Daniel S. Moura

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.

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.

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.

Systemic wound signaling in plants: A new perception

Over 100 species of plants exhibit systemic wound signaling that induces the production of defensive chemicals in leaves and stems (1). The signaling is caused by herbivore attacks and also occurs in response to some pathogens (2–5). The most intensively studied systemic signaling response is that found in species of the Solanaceae family, where a systemic wound signal that is graft transmissible regulates the expression of defensive proteinase inhibitors (PIs) and polyphenol oxidase genes (6) (Fig. 1). On wounding, an 18-aa polypeptide, called systemin (7), interacts with a cell-surface receptor (8, 9) to initiate a signaling cascade that includes the release of linolenic acid (18 carbon atoms) from plant cell membranes and its subsequent conversion to 12 oxo-phytodienoic acid (OPDA; 18 carbon atoms) and jasmonic acid (JA; 12 carbon atoms) (10) through the well known octadecanoid pathway (11). JA activates the expression of several signaling pathway genes that up-regulate JA synthesis and the production of H2O2 (12) leading to the synthesis of PIs. OPDA and JA synthesis is analogous to elements of the inflammatory pathway in animals in which arachidonic acid (20 carbon atoms) is converted to prostaglandins (13). Systemin is considered a mobile long-distance signal, and OPDA isomers (14) and JA have been considered to be localized signals produced in target cells. Li et al., in a recent issue of PNAS (15), use two mutants defective in the wound-signaling pathway to provide evidence that JA or a derivative may also act as a long-distance transmissible signal for wound signaling. Jasmonic acid or a derivative may act as a long-distance transmissible signal for wound signaling.

A conserved dibasic site is essential for correct processing of the peptide hormone AtRALF1 in Arabidopsis thaliana

Prohormone proteins in animals and yeast are typically processed at dibasic sites by convertases. Propeptide hormones are also found in plants but little is known about processing. We show for the first time that a dibasic site upstream of a plant peptide hormone, AtRALF1, is essential for processing. Overexpression of preproAtRALF1 causes semi‐dwarfism whereas overexpression of preproAtRALF1(R69A), the propeptide with a mutation in the dibasic site, shows a normal phenotype. RALF1(R69A) plants accumulate only the mutated proprotein and not the processed peptide. In vitro processing using microsomal fractions suggests that processing is carried out by a kexin‐like convertase.

Characterization and localization of a wound-inducible type I serine-carboxypeptidase from leaves of tomato plants (Lycopersicon esculentum Mill.)

Abstract. During the course of characterization of the wound-response related proteins from tomato (Lycopersicon esculentum Mill.) leaves, a serine carboxypeptidase (EC 3.4.16.1) was identified. An increase in peptidase activity in response to wounding, and the isolation of a protein with carboxypeptidase (CP) activity from tomato leaves had been reported previously, but the mRNA coding for the enzyme was not identified. We now report the isolation of a tomato leaf type I serine-CP cDNA whose corresponding mRNA is induced by wounding, systemin and methyl jasmonate. The protein sequence deduced from the cDNA exhibits homology to tomato CP, and barley and rice type I CPs. Southern blot results indicated that the CP gene is probably a member of a small gene family. Tomato CP mRNA was detected within 3 h after wounding, or treatment with systemin or methyl jasmonate. Employing Western blot analysis, CP protein was shown to increase 12 h after the treatments. Using the tomato def1 mutant, we have demonstrated that a functional octadecanoid pathway is necessary for CP transcription in response to wounding. Carboxypeptidase protein was immunolocalized as protein aggregates within the central vacuoles of palisade mesophyll cells as well as in vascular parenchyma where it had previously been found. Double labeling using antibodies specific for CP and inhibitor II indicated that the two proteins are co-localized in the vacuolar aggregates. Tomato CP is a member of the “late wound-inducible genes” whose mRNAs increase 4–12 h following wounding, in contrast to several “early wound-inducible genes”, whose mRNAs appear within 30 min. The data support a role for the enzyme in protein turnover that occurs systemically in leaf cells in response to wounding.

Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation

Summary The peptide RALF regulates cell expansion via an as-yet-unknown mechanism. Here, we provide evidence that AtRALF1 may be negatively regulating cell expansion by interfering with the brassinosteroid signalling pathway.

Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling

Timing a switch in tissue integrity In plants, sperm cells travel through the pollen tube as it grows toward the ovule. Successful fertilization depends on the pollen tube rupturing to release the sperm cells (see the Perspective by Stegmann and Zipfel). Ge et al. and Mecchia et al. elucidated the intercellular cross-talk that maintains pollen tube integrity during growth but destroys it at just the right moment. The signaling peptides RALF4 and RALF19, derived from the pollen tube, maintain its integrity as it grows. Once in reach of the ovule, a related signaling peptide, RALF34, which derives from female tissues, takes over and causes rupture of the pollen tube. Science, this issue p. 1596, p. 1600; see also p. 1544 A suite of signaling peptides and their receptors regulate tissue stability as the pollen tube navigates toward the ovule. In flowering plants, fertilization requires complex cell-to-cell communication events between the pollen tube and the female reproductive tissues, which are controlled by extracellular signaling molecules interacting with receptors at the pollen tube surface. We found that two such receptors in Arabidopsis, BUPS1 and BUPS2, and their peptide ligands, RALF4 and RALF19, are pollen tube–expressed and are required to maintain pollen tube integrity. BUPS1 and BUPS2 interact with receptors ANXUR1 and ANXUR2 via their ectodomains, and both sets of receptors bind RALF4 and RALF19. These receptor-ligand interactions are in competition with the female-derived ligand RALF34, which induces pollen tube bursting at nanomolar concentrations. We propose that RALF34 replaces RALF4 and RALF19 at the interface of pollen tube–female gametophyte contact, thereby deregulating BUPS-ANXUR signaling and in turn leading to pollen tube rupture and sperm release.

Danger-associated peptide signaling in Arabidopsis requires clathrin

Significance Plant endogenous molecules, such as the Arabidopsis thaliana elicitor peptides (AtPeps), activate defense responses by means of cell surface-located receptors that serve as an excellent model to study the implications of endomembrane trafficking in plant immunity. Here we used fluorescently labeled and bioactive pep1 to probe in vivo the intracellular dynamics and the fate of the active receptor-ligand complexes in the Arabidopsis root meristem. We show that AtPep1 internalization depends on its receptors and that clathrin-mediated endocytosis is essential for AtPep1-induced responses. The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H+-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor–ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.

Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter.