The genetic basis of constitutive and herbivore-induced ESP-independent nitrile formation in Arabidopsis

Abstract

Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system.

Figure 1.

Glucosinolate hydrolysis. Tissue damage as caused by chewing herbivores results in myrosinase-catalyzed hydrolysis of glucosinolates, yielding Glc and unstable agluca. Whereas the subsequent formation of simple nitriles, epithionitriles, and/or organic thiocyanates requires the action of specifier proteins, spontaneous rearrangement of the aglucone intermediates leads to isothiocyanates. The different types of hydrolysis products differ in their biological activities. Some products have been shown to act as chemical defenses against insect herbivores, but it had remained an open question whether herbivory affects the outcome of glucosinolate hydrolysis.

Figure 2.

Herbivore-induced simple nitrile formation in Arabidopsis Col-0 rosette leaves. Glucosinolate hydrolysis products were measured in rosette leaf homogenates of 6-week-old plants that had been infested with P. rapae. After 24 h of herbivory, all rosette leaves were harvested. Shown is the percentage of nitrile formed from the major leaf glucosinolate 4-methylsulfinylbutylglucosinolate in relation to the total amount of hydrolysis products (nanomoles) formed from this glucosinolate. Means ± sd from results obtained in two independent experiments (n = 6 per treatment class) are shown. Means were tested for significant differences by one-way ANOVA with a Tukey test (all pairwise comparisons). Means marked with a versus b were significantly different at P < 0.001, while the two means marked with a were not significantly different at P > 0.5. ctr., Control (no herbivore); dam., damaged; undam., undamaged.

Figure 3.

Expression analysis of specifier proteins by RT-PCR. Total RNA was isolated from rosette leaves of Arabidopsis Col-0 wild type (A) or SALK_072600 (B) that had been infested with P. rapae (control, no herbivore treatment). After 24 h of herbivory, all rosette leaves were harvested. Transcript levels of AtNSP1 and ESP were analyzed by RT-PCR using actin as a control for equal amounts of RNA. RT reactions were performed on 0.1, 0.3, and 1.0 μg of RNA. The experiment was conducted on two independent sets of plants with similar results. dam., Damaged; undam., undamaged.

Figure 4.

Predicted protein structure of ESP from Arabidopsis Ler and selected MBP-like proteins from Arabidopsis Col-0. Kelch domains (black boxes) and jacalin-like lectin domains (gray boxes) of the gene products of At1g54040 (ESP; GenBank accession no. NP_175806), At3g16400 (NP_566546), At2g33070 (NP_180866), At3g16390 (NP_566545), At3g16410 (NP_188262), At5g48180 (NP_568692), and At3g07720 (NM_556316) are presented as predicted by InterProScan (http://www.ebi.ac.uk/InterProScan/). aa, Amino acids.

Figure 5.

SALK_072600 is defective in constitutive and herbivore-induced simple nitrile formation. A to D, Glucosinolate hydrolysis products in homogenates of rosette leaves of Arabidopsis Col-0 wild type (A and B) or SALK_072600 (C and D). Leaves were harvested from control plants (no herbivore; A and C) or from plants infested with P. rapae larvae (B and D). Depicted are GC-FID traces. Peak 1, 5-Methylsulfanyl-pentanenitrile (nitrile product of 4-methylthiobutylglucosinolate); peak 2, 4-methanesulfinyl-butyronitrile (nitrile product of 3-methylsulfinylpropylglucosinolate); peak 3, 1-isothiocyanato-4-methylsulfanyl-butane (isothiocyanate product of 4-methylthiobutylglucosinolate); peak 4, 5-methanesulfinyl-pentanenitrile (nitrile product of 4-methylsulfinylbutylglucosinolate); peak 5, 1-isothiocyanato-3-methanesulfinyl-propane (isothiocyanate product of 3-methylsulfinylpropylglucosinolate); peak 6, 1-isothiocyanato-4-methanesulfinyl-butane (isothiocyanate product of 4-methylsulfinylbutylglucosinolate); peak IS, internal standard. E, Scheme of the AtNSP1 gene (gene model At3g16400.1; www.Arabidopsis.org). Black boxes represent coding regions, and black and gray lines indicate introns and untranslated regions, respectively. The SALK_072600 line carries a T-DNA insertion in the second coding region. Arrows indicate the positions of the primers used for RT-PCR.

Figure 6.

Effects of AtNSP1 on the hydrolysis of allylglucosinolate in vitro. A, Chemical structures of hydrolysis products formed from allylglucosinolate: 1, simple nitrile; 2, isothiocyanate; 3, epithionitrile. B to D, Hydrolysis products in enzyme assays carried out with purified AtNSP1 (B), purified ESP (C), or myrosinase alone (D) in 50 mm MES buffer, pH 6.0, containing 2 mm allylglucosinolate. Depicted are GC-MS chromatograms (total ion current traces) of dichloromethane extracts. IS, Internal standard; peak numbers refer to the structure numbers in A.

Figure 7.

Effects of iron salts and EDTA on AtNSP1 in vitro. Simple nitrile formation in assays with purified AtNSP1 and myrosinase (black bars) and in control assays with myrosinase alone (gray bars) was measured in 50 mm MES, pH 6.0, containing 2 mm allylglucosinolate. Fe²⁺ and Fe³⁺ were added as (NH₄)₂[Fe(SO₄)₂] and FeCl₃, respectively. Means ± sd of results obtained in three independent experiments are shown. ctr, Control without addition of iron; nd, not determined.

Figure 8.

Expression and phenotypic QTLs in the Arabidopsis Bay-0 × Sha RIL population. QTLs on chromosome III governing the expression of AtNSP1 (At3g16400) and ESM1 (At3g14210) and the formation of simple nitriles are shown. Different line styles (dashed, solid, etc.) show independent QTLs for each phenotype and the QTL significant conditional likelihood profiles as identified by the MIM module of QTL Cartographer. The vertical line through the three graphs shows the physical position of At3g16400 as determined by high-resolution mapping. This analysis focused only on chromosome III for clarity and precision. cM, Centimorgan position along chromosome III; LOD, logarithm of the odds.

Figure 9.

Phylogeny of the Kelch domain. For all higher plant ESP homologues as well as a bryophyte, an algal, and two fungal homologues, the portion of each protein sequence containing the Kelch domains was used to generate a phylogeny. The neighbor-joining tree is shown with bootstrap values over 600 out of 1,000. A distance scale is included at the bottom for the protein tree. An unrooted tree is presented to allow a focus on relatedness. Source organisms were Arabidopsis, Populus trichocarpa, Vitis vinifera, Oryza sativa, Hordeum vulgare, Aspergillus niger, Giberella zeae, Chlamydomonas reinhardtii, Physcomitrella patiens, Lepidium sativum, and Brassica oleracea.

Figure 10.

Phylogeny of the jacalin domain. Full-length protein sequences for all Arabidopsis, rice, and poplar proteins containing a jacalin domain homologous to the MBP1 (At1g52040) domain were used to generate a phylogeny. To identify the source of the second jacalin domain in At3g16410, the two jacalin domains were utilized separately, with At3g16410a being the domain at the N terminus. The neighbor-joining tree is shown with bootstrap values over 600 out of 1,000. A distance scale is included for the protein tree. An unrooted tree is presented to allow a focus on relatedness and to indicate that the identity of the ancestor is unknown.