|dc.description.abstract||People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing.
To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries.
The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.||en