isolating-incorporating-light-harvesting-antennas-from-diatom

Isolating and Incorporating Light-Harvesting Antennas from Diatom <em>Cyclotella Meneghiniana</em> in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

The consequences of ketocarotenoid production in transgenic tobacco (Nicotiana tabacum) plants expressing a Chlamydomonas reinhardtii gene encoding a β-carotene ketolase were examined concerning the functionality of the photosynthetic apparatus. T1 plants produced less photosynthetic pigments per dry weight, but Chl a/Chl b ratios remained unchanged. Almost as much ketocarotenoids as accessory xanthophylls accumulated per Chl a molecule. These ketocarotenoids were found mainly in the thylakoid membranes, but were not functionally bound to light-harvesting complexes, although LHCII is known to be able to bind astaxanthin. On the contrary, high amounts of ketocarotenoids probably changed the properties of the lipid phase of the thylakoids, thereby reducing the stability of photosystem II supercomplexes and LHCII trimers and ultimately decreasing grana formation. In addition, photosystem II function in electron transport was impaired, and plants exhibited less non-photochemical quenching compared to wild-type plants. Thus, in order not to disturb vital functions of the plants, production of astaxanthin and other nutritionally valuable ketocarotenoids apparently requires ways to sequester the additional carotenoids to plastoglobuli. 

Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability.

Phaeodactylum tricornutum like other diatoms synthesizes fucoxanthin and diadinoxanthin as major carotenoid end products. The genes involved have recently been assigned for early pathway steps. Beyond β-carotene, only gene candidates for β-carotene hydroxylase, zeaxanthin epoxidase and zeaxanthin de-epoxidase have been proposed from the available genome sequence. The two latter enzymes may be involved in the two different xanthophyll cycles which operate in P. tricornutum. The function of three putative zeaxanthin epoxidase genes (zep) was addressed by pathway complementation in the Arabidopsis thaliana Zep mutant npq2. Genes zep2 and zep3 were able to restore zeaxanthin epoxidation and a functional xanthophyll cycle but the corresponding enzymes exhibited different catalytic activities. Zep3 functioned as a zeaxanthin epoxidase whereas Zep2 exhibited a broader substrate specificity additionally converting lutein to lutein-5,6-epoxide. Although zep1 was transcribed and the protein could be identified after import into the chloroplast in A. thaliana, Zep1 was found not to be functional in zeaxanthin epoxidation. The non-photochemical quenching kinetics of wild type A. thaliana was only restored in transformant npq2-zep3. 

Identification of genes coding for functional zeaxanthin epoxidases in the diatom Phaeodactylum tricornutum.

The light reactions of photosynthesis, which include light-harvesting and charge separation, take place in the amphiphilic environment of the thylakoid membrane. The light-harvesting complex II (LHCII) is the main responsible for light absorption in plants and green algae and is involved in photoprotective mechanisms that regulate the amount of excited states in the membrane. The dual function of LHCII has been extensively studied in detergent micelles, but recent results have indicated that the properties of this complex differ in a lipid environment. In this work we checked these suggestions by studying LHCII in liposomes. By combining bulk and single molecule measurements, we monitored the fluorescence characteristics of liposomes containing single complexes up to densely packed proteoliposomes. We show that the natural lipid environment per se does not alter the properties of LHCII, which for single complexes remain very similar to that in detergent. However, we show that LHCII has the strong tendency to cluster in the membrane and that protein interactions and the extent of crowding modulate the lifetimes of the excited state in the membrane. Finally, the presence of LHCII monomers at low concentrations of complexes per liposome is discussed.

Light-harvesting Complexes (LHCs) Cluster Spontaneously in Membrane Environment Leading to Shortening of Their Excited State Lifetimes.

Regulation of photosynthetic light harvesting involves all major thylakoid membrane complexes. One important factor is the proton motive force (pmf) driving ATP production. Its proton gradient (ΔpH) component regulates the high energy quenching. Potassium ions largely contribute to the formation of the electric field (ΔΨ). ΔΨ and ΔpH partially compensate each other to form pmf. Whilst in plants considerable progress has been made in analyzing the interplay of H and K gradients, in diatoms knowledge in this field is still scarce. We relaxed cellular K gradients by valinomycin in Cyclotella meneghiniana. We observed a slow decrease of PSII maximum quantum yield in the dark upon valinomycin addition correlating with diatoxanthin accumulation which we attribute to the breakdown of organellar K gradients (either plastid or mitochondria) which might compensate for the loss of the K gradient by adjustment of the thylakoid pH in a secondary step. This response is reversible when ΔpH is relaxed. Similarly, we found higher non-photochemical quenching (NPQ) caused by higher DT accumulation in the steady state in valinomycin-treated cells. In vitro fucoxanthin chlorophyll a (FCPa) antenna complexes in liposomes with natural lipid composition showed a decrease in fluorescence yield if a K gradient is built up. The effect reversed by relaxing the gradient. We interpret these fluorescence changes with surface charge dynamics and FCPa organization in the membrane rather than a direct influence of K gradients on FCPa complexes. Both experiments reveal that K gradients might contribute to fine tuning of light harvesting capacity in relation to pmf in diatoms.

Relaxation of cellular K gradients by valinomycin induces diatoxanthin accumulation in Cyclotella meneghiniana cells and alters FCPa fluorescence yield in vitro.

Transient absorption spectroscopy has been applied to investigate the energy dissipation mechanisms in the nonameric fucoxanthin-chlorophyll-a,c-binding protein FCPb of the centric diatom Cyclotella meneghiniana. FCPb complexes in their unquenched state were compared with those in two types of quenching environments, namely aggregation-induced quenching by detergent removal, and clustering via incorporation into liposomes. Applying global and target analysis, in combination with a fluorescence lifetime study and annihilation calculations, we were able to resolve two quenching channels in FCPb that involve chlorophyll-a pigments for FCPb exposed to both quenching environments. The fast quenching channel operates on a timescale of tens of picoseconds and exhibits similar spectral signatures as the unquenched state. The slower quenching channel operates on a timescale of tens to hundreds of picoseconds, depending on the degree of quenching, and is characterized by enhanced population of low-energy states between 680 and 710 nm. The results indicate that FCPb is, in principle, able to function as a dissipater of excess energy and can do this in vitro even more efficiently than the homologous FCPa complex, the sole complex involved in fast photoprotection in these organisms. This indicates that when a complex displays photoprotection-related spectral signatures in vitro it does not imply that the complex participates in photoprotection in vivo. We suggest that FCPa is favored over FCPb as the sole energy-regulating complex in diatoms because its composition can more easily establish the balance between light-harvesting and quenching required for efficient photoprotection.

Article	Year
Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability. Photosynthesis research\| PubMed ID: 25366829	2015
Identification of genes coding for functional zeaxanthin epoxidases in the diatom Phaeodactylum tricornutum. Journal of plant physiology\| PubMed ID: 26851888	2016
Light-harvesting Complexes (LHCs) Cluster Spontaneously in Membrane Environment Leading to Shortening of Their Excited State Lifetimes. The Journal of biological chemistry\| PubMed ID: 27252376	2016
Relaxation of cellular K gradients by valinomycin induces diatoxanthin accumulation in Cyclotella meneghiniana cells and alters FCPa fluorescence yield in vitro. Physiologia plantarum\| PubMed ID: 28664565	2017
Energy dissipation mechanisms in the FCPb light-harvesting complex of the diatom Cyclotella meneghiniana. Biochimica et biophysica acta\| PubMed ID: 30056090	2018

Lars Dietzel

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