Paul Buschbeck (Johannes Gutenberg University Mainz)
β-Carotene is an obligate educt for the biosynthesis of the xanthophyll violaxanthin (A). The two hydroxyl groups in the structural formula of violaxanthin are highlighted in green and the two epoxide groups in red. In B, a leaf of the tobacco plant Nicotiana benthamiana infiltrated on the right half of the leaf with an agrobacteria suspension is shown. The agrobacteria mediated the transformation of leaf cells with genes of the unusual violaxanthin synthesis from chromalveolate algae, whose gene products catalyze the conversion of β-carotene to violaxanthin. Heterologous expression of these genes resulted in more than a doubling of the violaxanthin content compared to the untreated half of the leaf (C, HPLC chromatogram normalized to chlorophyll a). This bypassed the intermediate formation of zeaxanthin, which is required in endogenous violaxanthin synthesis of land plants. Graph: Paul Buschbeck
Paul Buschbeck's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Johannes Gutenberg University Mainz in the year 2021.
Title: Cloning and functional characterization of enzymes involved in the violaxanthin synthesis of chromalveolates
Transient implementation of a novel violaxanthin synthesis pathway in tobacco leaves by means of heterologous expression of algal enzymes resulted in a doubling of violaxanthin content compared to wild-type tobacco.
The superphylum Chromalveolata comprises a polyphyletic group of algae whose plastids originally arose from secondary endosymbiosis with an early red alga. With respect to the photosynthetic apparatus of chromalveolates, one of the most striking features compared to green algae or land plants is the high pigment diversity. This diversity is particularly striking for the carotenoids, which belong to the lipophilic pigments. An obligate intermediate for the biosynthesis of many important carotenoids of chromalveolates is the xanthophyll violaxanthin.
The synthesis of violaxanthin in land plants proceeds via the hydroxylation of β-carotene to zeaxanthin and the subsequent epoxidation of zeaxanthin to violaxanthin. Interestingly, previous studies led to the hypothesis that some chromalveolates may have an alternative violaxanthin biosynthetic pathway that differs from the pathway of land plants.
To investigate this hypothesis, in vivo characterizations of the substrate specificity of putative carotenogenic enzymes from different chromalveolates were performed. For that purpose, the corresponding algal genes were cloned and transiently expressed in tobacco leaves using Agrobacterium-mediated transformation. In tobacco leaves a certain amount of β-carotene is accessible to the heterologous enzymes when they are targeted to the plastids as the site of endogenous carotenoid biosynthesis. Thus, β-carotene could serve as a substrate of the heterologous enzymes and as an educt for novel carotenoid biosynthesis pathways catalyzed by the algal enzymes. Based on the pigment stoichiometries in the transformed tobacco leaves measured by HPLC, the substrate specificity of the heterologous enzymes could be deduced.
By this means, an alternative violaxanthin biosynthesis pathway catalyzed by algal enzymes was transiently implemented in tobacco leaves, bypassing zeaxanthin as an intermediate. Notably, the violaxanthin content in the transformed tobacco leaves was more than doubled compared to the wild type. Determination of the substrate specificities of the corresponding algal enzymes provided evidence that chromalveolate algae such as the well-known diatom Phaeodactylum tricornutum possess the genetic makeup to catalyze an alternative violaxanthin synthesis pathway that avoids the formation of zeaxanthin as intermediate. This finding opens further interesting avenues of research, such as the question of the selection pressure that led to the evolution of an alternative violaxanthin synthesis pathway in chromalveolates.
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Paul Buschbeck conducted this work at the Institute of Molecular Physiology in the group of Dr. Martin Lohr.