Abstract
Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes.
Highlights
Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests
To assess the systemic spread of tobacco rattle virus (TRV), RNA prepared from corollas of plants approximately 1 month after infection was used for reverse transcription (RT)-PCR analyses of TRV2 transcript
Meristem infection yielded the most reproducible results and corollas of plantlets inoculated with pTRV vectors by soaking, as shown in Figure 1A for petunia line B1, contained TRV2 transcript, which was absent from control mock-inoculated plants
Summary
Screening of Petunia Lines for Both Scent and Sensitivity to TRV Infection/Silencing. Leaves of petunia plantlets infected with a mixture of Agrobacterium transformed with pTRV1 and pTRV2 carrying the 369-bp PDS fragment (pTRV2-PDS) showed a strongly bleached phenotype throughout the plant for at least 3 months, first appearing approximately 2 to 3 weeks after inoculation (Fig. 2). Analyses of floral scent compounds in petunia B1 and P720 plants with suppressed CHS revealed that the levels of the emitted volatiles, with the exception of MeSA in line B1, were essentially unaffected by TRV infection (Fig. 3, G and H). Because MeSA levels in flowers of pTRV2-CHS-infected plants were similar to those in plants infected with pTRV2, we suggest that scent production was not affected by CHS suppression in line B1 either; rather, increased emission of MeSA was probably due to the plant’s response (as shown before in Deng et al, 2004) to viral infection
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