Item

Abstract

Background: Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse
roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Although nitrilases are present in all
higher plants, little is known about their function in trees. Upon herbivory, poplars produce considerable amounts of
toxic nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile. In addition, as byproduct of the
ethylene biosynthetic pathway upregulated in many plant species after herbivory, toxic β-cyanoalanine may
accumulate in damaged poplar leaves. In this work, we studied the nitrilase gene family in Populus trichocarpa and
investigated the potential role of the nitrilase PtNIT1 in the catabolism of herbivore-induced nitriles.
Results: A BLAST analysis revealed three putative nitrilase genes (PtNIT1, PtNIT2, PtNIT3) in the genome of P. trichocarpa.
While PtNIT1 was expressed in poplar leaves and showed increased transcript accumulation after leaf herbivory, PtNIT2
and PtNIT3 appeared not to be expressed in undamaged or herbivore-damaged leaves. Recombinant PtNIT1 produced
in Escherichia coli accepted biogenic nitriles such as β-cyanoalanine, benzyl cyanide, and indole-3-acetonitrile as
substrates in vitro and converted them into the corresponding acids. In addition to this nitrilase activity, PtNIT1 showed
nitrile hydratase activity towards β-cyanoalanine, resulting in the formation of the amino acid asparagine. The kinetic
parameters of PtNIT1 suggest that the enzyme utilizes β-cyanoalanine and benzyl cyanide as substrates in vivo. Indeed,
β-cyanoalanine and benzyl cyanide were found to accumulate in herbivore-damaged poplar leaves. The upregulation
of ethylene biosynthesis genes after leaf herbivory indicates that herbivore-induced β-cyanoalanine accumulation is
likely caused by ethylene formation.