Seasonal Variation of Defense Compounds

Years are no brothers, but so are spring and autumn siblings either –seasonal variation in defense chemistry may tell something of the herbivory as do high levels of hydrolysable tannins and low levels of proanthocyanidins

The whole set of phytochemist skills is required to do this properly

Figure 1. Chemical structures of the compounds quantified from different organs of Geranium sylvaticum several times during the season. Please see Tuominen & Salminen (2017) for details.

If there is something fascinating in plant chemistry, then this is it: to understand the structures of the defense compounds found in plants and to be able to follow their biogenesis by accurate quantifications as the growing season progresses, and perhaps to link all this further to the presence and performance of herbivore species feeding on the plant species during the season. This process as a whole is a huge black box that gets easily moistened with gallons of sweat from the phytochemist, without any promise of the hidden treasure inside.

Or do you think it is a simple task to first determine all the structures shown in Fig. 1, then create quantitative MS/MS methods, then collect 11 different types of tissue several times of a single season, then dry, grind, extract, freeze-dry the extract, dissove in water, filter, dilute, analyse, integrate and finally calculate the concentrations of the compounds in each sample. Quite a task and can only be achieved with the correct level of motivation. See yourself Tuominen & Salminen (2017) for this example.

Does it pay off to follow the seasonal variation in the first place?

If one wishes to follow the seasonal variation of defenses in some species, then one should first know the defensive chemistry well enough. As a rule of thumb it can be said that if the defense chemistry consists of a series of compounds that have clear linkages to each others via biosynthesis and/or have closely resembling structures, then a big go ahead can be given. Such structures are shown in Fig. 1 where clear structural patterns are seen with three main sets of compounds. But if the structures are not known or they are hugely different without any obvious linkages, then one can ask what is the point in studying their seasonal variation? Perhaps one knows that one of the compounds is a true defense compound, thus warranting its detailed inspection within the season? Or perhaps it would make sense to follow the variation of the most promising functional property of the foliage instead of some compounds that have no direct linkage to defense or that cannot be properly quantified in the first place?

The methods of quantitation – want to reveal detailed or average patterns?

Figure 2. Seasonal variation of ellagitannins in the foliage of Betula pubescens (left) and Quercus robur (right). Blue bars show the variation in total ellagitannins as the mean value of ten trees and red bars show the same for the main ellagitannin of B. pubescens (pedunculagin) and Q. robur (vescalagin). It is quite apparent that the main compound dictates the seasonal variation observed for total ellagitannin in both species. For more informative patterns observed with all of the individual tannins, please see Salminen et al. (2001) and Salminen et al. (2004).

An additional point that should hinder the attempts of quantification is the inadequate methods of quantitation. Having said this, we have to admit that we at the Natural Chemistry Research Group have studied seasonal variation of proanthocyanidins with the HCl-BuOH method only. As a result, we only revealed that “all proanthocyanidins” of oak leaves accumulate in old leaves. In fact, we think that such total methods are quite good in revealing only the patterns found with the major compounds or just one major compound in the group quantified. Examples of this are shown in Fig. 2.

Even the best of methods cannot guarantee exciting results

Our other studies dealing with hydrolysable tannins and flavonoid aglycones were much more exciting, since for those we both figured out the structures and quantified them individually. Especially with the hydrolysable tannin study we may consider us being lucky to have achieved such nice results with Betula pubescens. We could not replicate those seasonal variation results with Geranium sylvaticum, although we used even higher quality mass spectrometry quantitations. This must have to do with the life strategy differences of woody plants vs. herbs and the fact that from trees you can collect leaves multiple times during the season while the same is not possible for small herbal plants.

Look at that – that’s the opposite we learned from the literature!