Characterization of complex photosynthetic pigment profiles in European deciduous tree leaves by sequential extraction and reversed-phase high-performance liquid chromatography

Abstract

Leaf pigments, including chlorophylls and carotenoids, are important biochemical indicators of plant photosynthesis and photoprotection. In this study, we developed, optimized, and validated a sequential extraction and liquid chromatography-diode array detection method allowing for the simultaneous quantification of the main photosynthetic pigments, including chlorophyll a, chlorophyll b, β-carotene, lutein, neoxanthin, and the xanthophyll cycle (VAZ), as well as the characterization of plant pigment derivatives. Chromatographic separation was accomplished with the newest generation of core–shell columns revealing numerous pigment derivatives. The sequential extraction allowed for a better recovery of the main pigments (+25 % chlorophyll a, +30 % chlorophyll b, +42 % β-carotene, and 61% xanthophylls), and the characterization of ca. 5.3 times more pigment derivatives (i.e., up to 62 chlorophyll and carotenoid derivatives including isomers) than with a single-step extraction. A broad working range of concentrations (300–2,000 ng.mL−1) was achieved for most pigments and their derivatives and the limit of detection was as low as a few nanograms per milliliter. The method also showed adequate trueness (RSD < 1%) and intermediate precision (RSD < 5%). The method was developed and validated with spinach leaves and their extracts. The method was successfully performed on leaf pigment extracts of European deciduous tree species. Within a case study using Fagus sylvatica L. leaves, pigment derivatives revealed a high within-individual tree variability throughout the growing season that could not be detected using the main photosynthetic pigments alone, eventually showing that the method allowed for the monitoring of pigment dynamics at unprecedented detail.

Inoculation with Actinobacteria spp. Isolated from a Hyper-Arid Environment Enhances Tolerance to Salinity in Lettuce Plants (Lactuca sativa L.)

“…Chlorophylls and carotenoids are important plant pigments that play crucial roles in photosynthesis and photoprotection. These pigments can change in response to different stressors, such as salinity and drought, which can affect the plant’s ability to adapt and perform photosynthesis [ 58 ]. When there is an excess of Na + concentration in the cytosol, Na + takes over the K + binding sites, which results in chlorophyll degradation and interferes with the optimal functioning of proteins [ 48 ].…”

Leaf spectroscopy reveals drought response variation inFagus sylvaticasaplings from across the species’ range

“…For a subset of the collected samples (n=20), the pigment extraction and subsequent HPLC analysis was done according to the protocol proposed by Petibon and Wiesenberg (2022) and conducted under subdued light to prevent pigment degradation. We modified the gradient program and the eluent composition of the HPLC method to optimize peak separation (see supplementary methods).…”

Late-season biosynthesis of leaf fatty acids and n-alkanes of a mature beech (Fagus sylvatica) tree traced via13CO2 pulse-chase labelling and compound-specific isotope analysis

“…Furthermore, the ratio of the 13 C-excess of the bulk leaves to the 13 C-excess of the FA of the respective leaf suggests a larger investment of the assimilated C into FA and secondary metabolites in sun-exposed than in shaded leaves. One potential substance class that benefitted from this ongoing FA biosynthesis was potentially the cutin polymer (Kolattukudy and Walton, 1973) or plant pigments that also showed considerable changes in their composition during the same observation period for the whole tree (Petibon and Wiesenberg, 2022). As 13 C excess of FA exhibited a subsequent decrease until the end of the observation period and some variability of individual sampling dates, this supports our third hypothesis (iii) of a de-novo biosynthesis of leaf wax lipids in a mature beech tree of ~200 years even within the late phase of the growing season.…”