title: Differential elicitation of two processing proteases controls the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata.
abstract: Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.
authors.affiliation: Department of Protein Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague.
authors.forename: Martin
authors.initials: M
authors.lastname: Horn
authors.forename: Aparna G
authors.initials: AG
authors.lastname: Patankar
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Zavala
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Wu
authors.forename: Lucie
authors.initials: L
authors.lastname: Dolecková-Maresová
authors.forename: Milana
authors.initials: M
authors.lastname: Vujtechová
authors.forename: Michael
authors.initials: M
authors.lastname: Mares
authors.forename: Ian T
authors.initials: IT
authors.lastname: Baldwin
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000595:Amino Acid Sequence; D003517:Cyclopentanes / Q000494:pharmacology; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D054883:Oxylipins; D010447:Peptide Hydrolases / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D011480:Protease Inhibitors / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology* / Q000378:metabolism
chemical_list: D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; D010940:Plant Proteins; D011480:Protease Inhibitors; D011498:Protein Precursors; C072239:methyl jasmonate; D010447:Peptide Hydrolases