Previous sections of this chapter have described a mechanistic model for the transport of scent molecules between the site of biosynthesis and the site of evaporation into the headspace. Unfortunately very little direct experimental evidence has been published to date that can corroborate the steps detailed in this model. It would be particularly interesting to separately assess the permeability of the epidermal plasma membrane, cell wall, and cuticle for selected model compounds. The results could be used to calculate the resistance imposed by each transport segment. Once respective results become available, they will have to be compared with data on the overall resistance of the transport process in order to assess the contribution each step makes to the total transport barrier. The overall resistance in turn can be inferred from the mass flow of compounds, the surface area across which this flow occurs, and the concentration gradient driving transport. These quantities can be estimated based on literature data comparing the headspace accumulation of products with the biosyn-thetic capacities and the precursor pools for model plant species.
The headspace concentration of scent components has been reported for diverse plant species and is described in detail in Section 1.1. It should be noted that results are usually given as amounts per flower and time, or as amounts per tissue weight and time, but the permeability of the transport barriers can only be assessed using amounts per area and time. Thus the surface area per flower or per tissue weight would be necessary to transform the headspace results. For A. majus, the only species for which these data are available,52 it can be concluded that the petals emit up to 75 |g/mm2/day.
Next, the gradient between scent constituent concentrations inside and outside the tissue has to be taken into account. While the headspace amounts of these compounds have been reported for many plant species, relatively little is known about their internal concentrations. Essential oils were analyzed for diverse plant species, but in many cases only qualitative data were given and for only some species (and floral organs) have the amounts of essential oil constituents per flower (or surface area) been reported. Based on these results, the concentration of scent molecules inside epidermal cells can be inferred, assuming that only these cells biosynthesize and thus contain essential oils. Accordingly, the petal epidermis of A. majus should, for example, contain roughly 5 |g/mm3 of methyl benzoate (using published data52 and assuming an average epidermis thickness of 50 pm, as well as even distribution of methyl benzoate in all petal areas). This calculation demonstrates that scent components do accumulate inside the cell to substantial concentrations, and local concentrations in subcellular compartments must be even higher. As this internal concentration greatly exceeds the concentration of scent in the headspace surrounding the tissue, a gradient is established that acts as a driving force for the transport of the compounds toward the surface.
In the same study on A. majus flowers, Goodwin et al.52 also monitored the dynamics of essential oil quantities as a function of flower development. Their results have three important implications: (1) transport phenomena are relatively fast, occurring in time scales of hours; (2) significant transport barriers exist that cause transient buildup of internal material; and (3) these barriers are not rate limiting for emission of scent compounds.
Snapdragon petals represent the first system in which all the data have been acquired that are necessary to quantitatively describe the transport of scent compounds, assessing the overall flow rate, permeability, and resistance. Based on a flow rate for methyl benzoate of 0.7 ^.g/s/flower, surface areas of 750 mm2/flower, and concentration gradients between internal and external pools of 5 |J.g/mm3, an overall permeability on the order of 107 m/s can be predicted. This corresponds to an overall resistance of 10-7 s/m imposed on transport. Although these values represent only relatively rough estimates, calculated from various parameters using a number of assumptions, they give a quantitative description of the transport barrier. Hence this successful approach should be repeated by similar investigations on other plant species, allowing quantitative comparisons between the barriers involved in transport of scent compounds.
It should be noted that our efforts to describe the export and emission of scent molecules in quantitative terms might be complicated by (at least) one other factor. The absence of both appreciable internal pools of scent volatiles and the biosynthetic enzymes of such compounds in scent-emitting floral tissue of Jasminum species prompted Watanabe et al.68 to discover that fragrance components are stored as nonvolatile glycosides. However, Loughrin et al.69 found that the level of glycosid-ically bound volatiles in Nicotiana species was not correlated with the emission levels of such volatiles, but with the age of the flower; older, senescing flowers had higher levels of stored glycosides. It has been speculated that glycosides are either precursors, storage forms, or detoxification products of scent compounds. The role of these derivatives, as well as their amounts, may depend on plant species and physiology. Hence the (reversible?) formation of glycosides must be regarded side branch to the flux of scent material between biosynthesis and emission, and their amounts would accordingly have to be taken into account when quantifying transport phenomena and barrier properties. Unfortunately the presence and amounts of these derivatives have not been investigated in most of the studies on essential oils of flowers.
To further refine our understanding of transport mechanisms involved in scent emission, a number of approaches seem feasible. Among them, two appear especially promising: (1) The concentration of scent compounds within the mixture of cuticular waxes should be quantified, if possible as a function of flower development or diurnal rhythms. Respective data would help to distinguish between internal pools of scent compounds, showing where exactly they (transiently) accumulate and hence pointing to the rate-limiting step in transport. (2) Model species could be used to generate transgenic plants with altered capacity for scent biosynthesis. In one experiment, a volatile biosynthetic gene was introduced into petunia and the protein levels and enzyme activity of monoterpene synthase were detected.70 Nonetheless, the amounts of the target compound, linalool, in the floral scent bouquet were found to be only slightly changed. This result could be due to limited precursor availability, restricted capacities of pathway enzymes, mismatches in intracellular localization and trafficking, as well as rate-limiting barriers in epidermal cell export. Hence the transgenic plants are very interesting tools to investigate various aspects of scent production and test a number of related hypotheses, so it would be very interesting to characterize them in detail.
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