The role of phenolic compounds in apple bud dormancy

A number of extraction methods, and methods of analysis of “total phenolic compounds” or “sub-groups of phenolic compounds” or of individual phenolic compounds in apple bark samples were developed/tested.  The test samples used were bark samples, obtained from Granny Smith shoots, actually collected for a “chilling model project 2007” (no NC2-04), which represented 4 sites (2 cold, 2 warm) at different sampling dates (refer to that project for detail).  Three spectrophotometric methods typically used for the determination of “total phenolics” were implemented and tested, as well as one spectrophotometric method  for the determination of “total condensed tannins” and one for “total anthocyanins” (The last 2 methods were not required for this project, but at the time of the investigation it seemed sensible and necessary). The Absorbances measured in the respective methods represent the concentrations of “total phenolics”, “total anthocyanins” and “total condensed tannins”, respectively.

An 80min HPLC method of analysis of individual phenolic compounds was also developed which proved to be a good compromise between separation efficiency and time (and cost). Using available external standards, the main MeOH-extractable phenolic compounds were identified as Chlorogenic acid and Phloridzin. (-)-Epicatechin and Quercetin-galactoside were also shown to be present.  Based on spectral evidence other compounds (peaks in chromatogram) were identified by their spectra as belonging to certain sub-groups of phenolics (a common practice in chromatography).  Peak areas or peak heights were assumed to be linearly related to the concentration of the individual compounds.

The Absorbances measured spectrophotometrically as well as the peak areas (or heights) of individual compounds obtained by HPLC analysis were plotted against sampling dates.  These graphs showed compounds (or groups of compounds) either to be fairly constant, or eratic, or to obviously change seasonally, like chlorogenic acid, anthocyanin and some flavonols.  Thus prime candidates have been identified for investigating seasonal changes in phenolic compounds in future projects.  Even though only a relatively few samples have been analysed in this preliminary investigation, indications are, that the phenolic concentrations and seasonal variation might well differ between samples from the colder and the warmer areas, and therefore might well relate to dormancy.

The main aims (see below) of this project were accomplished. It has also been demonstrated that spectrophotometric assays, (although handy) used on their own, are certainly not revealing enough for studying the effect of phenolics on dormancy in future projects . The use of HPLC is imperative, since this enables the quantification of individual compounds that may or may not change during the season. Furthermore, ideas have been generated and more potential avenues of investigation discovered for future projects – this project being a start only, an important, and necessary stepping stone in the investigation of the role of phenolics in dormancy.  Even though basic methods of analysis (tools of the trade) have been established in this laboratory, it needs to be emphasized that the development of methods for the analysis of particular samples always is an ongoing process, since based on results from a particular analysis, the need typically arises to change or improve the method in some respect.  This should be taken into account when planning future projects.  These types of assays, and the lessons learnt, may be useful in other investigations, like those into post harvest browning of fruit too.