Gene_locus Report for: arath-HNL

Plant-pest interactions involve multifaceted processes encompassing a complex crosstalk of pathways, molecules, and regulators aimed at overcoming defenses developed by each interacting organism. Among plant defensive compounds against phytophagous arthropods, cyanide-derived products are toxic molecules that directly target pest physiology. Here, we identified the Arabidopsis (Arabidopsis thaliana) gene encoding hydroxynitrile lyase (AtHNL, At5g10300) as one gene induced in response to spider mite (Tetranychus urticae) infestation. AtHNL catalyzes the reversible interconversion between cyanohydrins and derived carbonyl compounds with free cyanide. AtHNL loss- and gain-of-function Arabidopsis plants showed that specific activity of AtHNL using mandelonitrile as substrate was higher in the overexpressing lines than in wild-type (WT) and mutant lines. Concomitantly, mandelonitrile accumulated at higher levels in mutant lines than in WT plants and was significantly reduced in the AtHNL overexpressing lines. After mite infestation, mandelonitrile content increased in WT and overexpressing plants but not in mutant lines, while hydrogen cyanide (HCN) accumulated in the three infested Arabidopsis genotypes. Feeding bioassays demonstrated that the AtHNL gene participated in Arabidopsis defense against T. urticae. The reduced leaf damage detected in the AtHNL overexpressing lines reflected the mite's reduced ability to feed on leaves, which consequently restricted mite fecundity. In turn, mites upregulated TuCAS1 encoding beta-cyanoalanine synthase to avoid the respiratory damage produced by HCN. This detoxification effect was functionally demonstrated by reduced mite fecundity observed when dsRNA-TuCAS-treated mites fed on WT plants and hnl1 mutant lines. These findings add more players in the Arabidopsis-T. urticae interplay to overcome mutual defenses.

Hydroxynitrile lyase (HNL)-catalysed stereoselective synthesis of beta-nitro alcohols from aldehydes and nitroalkanes is considered an efficient biocatalytic approach. However, only one S-selective HNL-Hevea brasiliensis (HbHNL)-exists that is appropriate for the synthesis of (S)-beta-nitro alcohols from the corresponding aldehydes. Further, synthesis catalysed by HbHNL is limited by low specific activity and moderate yields. We have prepared a number of (S)-beta-nitro alcohols, by kinetic resolution with the aid of an R-selective HNL from Arabidopsis thaliana (AtHNL). Optimization of the reaction conditions for AtHNL-catalysed stereoselective C-C bond cleavage of racemic 2-nitro-1-phenylethanol (NPE) produced (S)-NPE (together with benzaldehyde and nitromethane, largely from the R enantiomer) in up to 99 % ee and with 47 % conversion. This is the fastest HNL-catalysed route known so far for the synthesis of a series of (S)-beta-nitro alcohols. This approach widens the application of AtHNL for the synthesis not only of (R)- but also of (S)-beta-nitro alcohols from the appropriate substrates. Without the need for the discovery of a new enzyme, but rather by use of a retro-Henry approach, it was used to generate a number of (S)-beta-nitro alcohols by taking advantage of the substrate selectivity of AtHNL.

Plant-pest interactions involve multifaceted processes encompassing a complex crosstalk of pathways, molecules, and regulators aimed at overcoming defenses developed by each interacting organism. Among plant defensive compounds against phytophagous arthropods, cyanide-derived products are toxic molecules that directly target pest physiology. Here, we identified the Arabidopsis (Arabidopsis thaliana) gene encoding hydroxynitrile lyase (AtHNL, At5g10300) as one gene induced in response to spider mite (Tetranychus urticae) infestation. AtHNL catalyzes the reversible interconversion between cyanohydrins and derived carbonyl compounds with free cyanide. AtHNL loss- and gain-of-function Arabidopsis plants showed that specific activity of AtHNL using mandelonitrile as substrate was higher in the overexpressing lines than in wild-type (WT) and mutant lines. Concomitantly, mandelonitrile accumulated at higher levels in mutant lines than in WT plants and was significantly reduced in the AtHNL overexpressing lines. After mite infestation, mandelonitrile content increased in WT and overexpressing plants but not in mutant lines, while hydrogen cyanide (HCN) accumulated in the three infested Arabidopsis genotypes. Feeding bioassays demonstrated that the AtHNL gene participated in Arabidopsis defense against T. urticae. The reduced leaf damage detected in the AtHNL overexpressing lines reflected the mite's reduced ability to feed on leaves, which consequently restricted mite fecundity. In turn, mites upregulated TuCAS1 encoding beta-cyanoalanine synthase to avoid the respiratory damage produced by HCN. This detoxification effect was functionally demonstrated by reduced mite fecundity observed when dsRNA-TuCAS-treated mites fed on WT plants and hnl1 mutant lines. These findings add more players in the Arabidopsis-T. urticae interplay to overcome mutual defenses.

Hydroxynitrile lyase (HNL)-catalysed stereoselective synthesis of beta-nitro alcohols from aldehydes and nitroalkanes is considered an efficient biocatalytic approach. However, only one S-selective HNL-Hevea brasiliensis (HbHNL)-exists that is appropriate for the synthesis of (S)-beta-nitro alcohols from the corresponding aldehydes. Further, synthesis catalysed by HbHNL is limited by low specific activity and moderate yields. We have prepared a number of (S)-beta-nitro alcohols, by kinetic resolution with the aid of an R-selective HNL from Arabidopsis thaliana (AtHNL). Optimization of the reaction conditions for AtHNL-catalysed stereoselective C-C bond cleavage of racemic 2-nitro-1-phenylethanol (NPE) produced (S)-NPE (together with benzaldehyde and nitromethane, largely from the R enantiomer) in up to 99 % ee and with 47 % conversion. This is the fastest HNL-catalysed route known so far for the synthesis of a series of (S)-beta-nitro alcohols. This approach widens the application of AtHNL for the synthesis not only of (R)- but also of (S)-beta-nitro alcohols from the appropriate substrates. Without the need for the discovery of a new enzyme, but rather by use of a retro-Henry approach, it was used to generate a number of (S)-beta-nitro alcohols by taking advantage of the substrate selectivity of AtHNL.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 A. The structure exhibits an alpha/beta-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an alpha-helix dipole very similar to alpha/beta-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.

We report the 207-Mb genome sequence of the North American Arabidopsis lyrata strain MN47 based on 8.3x dideoxy sequence coverage. We predict 32,670 genes in this outcrossing species compared to the 27,025 genes in the selfing species Arabidopsis thaliana. The much smaller 125-Mb genome of A. thaliana, which diverged from A. lyrata 10 million years ago, likely constitutes the derived state for the family. We found evidence for DNA loss from large-scale rearrangements, but most of the difference in genome size can be attributed to hundreds of thousands of small deletions, mostly in noncoding DNA and transposons. Analysis of deletions and insertions still segregating in A. thaliana indicates that the process of DNA loss is ongoing, suggesting pervasive selection for a smaller genome. The high-quality reference genome sequence for A. lyrata will be an important resource for functional, evolutionary and ecological studies in the genus Arabidopsis.