Ökophysiologie der Pflanzen

Optical indicators

Chlorophyll fluorescence can be used for an extensive characterization of the photosynthetic apparatus, in isolated chloroplasts as well as, and even more important, in intact leaves. The relative efficiency of fluorescence excitation at different wavelengths can also be used to determine compounds besides chlorophyll in the intact leaves.

1. Epidermal transmittance for UV radiation

Plants protect themselves against UV radiation (UV-B, 280 - 315 nm, UV-A, 315 - 400 nm) by accumulating UV absorbing compounds in their outer tissues (usually the epidermis). Since they are dependent on photosynthesis, they are using pigments which do not absorb in the photosynthetically active wavelength range (400 - 700 nm). These can be flavonols or hydroxycinnamic acid derivatives. Since they don't have a color, we can determine by our eyes if a leaf is protected or not.

We have developed a method by which the extent of UV protection can be determined rapidly and non-destructively. The principle of the technique is shown schematically in this figure UVB-1 . The method is based on the fact that chlorophyll located in the mesophyll absorbs UV radiation and emits a small part of the absorbed energy again as fluorescence in the long wavelength range. The more radiation is absorbed by chlorophyll, the more fluorescence is emitted. In this context it is important to note that the measuring beam must be weak enough not to excite variable fluorescence. Therefore, the fluorescence signal is proportional to the radiation passing the epidermis. As a reference, a beam is used which is not absorbed by the UV screening pigments. In the figure, a blue beam is shown, but it has turned out that a red beam is better (see below). Setting the determined fluorescence excitation ratio in relation to the ratio determined with a leaf whose epidermis was removed, the transmittance for UV radiation can be calculated. When calculating absorbance from the transmittance one obtains a parameter which is proportional to the contents of the screening compounds.

Portable apparatus such as the UV-A-PAM (Gademann Instruments) or the Dualex (Force-A) can be used on attached leaves, allowing repeated measurements over several days on the same leaf, even under field conditions. However, in these instruments, LEDs emitting in the near UV-A are used for fluorescence excitation, which causes a bias in the detection in favor of flavonols and flavones. Hydroxycinnamic acids are better detected using a Xe-PAM apparatus. Here, the choice of the excitation wavelength is more flexible and filter sets for excitation at 313 nm are readily available. This way, also UV-B screening can be addressed, with the drawback of destructive sampling.

2. Detection of the violaxanthin cycle pool size

Using the same measuring principle as for UV transmittance, carotenoid contents of intact leaves can be estimated (see publication by Nichelmann et al. 2016). Here, a blue excitation beam is used with a red beam as reference. The rationale of the measurements is that some carotenoids, especially those of the violaxanthin (V) cycle, do not transfer absorbed light energy to chlorophyll. Hence, they act as a kind of internal shielding pigments. Since the V cycle pool size changes strongly with light acclimation of the photosynthetic apparatus, F(blue) / F(red) excitation ratios change accordingly.

3. Detection of anthocyanins

As anthocyanins have their main absorption at wavelengths between 450 to 600 nm, they can be detected by chlorophyll fluorescence excitation in this wavelength range. Again, a red measuring beam serves as a reference beam. Screening of light of the wavelength 545 nm of up to 90% was detected (Nichelmann and Bilger, in preparation).