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MALDI time-of-flight mass spectrometry (MALDI-TOF MS) has become a widely used tool for the classification of biological samples. The complex chemical composition of pollen grains leads to highly specific, fingerprint-like mass spectra, with respect to the pollen species. Beyond the species-specific composition, the variances in pollen chemistry can be hierarchically structured, including the level of different populations, of environmental conditions or different genotypes. We demonstrate here the sensitivity of MALDI-TOF MS regarding the adaption of the chemical composition of three Poaceae (grass) pollen for different populations of parent plants by analyzing the mass spectra with partial least squares discriminant analysis (PLS-DA) and principal component analysis (PCA). Thereby, variances in species, population and specific growth conditions of the plants were observed simultaneously. In particular, the chemical pattern revealed by the MALDI spectra enabled discrimination of the different populations of one species. Specifically, the role of environmental changes and their effect on the pollen chemistry of three different grass species is discussed. Analysis of the Group formation within the respective populations showed a varying influence of plant genotype on the classification, depending on the species, and permits conclusions regarding the respective rigidity or plasticity towards environmental changes.
1. Fourier transform infrar ed (FTIR) spectroscopy enables chemical analysis of pollen samples for plant phenotyping to stud y plant–environment interactions, such as influence of climate change or pathogens. However, current approach, such as microspectrosc opy and attenuated total refle ction spectrosco py, doe s not allow fo r high-throughput protocols. This st udy at hand suggests a new sp ectroscopic method for high-throughp ut characterization of pollen.
2. Samples were measured as thin films of pollen fragments using a Bruker FTIR spe ctrometer with a high-throughput eXTension (HTS-XT) unit employing 384-well plates. In total, 146 pollen samples, belonging to 31 different pollen species of Fagaceae and Betulaceae and collected during three consecutive years (2012–2014) at locations in Croatia, Germany and Norway, were analysed. Critical steps in the sample preparation and measurement, such as variabilities between technical replicates, between microplates and between spectrometers, were studied.
3. Measurement variations due to sample preparation, microplate holders and instrumentation were low, and thus allowed differentiation of samples with respect to phylogeny and biogeography. The spectral variability for a ran ge of Fagales spec ies (Fagus, Quercus, Betula, Corylus, Alnus and Ostrya) showed high-species-specific differences in pollen’s chemical composition due to eithe r location or year. Statistically significant inter-annual and locational differences in the pollen spectra indicate that pollen chemical composition has high phenotypic plasticity and is influenced by local climate conditions. The variations in composition are connected to lipids, proteins, carbohydrates and sporopollenins that play crucial ro les in cold and desiccation tolerance, protection against UV radiation and as material and energy reserves.
4. The results of this study demonstrate the value of high-throughput FTIR approach for the systematic collection of data on ecosystems. The novel FTIR approach offers fast, reliable and economical screening of large number of samples by semi-automated methodology. The high-throughput approach could provide crucial understanding on plant–climate interactions with respect to biochemical variation within genera, species and populations.