Analysis of inducible anti-herbivore defenses and signals in Populus




Haruta, Miyoshi

Journal Title

Journal ISSN

Volume Title



Plants are continuously subjected to biotic stresses such as herbivory and pathogens. Consequently they have evolved many defense mechanisms. Inducible defenses that are activated only after insect infestation are one type of plant adaptation to herbivory. Many plant species possess arrays of inducible defenses, including the accumulation of toxic phytochemicals and antinutritive proteins that function to deter herbivory. Inducible defenses are generally activated at the transcriptional level and they can occur at the whole plant level, which presumably protects the plant from future herbivory. The genus Populus which includes both aspens and poplars, is an important tree for forestry but often undergoes severe defoliation by herbivores. Outbreaks of forest tent caterpillar (Malacosoma disstria, FTC) and the subsequent massive defoliation of its natural host, trembling aspen (Populus tremuloides Michx.), are known to periodically occur in North America. Within aspen populations, however, individual clones show variation in susceptibility to FTC, and this suggests the importance of innate defenses of aspen. Although it has been known that aspen leaves contain phenolic phytochemicals as defensive compounds, the involvement of defensive proteins was not known when this work began. Therefore, one aim of this study was to investigate protein-based induced defenses in trembling aspen, using a molecular approach. In order to initiate investigation of protein-based induced defenses in trembling aspen, genes for polyphenol oxidase (PPO) and trypsin inhibitor (Tl), known defense-related genes in other plant species, were isolated and characterized. Both PPO and TI were transcriptionally activated in aspen foliage by FTC herbivory, artificial tissue damage, and methyl jasmonate, a signal molecule for inducible defenses. In time course analyses, it was demonstrated that PPO and TI mRNAs accumulated within several hours in both wounded leaves and unwounded leaves of the same plant. This was consistent with the wound response previously reported from other plant species including hybrid poplar (Populus trichocarpa x P. deltoides) and is indicative of the presence of signaling mechanisms for systemic induction of defense proteins in trembling aspen. To further obtain insight into mechanisms for inducible defenses, signal molecules for induction of defenses were investigated using a model system, poplar suspension cultures, based on the observation that plant cell cultures often show rapid alkalinization of the medium in response to defense-related signal molecules. Using the alkalinization assay system, two different alkalinization factors were purified from poplar leaf extracts. First, three 5 kD peptides causing rapid alkalinization, the rapid alkalinization factors (RALFs), were isolated and further characterized at the molecular level. RALF appears to be a novel hormone-like peptide that was also recently characterized from tobacco. In contrast to other known alkalinization factors, RALF did not induce defenses such as the expression of phenylalanine ammonia lyase. Based on the expression profile of RALF genes, it was predicted that RALF may be involved in general cellular signaling such as growth and development rather than defense signaling. A second alkalinization peptide causing slower alkalinization, slow alkalinization factor (SALF), was also isolated and partially sequenced by Edman degradation. Database searches of the obtained peptide sequence revealed that SALF seems to be derived from the N-terminus of a known protein, photosystem 1 centre protein subunit D. Although it is not yet clear whether the SALF peptide is a defense-related signal in poplar, it is hypothesized that this breakdown product of a known protein may act as a biologically active signal in plants. Overall, this thesis presents: 1) the first demonstration of protein-based inducible defenses in trembling aspen at molecular level; 2) the discovery of novel peptide molecules with alkalinization activity in suspension cultures of poplar cells.



Poplar, Aspen