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.

Bulb color is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

The significance of the onset and symmetry of pollen mitosis I (PMI) for the subsequent differentiation of the vegetative and generative cells was investigated by the in vitro maturation of isolated microspores of transgenic tobacco. Free uninucleate microspores of transgenic plants harboring the vegetative cell (VC)-specific late anther tomato lat52 promoter fused to the [beta]-glucuronidase (gus) gene showed normal asymmetric cell division at PMI and activated the lat52 promoter specifically in the nascent VC during in vitro maturation. In vitro maturation in the presence of high levels of colchicine effectively blocked PMI, resulting in the formation of uninucleate pollen grains in which the lat52 promoter was activated. Furthermore, matured uninucleate pollen grains were capable of germination and pollen tube growth despite the absence of a functional generative cell (GC). Lower levels of colchicine induced symmetric division at PMI, producing two similar daughter cells in which typical GC chromatin condensation was prevented. Similar cultures of transgenic microspores harboring the lat52 promoter driving the expression of a nuclear-targeted GUS fusion protein showed that lat52 promoter activation occurred in both symmetric daughter cells. These results directly demonstrate that division asymmetry at PMI is essential for correct GC differentiation and that activation of VC-specific transcription and functional VC maturation may be uncoupled from cytokinesis at PMI. These results are discussed in relation to models proposed to account for the role and distribution of factors controlling the differing fates of the vegetative and generative cells.

The significance of the onset and symmetry of pollen mitosis I (PMI) for the subsequent differentiation of the vegetative and generative cells was investigated by the in vitro maturation of isolated microspores of transgenic tobacco. Free uninucleate microspores of transgenic plants harboring the vegetative cell (VC)-specific late anther tomato lat52 promoter fused to the [beta]-glucuronidase (gus) gene showed normal asymmetric cell division at PMI and activated the lat52 promoter specifically in the nascent VC during in vitro maturation. In vitro maturation in the presence of high levels of colchicine effectively blocked PMI, resulting in the formation of uninucleate pollen grains in which the lat52 promoter was activated. Furthermore, matured uninucleate pollen grains were capable of germination and pollen tube growth despite the absence of a functional generative cell (GC). Lower levels of colchicine induced symmetric division at PMI, producing two similar daughter cells in which typical GC chromatin condensation was prevented. Similar cultures of transgenic microspores harboring the lat52 promoter driving the expression of a nuclear-targeted GUS fusion protein showed that lat52 promoter activation occurred in both symmetric daughter cells. These results directly demonstrate that division asymmetry at PMI is essential for correct GC differentiation and that activation of VC-specific transcription and functional VC maturation may be uncoupled from cytokinesis at PMI. These results are discussed in relation to models proposed to account for the role and distribution of factors controlling the differing fates of the vegetative and generative cells.

Plasmodiophora brassicae is an intracellular pathogen that infects plants in the Brassicaceae family. Although an important pathogen group, information on the genomic makeup of the plasmodiophorids is almost completely lacking. We performed suppression subtractive hybridization (SSH) between RNA from P. brassicae-infected and uninfected Arabidopsis tissue, then screened 232 clones from the resulting SSH library. In addition, we used an oligo-capping procedure to screen 305 full-length cDNA clones from the infected tissue. A total of 76 new P. brassicae gene sequences were identified, the majority of which were extended to full length at the 5' end by the use of RACE amplification. Many of the unisequences were predicted to contain signal peptides for ER translocation. Although we located few sequences in total, these markedly increase available data from the plasmodiophorids, and provide new opportunities to examine plasmodiophorid biology. Our study also points towards the best methods for future plasmodiophorid gene discovery.