Functional Analysis of the Kunitz Trypsin Inhibitor Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against Herbivores      

Abstract

We investigated the functional and biochemical variability of Kunitz trypsin inhibitor (KTI) genes of Populus trichocarpa 3 Populus deltoides. Phylogenetic analysis, expressed sequence tag databases, and western-blot analysis confirmed that these genes belong to a large and diverse gene family with complex expression patterns. Five wound-and herbivore-induced genes representing the diversity of the KTI gene family were selected for functional analysis and shown to produce active KTI proteins in Escherichia coli. These recombinant KTI proteins were all biochemically distinct and showed clear differences in efficacy against trypsin-, chymotrypsin-, and elastase-type proteases, suggesting functional specialization of different members of this gene family. The in vitro stability of the KTIs in the presence of reducing agents and elevated temperature also varied widely, emphasizing the biochemical differences of these proteins. Significantly, the properties of the recombinant KTI proteins were not predictable from primary amino acid sequence data. Proteases in midgut extracts of Malacosoma disstria, a lepidopteran pest of Populus, were strongly inhibited by at least two of the KTI gene products. This study suggests that the large diversity in the poplar (Populus spp.) KTI family is important for biochemical and functional specialization, which may be important in the maintenance of pest resistance in long-lived plants such as poplar.

Plant Interactions with Arthropod Herbivores: State of the Field

“…A research article by Goulet et al (2008) describes targeted engineering of plant cystatins to provide greater efficacy against insect digestive proteases. Major and Constabel (2008) describe the functional and biochemical variability of Kunitz trypsin inhibitor genes of hybrid poplar, which may provide a starting point for similar engineering efforts in tree species.…”

Pathogenesis-related genes and proteins in forest tree species

“…In hybrid poplar (P. trichocarpa 9 P. deltoides), diverse group of KPIs were involved during insect attack and herbivory. Gene duplication followed by strong positive selection on these genes was suggested to provide ecological advantage for survival of the poplar species and neutralized the fast-evolving pest and pathogen invasion (Major and Constabel 2008). A comparative analysis of sequenced plant genomes revealed the presence of high number PIs in poplar (24) in comparison with Arabidopsis where it was limited to 3 and no PI was reported from rice genome (Duplessis et al 2009).…”

Heavy Metals Stress on Poplar: Molecular and Anatomical Modifications

“…This kind of proteins were upregulated also in response to different biotic and abiotic stresses including insects (Major and Constabel 2008), pathogenic fungus (Rinaldi et al 2007), wounding (Christopher et al 2004), and ozone (Gupta et al 2005), suggesting that this upregulation depends on the crosstalk among the stress-related signalling pathways, such as those induced by salicylate (SA) and nitric oxide (NO) (Poschenrieder et al 2006 Defence genes differentially expressed by Cu stress also included several enzymes involved in ROS scavenging such as peroxidases, Cu/Zn superoxide dismutases and catalases that have shown a downregulation under this condition. This phenomenon has been observed also in Cdstressed roots of P. x canescens (Schützendübel et al 2002) suggesting conserved responses of these enzymes in different poplar clones subjected to heavy metal stress.…”