An unexpected hydratase synthesizes the green light-absorbing pigment fucoxanthin.

The ketocarotenoid fucoxanthin and its derivatives can absorb blue-green light enriched in marine environments. Fucoxanthin is widely adopted by phytoplankton species as a main light-harvesting pigment, in contrast to land plants that primarily employ chlorophylls. Despite its supreme abundance in the oceans, the last steps of fucoxanthin biosynthesis have remained elusive. Here, we identified the carotenoid isomerase-like protein CRTISO5 as the diatom fucoxanthin synthase that is related to the carotenoid cis-trans isomerase CRTISO from land plants but harbors unexpected enzymatic activity. A crtiso5 knockout mutant in the model diatom Phaeodactylum tricornutum completely lacked fucoxanthin and accumulated the acetylenic carotenoid phaneroxanthin. Recombinant CRTISO5 converted phaneroxanthin into fucoxanthin in vitro by hydrating its carbon-carbon triple bond, instead of functioning as an isomerase. Molecular docking and mutational analyses revealed residues essential for this activity. Furthermore, a photophysiological characterization of the crtiso5 mutant revealed a major structural and functional role of fucoxanthin in photosynthetic pigment-protein complexes of diatoms. As CRTISO5 hydrates an internal alkyne physiologically, the enzyme has unique potential for biocatalytic applications. The discovery of CRTISO5 illustrates how neofunctionalization leads to major diversification events in evolution of photosynthetic mechanisms and the prominent brown coloration of most marine photosynthetic eukaryotes.

Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin.

Fucoxanthin is a major light-harvesting pigment in ecologically important algae such as diatoms, haptophytes, and brown algae (Phaeophyceae). Therefore, it is a major driver of global primary productivity. Species of these algal groups are brown colored because the high amounts of fucoxanthin bound to the proteins of their photosynthetic machineries enable efficient absorption of green light. While the structure of these fucoxanthin-chlorophyll proteins has recently been resolved, the biosynthetic pathway of fucoxanthin is still unknown. Here, we identified two enzymes central to this pathway by generating corresponding knockout mutants of the diatom Phaeodactylum tricornutum that are green due to the lack of fucoxanthin. Complementation of the mutants with the native genes or orthologs from haptophytes restored fucoxanthin biosynthesis. We propose a complete biosynthetic path to fucoxanthin in diatoms and haptophytes based on the carotenoid intermediates identified in the mutants and in vitro biochemical assays. It is substantially more complex than anticipated and reveals diadinoxanthin metabolism as the central regulatory hub connecting the photoprotective xanthophyll cycle and the formation of fucoxanthin. Moreover, our data show that the pathway evolved by repeated duplication and neofunctionalization of genes for the xanthophyll cycle enzymes violaxanthin de-epoxidase and zeaxanthin epoxidase. Brown algae lack diadinoxanthin and the genes described here and instead use an alternative pathway predicted to involve fewer enzymes. Our work represents a major step forward in elucidating the biosynthesis of fucoxanthin and understanding the evolution, biogenesis, and regulation of the photosynthetic machinery in algae.

Integration of physiologically relevant photosynthetic energy flows into whole genome models of light-driven metabolism.

Characterizing photosynthetic productivity is necessary to understand the ecological contributions and biotechnology potential of plants, algae, and cyanobacteria. Light capture efficiency and photophysiology have long been characterized by measurements of chlorophyll fluorescence dynamics. However, these investigations typically do not consider the metabolic network downstream of light harvesting. By contrast, genome-scale metabolic models capture species-specific metabolic capabilities but have yet to incorporate the rapid regulation of the light harvesting apparatus. Here, we combine chlorophyll fluorescence parameters defining photosynthetic and non-photosynthetic yield of absorbed light energy with a metabolic model of the pennate diatom Phaeodactylum tricornutum. This integration increases the model predictive accuracy regarding growth rate, intracellular oxygen production and consumption, and metabolic pathway usage. Through the quantification of excess electron transport, we uncover the sequential activation of non-radiative energy dissipation processes, cross-compartment electron shuttling, and non-photochemical quenching as the rapid photoacclimation strategy in P. tricornutum. Interestingly, the photon absorption thresholds that trigger the transition between these mechanisms were consistent at low and high incident photon fluxes. We use this understanding to explore engineering strategies for rerouting cellular resources and excess light energy towards bioproducts in silico. Overall, we present a methodology for incorporating a common, informative data type into computational models of light-driven metabolism and show its utilization within the design-build-test-learn cycle for engineering of photosynthetic organisms.

Characterizing compensatory mechanisms in the absence of photoprotective qE in Chlamydomonas reinhardtii.

Rapid fluctuations in the quantity and quality of natural light expose photosynthetic organisms to conditions when the capacity to utilize absorbed quanta is insufficient. These conditions can result in the production of reactive oxygen species and photooxidative damage. Non-photochemical quenching (NPQ) and alternative electron transport are the two most prominent mechanisms which synergistically function to minimize the overreduction of photosystems. In the green alga Chlamydomonas reinhardtii, the stress-related light-harvesting complex (LHCSR) is a required component for the rapid induction and relaxation of NPQ in the light-harvesting antenna. Here, we use simultaneous chlorophyll fluorescence and oxygen exchange measurements to characterize the acclimation of the Chlamydomonas LHCSR-less mutant (npq4lhcsr1) to saturating light conditions. We demonstrate that, in the absence of NPQ, Chlamydomonas does not acclimate to sinusoidal light through increased light-dependent oxygen consumption. We also show that the npq4lhcsr1 mutant has an increased sink capacity downstream of PSI and this energy flow is likely facilitated by cyclic electron transport. Furthermore, we show that the timing of additions of mitochondrial inhibitors has a major influence on plastid/mitochondrial coupling experiments.

A Chlorophyte Alga Utilizes Alternative Electron Transport for Primary Photoprotection.

The green alga Desmodesmus armatus is an emerging biofuel platform that produces high amounts of lipids and biomass in mass culture. We observed D. armatus in light-limiting, excess-light, and sinusoidal-light environments to investigate its photoacclimation behaviors and the mechanisms by which it dissipates excess energy. Chlorophyll a/b ratios and the functional absorption cross section of PSII suggested a constitutively small light-harvesting antenna size relative to other green algae. In situ and ex situ measurements of photo-physiology revealed that nonphotochemical quenching is not a significant contributor to photoprotection; however, cells do not suffer substantial photoinhibition despite its near absence. We performed membrane inlet mass spectrometry analysis to show that D. armatus has a very high capacity for alternative electron transport (AET) measured as light-dependent oxygen consumption. Up to 90% of electrons generated at PSII can be dissipated by AET in a water-water cycle during growth in rapidly fluctuating light environments, like those found in industrial-scale photobioreactors. This work highlights the diversity of photoprotective mechanisms present in algal systems, indicating that nonphotochemical quenching is not necessarily required for effective photoprotection in some algae, and suggests that engineering AET may be an attractive target for increasing the biomass productivity of some strains.

Cross-compartment metabolic coupling enables flexible photoprotective mechanisms in the diatom Phaeodactylum tricornutum.

Photoacclimation consists of short- and long-term strategies used by photosynthetic organisms to adapt to dynamic light environments. Observable photophysiology changes resulting from these strategies have been used in coarse-grained models to predict light-dependent growth and photosynthetic rates. However, the contribution of the broader metabolic network, relevant to species-specific strategies and fitness, is not accounted for in these simple models. We incorporated photophysiology experimental data with genome-scale modeling to characterize organism-level, light-dependent metabolic changes in the model diatom Phaeodactylum tricornutum. Oxygen evolution and photon absorption rates were combined with condition-specific biomass compositions to predict metabolic pathway usage for cells acclimated to four different light intensities. Photorespiration, an ornithine-glutamine shunt, and branched-chain amino acid metabolism were hypothesized as the primary intercompartment reductant shuttles for mediating excess light energy dissipation. Additionally, simulations suggested that carbon shunted through photorespiration is recycled back to the chloroplast as pyruvate, a mechanism distinct from known strategies in photosynthetic organisms. Our results suggest a flexible metabolic network in P. tricornutum that tunes intercompartment metabolism to optimize energy transport between the organelles, consuming excess energy as needed. Characterization of these intercompartment reductant shuttles broadens our understanding of energy partitioning strategies in this clade of ecologically important primary producers.

A siphonous morphology affects light-harvesting modulation in the intertidal green macroalga Bryopsis corticulans (Ulvophyceae).

MAIN CONCLUSION: The macroalga Bryopsis corticulans relies on a sustained protective NPQ and a peculiar body architecture to efficiently adapt to the extreme light changes of intertidal shores. During low tides, intertidal algae experience prolonged high light stress. Efficient dissipation of excess light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is therefore required to avoid photodamage. Light-harvesting regulation was studied in the intertidal macroalga Bryopsis corticulans, during high light and air exposure. Photosynthetic capacity and NPQ kinetics were assessed in different filament layers of the algal tufts and in intact chloroplasts to unravel the nature of NPQ in this siphonous green alga. We found that the morphology and pigment composition of the B. corticulans body provides functional segregation between surface sunlit filaments (protective state) and those that are underneath and undergo severe light attenuation (light-harvesting state). In the surface filaments, very high and sustained NPQ gradually formed. NPQ induction was triggered by the formation of transthylakoid proton gradient and independent of the xanthophyll cycle. PsbS and LHCSR proteins seem not to be active in the NPQ mechanism activated by this alga. Our results show that B. corticulans endures excess light energy pressure through a sustained protective NPQ, not related to photodamage, as revealed by the unusually quick restoration of photosystem II (PSII) function in the dark. This might suggest either the occurrence of transient PSII photoinactivation or a fast rate of PSII repair cycle.

Dynamic interplay between photodamage and photoprotection in photosystem II.

Photoinhibition is the light-induced reduction in photosynthetic efficiency and is usually associated with damage to the D1 photosystem II (PSII) reaction centre protein. This damage must either be repaired, through the PSII repair cycle, or prevented in the first place by nonphotochemical quenching (NPQ). Both NPQ and D1 repair contribute to light tolerance because they ensure the long-term maintenance of the highest quantum yield of PSII. However, the relative contribution of each of these processes is yet to be elucidated. The application of a pulse amplitude modulation fluorescence methodology, called protective NPQ, enabled us to evaluate of the protective effectiveness of the processes. Within this study, the contribution of NPQ and D1 repair to the photoprotective capacity of Arabidopsis thaliana was elucidated by using inhibitors and mutants known to affect each process. We conclude that NPQ contributes a greater amount to the maintenance of a high PSII yield than D1 repair under short periods of illumination. This research further supports the role of protective components of NPQ during light fluctuations and the value of protective NPQ and qPd as unambiguous fluorescence parameters, as opposed to qI and Fv /Fm , for quantifying photoinactivation of reaction centre II and light tolerance of photosynthetic organisms.

Effects of periodic photoinhibitory light exposure on physiology and productivity of Arabidopsis plants grown under low light.

This work examined the long-term effects of periodic high light stress on photosynthesis, morphology, and productivity of low-light-acclimated Arabidopsis plants. Significant photoinhibition of Arabidopsis seedlings grown under low light (100 µmol photons m-2 s-1) was observed at the beginning of the high light treatment (three times a day for 30 min at 1800 µmol photons m-2 s-1). However, after 2 weeks of treatment, similar photosynthesis yields (Fv/Fm) to those of control plants were attained. The daily levels of photochemical quenching measured in the dark (qPd) indicated that the plants recovered from photoinhibition within several hours once transferred back to low light conditions, with complete recovery being achieved overnight. Acclimation to high light stress resulted in the modification of the number, structure, and position of chloroplasts, and an increase in the average chlorophyll a/b ratio. During ontogenesis, high-light-exposed plants had lower total leaf areas but higher above-ground biomass. This was attributed to the consumption of starch for stem and seed production. Moreover, periodic high light exposure brought forward the reproductive phase and resulted in higher seed yields compared with control plants grown under low light. The responses to periodic high light exposure of mature Arabidopsis plants were similar to those of seedlings but had higher light tolerance.

Photoprotective capacity of non-photochemical quenching in plants acclimated to different light intensities.

Arabidopsis plants grown at low light were exposed to a gradually increasing actinic light routine. This method allows for the discerning of the photoprotective component of NPQ, pNPQ and photoinhibition. They exhibited lower values of Photosystem II (PSII) yield in comparison to high-light grown plants, and higher calculated dark fluorescence level (F'o calc.) than the measured one (F'o act.). As a result, in low-light grown plants, the values of qP measured in the dark appeared higher than 1. Normally, F'o act. and F'o calc. match well at moderate light intensities but F'o act. becomes higher at increasing intensities due to reaction centre (RCII) damage; this indicates the onset of photoinhibition. To explain the unusual increase of qP in the dark in low-light grown plants, we have undertaken an analysis of PSII antenna size using biochemical and spectroscopic approaches. Sucrose gradient separation of thylakoid membrane complexes and fast fluorescence induction experiments illustrated that the relative PSII cross section does not increase appreciably with the rise in PSII antenna size in the low-light grown plants. This suggests that part of the increased LHCII antenna is less efficiently coupled to the RCII. A model based upon the existence of an uncoupled population LHCII is proposed to explain the discrepancies in calculated and measured values of F'o.

Assessment of the impact of photosystem I chlorophyll fluorescence on the pulse-amplitude modulated quenching analysis in leaves of Arabidopsis thaliana.

In their natural environment, plants are exposed to varying light conditions, which can lead to a build-up of excitation energy in photosystem (PS) II. Non-photochemical quenching (NPQ) is the primary defence mechanism employed to dissipate this excess energy. Recently, we developed a fluorescence-quenching analysis procedure that enables the protective effectiveness of NPQ in intact Arabidopsis leaves to be determined. However, pulse-amplitude modulation measurements do not currently allow distinguishing between PSII and PSI fluorescence levels. Failure to account for PSI contribution is suggested to lead to inaccurate measurements of NPQ and, particularly, maximum PSII yield (F v/F m). Recently, Pfündel et al. (Photosynth Res 114:189-206, 2013) proposed a method that takes into account PSI contribution in the measurements of F o fluorescence level. However, when PSI contribution was assumed to be constant throughout the induction of NPQ, we observed lower values of the measured minimum fluorescence level ([Formula: see text]) than those calculated according to the formula of Oxborough and Baker (Photosynth Res 54:135-142 1997) ([Formula: see text]), regardless of the light intensity. Therefore, in this work, we propose a refined model to correct for the presence of PSI fluorescence, which takes into account the previously observed NPQ in PSI. This method efficiently resolves the discrepancies between measured and calculated F o' produced by assuming a constant PSI fluorescence contribution, whilst allowing for the correction of the maximum PSII yield.

Quantifying the dynamics of light tolerance in Arabidopsis plants during ontogenesis.

The amount of light plants can tolerate during different phases of ontogenesis remains largely unknown. This was addressed here employing a novel methodology that uses the coefficient of photochemical quenching (qP) to assess the intactness of photosystem II reaction centres. Fluorescence quenching coefficients, total chlorophyll content and concentration of anthocyanins were determined weekly during the juvenile, adult, reproductive and senescent phases of plant ontogenesis. This enabled quantification of the protective effectiveness of non-photochemical fluorescence quenching (NPQ) and determination of light tolerance. The light intensity that caused photoinhibition in 50% of leaf population increased from ∼70 µmol m(-2) s(-1) , for 1-week-old seedlings, to a maximum of 1385 µmol m(-2) s(-1) for 8-week-old plants. After 8 weeks, the tolerated light intensity started to gradually decline, becoming only 332 µmol m(-2) s(-1) for 13-week-old plants. The dependency of light tolerance on plant age was well-related to the amplitude of protective NPQ (pNPQ) and the electron transport rates (ETRs). Light tolerance did not, however, show a similar trend to chlorophyll a/b ratios and content of anthocyanins. Our data suggest that pNPQ is crucial in defining the capability of high light tolerance by Arabidopsis plants during ontogenesis.

Comparison of the protective effectiveness of NPQ in Arabidopsis plants deficient in PsbS protein and zeaxanthin.

The efficiency of protective energy dissipation by non-photochemical quenching (NPQ) in photosystem II (PSII) has been recently quantified by a new non-invasive photochemical quenching parameter, qPd. PSII yield (ФPSII) was expressed in terms of NPQ, and the extent of damage to the reaction centres (RCIIs) was calculated via qPd as: ФPSII=qPd×(F v/F m)/{1+[1-(F v/F m)]×NPQ}. Here this approach was used to determine the amount of NPQ required to protect all PSII reaction centres (pNPQ) under a gradually increasing light intensity, in the zeaxanthin-deficient (npq1) Arabidopsis mutant, compared with PsbS protein-deficient (npq4) and wild-type plants. The relationship between maximum pNPQ and tolerated light intensity for all plant genotypes followed similar trends. These results suggest that under a gradually increasing light intensity, where pNPQ is allowed to develop, it is only the amplitude of pNPQ which is the determining factor for protection. However, the use of a sudden constant high light exposure routine revealed that the presence of PsbS, not zeaxanthin, offered better protection for PSII. This was attributed to a slower development of pNPQ in plants lacking PsbS in comparison with plants that lacked zeaxanthin. This research adds further support to the value of pNPQ and qPd as effective parameters for assessing NPQ effectiveness in different types of plants.

Recovery after deficiency: systemic copper prioritization and partitioning in the leaves and stems of hybrid poplar.

Copper (Cu) is important for many aspects of plant function including photosynthesis. It has been suggested that photosynthesis, especially in young leaves, is prioritized for Cu delivery after deficiency in hybrid poplar. To determine relative Cu delivery prioritization, we enriched hydroponic plant growth media of Cu-deficient poplar with 98% 65Cu and tracked Cu delivery after deficiency to young leaves, mature leaves and stems. Young leaves acquired ~58% more 65Cu on Day 1 and ~65% more 65Cu by Day 3 compared with mature leaves. Additionally, stomatal conductance (gs) was measured on leaves for 6 weeks and during a 3-day 65Cu pulse resupply period. During deficiency, mature leaves maintained a higher gs than younger leaves but 3 days after Cu resupply the younger leaves that had recovered showed the highest gs. In conclusion, these results provide a quantitative understanding of how Cu is systemically transported and distributed to photosynthetic and stem tissues.

An In Vivo Quantitative Comparison of Photoprotection in Arabidopsis Xanthophyll Mutants.

Contribution of different LHCII antenna carotenoids to protective NPQ (pNPQ) were tested using a range of xanthophyll biosynthesis mutants of Arabidopsis: plants were either devoid of lutein (lut2), violaxanthin (npq2), or synthesized a single xanthophyll species, namely violaxanthin (aba4npq1lut2), zeaxanthin (npq2lut2), or lutein (chy1chy2lut5). A novel pulse amplitude modulated (PAM) fluorescence analysis procedure, that used a gradually increasing actinic light intensity, allowed the efficiency of pNPQ to be tested using the photochemical quenching (qP) parameter measured in the dark (qPd). Furthermore, the yield of photosystem II (ΦPSII) was calculated, and the light intensity which induces photoinhibition in 50% of leaves for each mutant was ascertained. Photoprotective capacities of each xanthophyll were quantified, taking into account chlorophyll a/b ratios and excitation pressure. Here, light tolerance, pNPQ capacity, and ΦPSII were highest in wild type plants. Of the carotenoid mutants, lut2 (lutein-deficient) plants had the highest light tolerance, and the joint the highest ΦPSII with violaxanthin only plants. We conclude that all studied mutants possess pNPQ and a more complete composition of xanthophylls in their natural binding sites is the most important factor governing photoprotection, rather than any one specific xanthophyll suggesting a strong structural effect of the molecules upon the LHCII antenna organization and discuss the results significance for future crop development.

PsbS protein modulates non-photochemical chlorophyll fluorescence quenching in membranes depleted of photosystems.

Plants with varying levels of PsbS protein were grown on lincomycin. Enhanced levels of non-photochemical fluorescence quenching (NPQ) in over-expressers of the protein have been observed. This was accompanied by increased amplitude of the irreversible NPQ component, qI, previously considered to reflect mainly photoinhibition of PSII reaction centres (RCII). However, since RCIIs were largely absent the observed qI is likely to originate from the LHCII antenna. In chloroplasts of over-expressers of PsbS grown on lincomycin an abnormally large NPQ (∼7) was characterised by a 0.34 ns average chlorophyll fluorescence lifetime. Yet the lifetime in the Fm state was similar to that of wild-type plants. 77K fluorescence emission spectra revealed a specific 700 nm peak typical of LHCII aggregates as well as quenching of the PSI fluorescence at 730 nm. The aggregated state manifested itself as a clear change in the distance between LHCII complexes detected by freeze-fracture electron microscopy. Grana thylakoids in the quenched state revealed 3 times more aggregated LHCII particles compared to the dark-adapted state. Overall, the results directly demonstrate the importance of LHCII aggregation in the NPQ mechanism and show that the PSII supercomplex structure plays no role in formation of the observed quenching.

An intact light harvesting complex I antenna system is required for complete state transitions in Arabidopsis.

Efficient photosynthesis depends on maintaining balance between the rate of light-driven electron transport occurring in photosystem I (PSI) and photosystem II (PSII), located in the chloroplast thylakoid membranes. Balance is achieved through a process of 'state transitions' that increases energy transfer towards PSI when PSII is overexcited (state II), and towards PSII when PSI is overexcited (state I). This is achieved through redox control of the phosphorylation state of light-harvesting antenna complex II (LHCII). PSI is served by both LHCII and four light-harvesting antenna complex I (LHCI) subunits, Lhca1, 2, 3 and 4. Here we demonstrate that despite unchanged levels of LHCII phosphorylation, absence of specific Lhca subunits reduces state transitions in Arabidopsis. The severest phenotype-observed in a mutant lacking Lhca4 (ΔLhca4)-displayed a 69% reduction compared with the wild type. Yet, surprisingly, the amounts of the PSI-LHCI-LHCII supercomplex isolated by blue native polyacrylamide gel electrophoresis (BN-PAGE) from digitonin-solubilized thylakoids were similar in the wild type and ΔLhca mutants. Fluorescence excitation spectroscopy revealed that in the wild type this PSI-LHCI-LHCII supercomplex is supplemented by energy transfer from additional LHCII trimers in state II, whose binding is sensitive to digitonin, and which are absent in ΔLhca4. The grana margins of the thylakoid membrane were found to be the primary site of interaction between this 'extra' LHCII and the PSI-LHCI-LHCII supercomplex in state II. The results suggest that the LHCI complexes mediate energetic interactions between LHCII and PSI in the intact membrane.