides have been identified that can be classed as hormones (intercellular chemical messengers) but none are known to be produced as multiple copies from a single precursor. Here we describe a polyprotein hormone precursor, present in tobacco plants, that gives rise to two polypeptide hormones, as often found in animals and yeast. The tobacco polypeptides activate the synthesis of defensive proteinase-inhibitor proteins in a manner similar to that of systemin, an 18-amino-acid polypeptide found in tomato plants. The two tobacco polypeptides are derived from each end of a 165-amino-acid precursor that bears no homology to tomato prosystemin. The data show that structurally diverse polypeptide hormones in different plant species can serve similar signalling roles, a condition not found in animals or yeast.

Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects

Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin (35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.

Convergence of Signaling Pathways Induced by Systemin, Oligosaccharide Elicitors, and Ultraviolet-B Radiation at the Level of Mitogen-Activated Protein Kinases in Lycopersicon peruvianum Suspension-Cultured Cells

We tested whether signaling pathways induced by systemin, oligosaccharide elicitors (OEs), and ultraviolet (UV)-B radiation share common components in Lycopersicon peruvianum suspension-cultured cells. These stress signals all induce mitogen-activated protein kinase (MAPK) activity. In desensitization assays, we found that pretreatment with systemin and OEs transiently reduced the MAPK response to a subsequent treatment with the same or a different elicitor. In contrast, MAPK activity in response to UV-B increased after pretreatment with systemin and OEs. These experiments demonstrate the presence of signaling components that are shared by systemin, OEs, and UV-B. Based on desensitization assays, it is not clear if the same or different MAPKs are activated by different stress signals. To identify specific stress-responsive MAPKs, we cloned three MAPKs from a tomato (Lycopersicon esculentum) leaf cDNA library, generated member-specific antibodies, and performed immunocomplex kinase assays with extracts from elicited L. peruvianum cells. Two highly homologous MAPKs, LeMPK1 and LeMPK2, were activated in response to systemin, four different OEs, and UV-B radiation. An additional MAPK, LeMPK3, was only activated by UV-B radiation. The common activation of LeMPK1 and LeMPK2 by many stress signals is consistent with the desensitization assays and may account for substantial overlaps among stress responses. On the other hand, MAPK activation kinetics in response to elicitors and UV-B differed substantially, and UV-B activated a different set of LeMPKs than the elicitors. These differences may account for UV-B-specific responses.

Long distance run in the wound response ¿ jasmonic acid is pulling ahead

When plants are wounded, a long-distance signal is produced that activates the wound response in unwounded leaves. Recent work by Gregg Howes group suggests that jasmonic acid is the long-distance wound signal. Previously, many data indicated that the signaling peptide systemin might function as the systemic wound signal. According to the new data, systemin is crucial for the production of the systemic wound signal in the wounded leaf, but might not travel long distances.

Tomato Mitogen-Activated Protein Kinases LeMPK1, LeMPK2, and LeMPK3 Are Activated during the Cf-4/Avr4-Induced Hypersensitive Response and Have Distinct Phosphorylation Specificities

Tomato (Solanum lycopersicum) plants with the Cf-4 resistance gene recognize strains of the pathogenic fungus Cladosporium fulvum that secrete the avirulence protein Avr4. Transgenic tomato seedlings coexpressing Cf-4 and Avr4 mount a hypersensitive response (HR) at 20°C, which is suppressed at 33°C. Within 120 min after a shift from 33°C to 20°C, tomato mitogen-activated protein (MAP) kinase (LeMPK) activity increases in Cf-4/Avr4 seedlings. Searching tomato genome databases revealed at least 16 LeMPK sequences, including the sequence of LeMPK1, LeMPK2, and LeMPK3 that cluster with biotic stress-related MAP kinase orthologs from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). LeMPK1, LeMPK2, and LeMPK3 are simultaneously activated in Cf-4/Avr4 seedlings, and, to reveal whether they are functionally redundant or not, recombinant LeMPKs were incubated on PepChip Kinomics slides carrying peptides with potential phosphorylation sites. Phosphorylated peptides and motifs present in them discriminated between the phosphorylation specificities of LeMPK1, LeMPK2, and LeMPK3. LeMPK1, LeMPK2, or LeMPK3 activity was specifically suppressed in Cf-4-tomato by virus-induced gene silencing and leaflets were either injected with Avr4 or challenged with C. fulvum-secreting Avr4. The results of these experiments suggested that the LeMPKs have different but also overlapping roles with regard to HR and full resistance in tomato.

Systemin and jasmonic acid regulate constitutive and herbivore-induced systemic volatile emissions in tomato, Solanum lycopersicum.

Transgenic tomato (Solanum lycopersicum) plants that overexpress the Prosystemin gene (35S::PS) and plants with a mutation in the JA biosynthetic pathway (def1) are known to exhibit a constitutive or reduced wound response, respectively. Here it is demonstrated that several independent 35S::PS lines emit high levels of specific volatiles in addition to increased accumulation of proteinase inhibitors (PIs). Furthermore, the temporal dynamics of systemically induced volatile compounds including green-leaf volatiles, terpenes, and shikimic acid-derivatives from 35S::PS and def1 plants in response to herbivore wounding and treatment with jasmonic acid (JA) are described. Application of JA induced defense protein accumulation and volatile emissions in wild type plants, but did not further increase systemic volatile emissions from 35S::PS plants. Wounding by Manduca sexta larvae induced synthesis of defense proteins and emission of volatiles in wild type plants, but not in def1 plants. Application of jasmonic acid restored the local and systemic accumulation of defense proteins in def1, as well as enhanced herbivore-induced volatile emissions. These results provide strong support for the role of prosystemin- and JA-signaling in the regulation of volatile emissions in tomato plants.

UVB/UVA Radiation Activates a 48 kDa Myelin Basic Protein Kinase and Potentiates Wound Signaling in Tomato Leaves

We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet‐C (<280 nm) and UVB/UVA (280–390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen‐activated protein kinase family. Ultraviolet‐C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826–829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA‐irradiated plants that were subsequently wounded accumulated 2.5–4‐fold higher levels of proteinase inhibitor I when compared to wounded non‐irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12‐oxo‐phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.

The tomato brassinosteroid receptor BRI1 increases binding of systemin to tobacco plasma membranes, but is not involved in systemin signaling

The tomato wound signal systemin is perceived by a specific high-affinity, saturable, and reversible cell surface receptor. This receptor was identified as the receptor-like kinase SR160, which turned out to be identical to the brassinosteroid receptor BRI1. Recently, it has been shown that the tomato bri1 null mutant cu3 is as sensitive to systemin as wild type plants. Here we explored these contradictory findings by studying the responses of tobacco plants (Nicotiana tabacum) to systemin. A fluorescently-labeled systemin analog bound specifically to plasma membranes of tobacco suspension-cultured cells that expressed the tomato BRI1-FLAG transgene, but not to wild type tobacco cells. On the other hand, signaling responses to systemin, such as activation of mitogen-activated protein kinases and medium alkalinization, were neither increased in BRI1-FLAG-overexpressing tobacco cells nor decreased in BRI1-silenced cells as compared to levels in untransformed control cells. Furthermore, in transgenic tobacco plants BRI1-FLAG became phosphorylated on threonine residues in response to brassinolide application, but not in response to systemin. When BRI1 transcript levels were reduced by virus-induced gene silencing in tomato plants, the silenced plants displayed a phenotype characteristic of bri1 mutants. However, their response to overexpression of the Prosystemin transgene was the same as in control plants. Taken together, our data suggest that BRI1 can function as a systemin binding protein, but that binding of the ligand does not transduce the signal into the cell. This unusual behavior and the nature of the elusive systemin receptor will be discussed.