Targeted Metabolome Profiling of Indonesian Shallots and Japanese Long-Day/Short-Day Bulb Onions.

In this study, targeted metabolome analysis was applied to identify the discriminative metabolites between Indonesian shallot landraces, Japanese long-day onion (LDO) varieties, and Japanese short-day onion (SDO) varieties. In total, 172 metabolite signal intensities were subjected to multivariate PLS-DA, VIP, and random forest modeling to gain further insight into genotype-specific metabolites. PLS-DA divides the examined genotypes into three different clusters, implying that shallot landraces exhibited a distinct metabolite profile compared with Japanese LDO and SDO varieties. The PLS-DA, VIP, and random forest results indicated that the shallot and LDO are richer in metabolite constituents in comparison with the SDO. Specifically, amino acids and organosulfur compounds were the key characteristic metabolites in shallot and LDO genotypes. The analysis of S-alk(en)yl-L-cysteine sulfoxide (ACSO) compounds showed higher accumulation in the shallot landraces relative to LDO and SDO varieties, which explains the stronger pungency and odor in shallots. In addition, the LDO showed higher ACSO content compared with the SDO, implying that long-day cultivation might enhance sulfur assimilation in the Japanese onion. The LDO 'Super Kitamomiji' and the shallots 'Probolinggo' and 'Thailand' showed higher ACSO content than other varieties, making it useful for Allium breeding to improve the flavor and stress tolerance of onions.

Metabolome-Based Discrimination Analysis of Shallot Landraces and Bulb Onion Cultivars Associated with Differences in the Amino Acid and Flavonoid Profiles.

Shallot landraces and varieties are considered an important genetic resource for Allium breeding due to their high contents of several functional metabolites. Aiming to provide new genetic materials for the development of a novel bulb onion cultivar derived from intraspecific hybrids with useful agronomic traits from shallots, the metabolic profiles in the bulbs of 8 Indonesian shallot landraces and 7 short-day and 3 long-day bulb onion cultivars were established using LC-Q-TOF-MS/MS. Principal component analysis, partial least squares discriminant analysis, and dendrogram clustering analysis showed two major groups; group I contained all shallot landraces and group II contained all bulb onion cultivars, indicating that shallots exhibited a distinct metabolic profile in comparison with bulb onions. Variable importance in the projection and Spearman's rank correlation indicated that free and conjugated amino acids, flavonoids (especially metabolites having flavonol aglycone), and anthocyanins, as well as organic acids, were among the top metabolite variables that were highly associated with shallot landraces. The absolute quantification of 21 amino acids using conventional HPLC analysis showed high contents in shallots rather than in bulb onions. The present study indicated that shallots reprogrammed their metabolism toward a high accumulation of amino acids and flavonoids as an adaptive mechanism in extremely hot tropical environments.

A region corresponding to second aspartate-rich motif in tryptophan isoprenylating enzyme, ComQ, serves as a substrate-binding site.

Posttranslational isoprenylation of a tryptophan residue identified from Bacillus quorum sensing pheromone, ComX pheromone, is unique and essential for the bioactivity. A modifying enzyme, ComQ, forms ComX pheromone from the ComX precursor and isoprenyl pyrophosphate and exhibits moderate similarity to isoprenyl pyrophosphate synthases. We investigated non-conserved region in ComQ, corresponding to isopentenyl pyrophosphate binding region of the synthases, using in vitro cell-free isoprenylation. These results suggested that the only conserved aspartic acid residue in the region of ComQ is critical for enzyme activity and responsible for ComX binding. Our findings should contribute to basic understanding of the mechanism of tryptophan isoprenylation.

Lack of the consensus sequence necessary for tryptophan prenylation in the ComX pheromone precursor.

ComX, an oligopeptide pheromone that stimulates the natural genetic competence controlled by quorum sensing in Bacillus subtilis and related bacilli, contains a prenyl-modified tryptophan residue. Since ComX is the only protein known to contain prenylated tryptophan, the universality of this unique posttranslational modification has yet to be determined. Recently, we developed a cell-free assay system in which the tryptophan residue in the ComX(RO-E-2) pheromone precursor derived from B. subtilis strain RO-E-2 can be geranylated by the ComQ(RO-E-2) enzyme. We report here our attempt to identify the consensus sequence surrounding the geranylated tryptophan residue by using the cell-free system with various ComX(RO-E-2) pheromone precursor analogs. We found that [47-58]ComX(RO-E-2), corresponding to the C-terminal 12-residue peptide of the pheromone precursor, contained a short sequence essential for geranylation. We also found that the length of the sequence between the tryptophan residue and the C-terminus was important for geranylation, and that some [47-58]ComX(RO-E-2) pheromone precursor amino acids were involved in the geranylation reaction. However, we could not identify a consensus sequence surrounding the geranylated tryptophan. Our evidence suggests that, like Rab which lacks a consensus sequence yet is geranylgeranyl-modified on a cysteine residue, the ComX pheromone and its precursor also lack a consensus sequence.

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system.

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

The geranyl-modified tryptophan residue is crucial for ComXRO-E-2 pheromone biological activity.

The ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in gram-positive bacteria, Bacillus. We have reported the structure of the ComX(RO-E-2) pheromone, which is produced by the RO-E-2 strain of Bacillus subtilis. ComX(RO-E-2) analogs with substituted amino acids and isoprenoid modified tryptophan residues (e.g., prenyl, geranyl, and farnesyl), were synthesized and examined for biological activity. These results indicate that Phe-Trp(∗)(Ger)-NH(2) is the minimum pharmacophore of the ComX(RO-E-2) pheromone. Furthermore, the length of the isoprenoid moiety (i.e., modification style), and the presence of double bonds, are crucial for biological activity. The modification style of the ComX pheromone is more important than the peptide sequence with respect to biological activity.

Chemical structure of posttranslational modification with a farnesyl group on tryptophan.

Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide, the ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX(RO-C-2) pheromone from strain RO-C-2 must be modified with a farnesyl group on the Trp residue, but the precise structure is not known. Here we report the precise nature of posttranslational farnesylation of ComX(RO-C-2) pheromone on the Trp residue, resulting in the formation of a tricyclic structure. The ComX(168) pheromone, produced by the standard laboratory strain used in the study of B. subtilis, is also posttranslationally farnesylated according to phylogenetic resemblance.

Acid labile ComX pheromone from Bacillus mojavensis RO-H-1.

Bacillus mojavensis strain RO-H-1 produces a posttranslationally modified hexapeptide, the ComX(RO-H-1) pheromone, that stimulates natural genetic competence controlled by quorum sensing. LC/ESI-MS analysis of partially purification of the ComX(RO-H-1) pheromone suggested a precise modification in its tryptophan residue. The corresponding ComX(RO-H-1) pheromone prepared by solid-phase synthesis was identical to the natural pheromone, and showed significant biological activity. These results indicated that the posttranslational modification of the ComX(RO-H-1) pheromone was geranylation on the tryptophan residue, resulting in the formation of a tricyclic structure. The ComX(RO-H-1) pheromone was immediately dehydrated by acid because of its extreme acid lability.