Abstract
BackgroundNitrilases 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.ResultsA 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.ConclusionsOur data suggest a role for PtNIT1 in the catabolism of herbivore-induced β-cyanoalanine and benzyl cyanide in poplar leaves.
Highlights
Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis
Results β-Cyanoalanine and benzyl cyanide accumulate upon herbivory in poplar leaves Recently it has been shown that caterpillar feeding induces the formation and emission of volatile nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile in poplars [16, 17]
Β-cyanoalanine and benzyl cyanide were barely detectable in undamaged control leaves, but showed a significantly increased accumulation in leaves after feeding of Lymantria dispar caterpillars (Fig. 1). 2-Methylbutyronitrile and 3-methylbutyronitrile, could not be detected in the extracts of damaged or undamaged leaves, suggesting that these aliphatic nitriles are more volatile than benzyl cyanide and do not accumulate in detectable concentrations in the plant
Summary
Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Nitrilases are thiol enzymes that have gained attention in plant biology over the last 50 years for their roles in plant development [1], detoxification of nitriles originating from other metabolic processes [2], and nitrogen recycling [3]. Plant nitrilases are members of the nitrilase superfamily that is classified into 13 branches of enzymes [4]. Most of these branches consist of enzymes that catalyze the hydrolysis of nitrile or amide bonds. In the Brassicaceae and Poaceae, NIT1 homologues were shown to be involved in the metabolism of breakdown products of glucosinolates [6] and cyanogenic glycosides
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