NEW YORK (GenomeWeb) – An analysis of the cucumber genome has narrowed in on the genes responsible for the bitter taste in wild plants and gives a glimpse into how non-bitter cucumbers were derived during domestication.
As they reported in Science last week, researchers led by Sanwen Huang from the Chinese Academy of Agricultural Sciences combined genomic and biochemical approaches to tease out nine genes involved in the curcurbitacin C biosynthesis pathway. Curcurbitacins are triterpenoids that give rise to the bitter taste of curcubits like cucumber, melon, and squash.
"You don't eat wild cucumber, unless you want to use it as a purgative," co-author William Lucas, a plant biologist at the University of California, Davis, said in a statement.
Additionally, Huang, Lucas, and their colleagues report that two transcription factors — Bl and Bt — regulate the curcurbitacin C biosynthesis pathway in plant leaves and fruit, and that selection on these regulators during domestication appears to be how non-bitter curcubits were derived.
Even though they are bitter, the researchers noted that curcurbitacins have been used as anti-inflammatories and to treat liver disease in traditional herbal medicine and recently have been found to have anti-cancer properties.
Additionally, previous work has found that the Mendelian Bi gene confers bitterness in the cucumber plant, though the Bt gene is also needed for the fruit to be bitter. Non-bitterness, meanwhile, is linked to the recessive bt gene, the researchers noted.
In this study, Huang, Lucas, and their colleagues first performed a genome-wide association study in 115 diverse cucumber lines to search for genetic variants linked with Bi.
The most significant SNP they found mapped to the cucumber gene Csa6G088690, in which residue 393 was changed from a cysteine to a tyrosine, and that SNP explained plant bitterness in all but one of the cell lines. In that line, though, there was a one-base deletion at Csa6G088690 that led to a frameshift mutation.
Csa6G088690, the researchers argued, is the Mendelian Bi gene. This gene, they added, is an ortholog of the squash cucurbitadienol synthase gene CPQ.
By expressing Bi and its two mutant alleles in yeast, the researchers examined its biochemical function. Using GC-MS, they noted that cucurbitadienol was only formed in the yeast expressing the wild-type Bi gene. This cucurbitadienol synthase, they added, catalyzes the first step of the CuC biosynthesis pathway.
The researchers searched for and uncovered two naturally occurring non-bitter cucumber mutants. By examining these two lines, they found that the expression of Bi in the foliage of one mutant was reduced, as compared to an isogenic line. By resequencing the genomes of the mutant and isogenic lines, the researchers uncovered a SNP in Csa5G156220, which encodes a putative basic helix-loop-helix transcription factor.
Similarly, the other naturally occurring mutant they found also had a SNP in Csa5G156220 affecting the same transcription factor, suggesting that transcription factor binding and gene expression regulation may be disrupted in these mutants.
By turning to a transient agro-infiltration expression system in cucumber cotyledons, the researchers noted that increased expression of Csa5G156220 in mutant plant cotyledons up-regulates Bi, which the researchers said functionally complemented the non-bitter phenotype.
As such, they dubbed Csa5G156220 Bitter leaf, or Bl, as it regulates bitterness biosynthesis in cucumber leaves.
Additionally, through yeast one-hybrid assays, the researchers found that Bl binds the Bi promoter region and regulates curcurbitacin biosynthesis by activating Bi transcription in plant leaves.
Bt, the researchers noted, maps to a 442-kilobase region of the cucumber genome that contains some 67 other predicted genes. Bl and its two homologs — Csa5G156220 and Csa5G157230 — they added, are among that number, clustered in an 8.5-kb region.
Because Csa5G157230 was expressed in the fruit of wild, bitter plants and because its expression was positively correlated with Bi in a number of cucumber lines, they suspected that Csa5G157230 was Bt, the Mendelian gene needed for the fruit to be bitter.
Through a local association analysis of that 442-kilobase region, Huang, Lucas, and their colleagues uncovered 10 SNPs and one structural variant linked to extreme bitterness.
This also indicated that selection at the Csa5G157230 regulatory region may down-regulate Csa5G157230 in cultivated lines, resulting in less bitterness in domesticated lines.
Additional in vivo functional analyses further underscored the role of Csa5G157230 in activating Bi expression, and the likelihood that it is the Bt gene.
Based on their findings of the Bi and Bt genes, the researchers set out to uncover other players in the curcurbitacin C biosynthesis pathway.
Bi, they noted, is co-localized with four P450 genes and an acyltransferase gene, four of which share expression patterns in the leaves and fruit of multiple cucumber lines. Bl and Bt, they added, could bind each of those four co-expressed genes to activate them.
Additionally, using an integrative bioinformatic and molecular biology-based approach, the researchers identified a further four P450 genes that are co-expressed with that Bi cluster and are activated by Bl and Bt in the fruits and leaves.
RNAi knockdown of the candidate genes yield reduced CuC content in the cotyledons. This, the researcher said, indicates that Bl and Bt regulate bitterness in leaves and fruit by directly acting on the nine genes of the CuC pathway.
Expressing each of the candidate P450s in an engineered yeast strain led to the expression of 10 times as much CuC as the original strain, the researchers noted.
Using LC-MS, they teased out three more steps in the CuC pathway: Csa3G903540 yields 19-hydroxy cucurbitadienol; Csa6G088160 catalyzes it to 19, 25-dihydroxy cucurbitadienol; and the ACT enzyme Csa6G088700 acetylates the deacetyl CuC to yield CuC.
"The new knowledge on cucurbitacin biosynthesis will open a door for biological manufacturing and engineering of these triterpenoids as anti-tumor drugs, for example, in a manner similar to the biosynthesis of artemisinic acid, the anti- malarial drug precursor, " Huang, Lucas, and their colleagues wrote in Science.