Induction of anthocyanin in the inner epidermis of red onion leaves by environmental stimuli and transient expression of transcription factors.

KEY MESSAGE: Novel imaging approaches have allowed measurements of the anthocyanin induction in onion epidermal cells that can be induced through water stress or transient expression of exogenous transcription factors. Environmental and genetic mechanisms that allow the normally colourless inner epidermal cells of red onion (Allium cepa) bulbs to accumulate anthocyanin were quantified by both absorbance ratios and fluorescence. We observed that water-stressing excised leaf segments induced anthocyanin formation, and fluorescence indicated that this anthocyanin was spectrally similar to the anthocyanin in the outer epidermal cells. This environmental induction may require a signal emanating from the leaf mesophyll, as induction did not occur in detached epidermal peels. Exogenous transcription factors that successfully drive anthocyanin biosynthesis in other species were also tested through transient gene expression using particle bombardment. Although the petunia R2R3-MYB factor AN2 induced anthocyanin in both excised leaves and epidermal peels, several transcription factors including maize C1 and Lc inhibited normal anthocyanin development in excised leaves. This inhibition may be due to moderate levels of conservation between the exogenous transcription factors and endogenous Allium transcription factors. The over-expressed exogenous transcription factors cannot drive anthocyanin biosynthesis themselves, but bind to the endogenous transcription factors and prevent them from driving anthocyanin biosynthesis.

Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

Fast phenotyping of LFS-silenced (tearless) onions by desorption electrospray ionization mass spectrometry (DESI-MS).

Fast MS techniques have been applied to the analysis of sulfur volatiles in Allium species and varieties to distinguish phenotypes. Headspace sampling by proton transfer reaction (PTR) MS and surface sampling by desorption electrospray ionization (DESI) MS were used to distinguish lachrymatory factor synthase (LFS)-silenced (tearless; LFS-) onions from normal, LFS-active (tear-inducing; LFS+), onions. PTR-MS showed lower concentrations of the lachrymatory factor (LF, 3) and dipropyl disulfide 12 from tearless onions. DESI-MS of the tearless onions confirmed the decreased LF 3 and revealed much higher concentrations of the sulfenic acid condensates. Using DESI-MS with MS(2) could distinguish zwiebelane ions from thiosulfinate ions. DESI-MS gave reliable fast phenotyping of LFS+ versus LFS- onions by simply scratching leaves and recording the extractable ions for <0.5 min. DESI-MS leaf compound profiles also allowed the rapid distinction of a variety of Allium cultivars to aid plant breeding selections.

Genomics of biotrophic, plant-infecting plasmodiophorids using in vitro dual cultures.

The plasmodiophorids are a phylogenetically distinct group of parasitic protists that infect plants and stramenopiles, causing several important agricultural diseases. Because of the obligate intracellular part of their lifecycle, none of the plasmodiophorids has been axenically cultured. Further, the molecular biology of the plasmodiophorids is poorly understood because pure cultures are not available from any species. We report on an in-vitro dual culture system of the plasmodiophorids Plasmodiophora brassicae and Spongospora subterranea with their respective plant hosts, Brassica rapa and Solanum tuberosum. We show that these plasmodiophorids are capable of initiating and maintaining stable, long-term plant cell callus cultures in the absence of exogenous plant growth regulators. We show that callus cultures harbouring S. subterranea provide an excellent starting material for gene discovery from this organism by constructing a pilot-scale DNA library. Bioinformatic analysis of the sequences established that almost all of the DNA clones from this library were from S. subterranea rather than the plant host. The Spongospora genome was found to be rich in retrotransposable elements, and Spongospora protein-coding genes were shown to contain introns. The sequence of a near full-length non-LTR retrotransposon was obtained, the first transposable element reported from a cercozoan protist.

Silencing onion lachrymatory factor synthase causes a significant change in the sulfur secondary metabolite profile.

Through a single genetic transformation in onion (Allium cepa), a crop recalcitrant to genetic transformation, we suppressed the lachrymatory factor synthase gene using RNA interference silencing in six plants. This reduced lachrymatory synthase activity by up to 1,544-fold, so that when wounded the onions produced significantly reduced levels of tear-inducing lachrymatory factor. We then confirmed, through a novel colorimetric assay, that this silencing had shifted the trans-S-1-propenyl-l-cysteine sulfoxide breakdown pathway so that more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of this raised thiosulfinate level was a marked increase in the downstream production of a nonenzymatically produced zwiebelane isomer and other volatile sulfur compounds, di-1-propenyl disulfide and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene, which had previously been reported in trace amounts or had not been detected in onion. The consequences of this dramatic simultaneous down- and up-regulation of secondary sulfur products on the health and flavor attributes of the onion are discussed.

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum.

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.