Biochemical and molecular properties of LHCX1, the essential regulator of dynamic photoprotection in diatoms.

Light harvesting is regulated by a process triggered by the acidification of the thylakoid lumen, known as nonphotochemical "energy-dependent quenching" (qE). In diatoms, qE is controlled by the light-harvesting complex (LHC) protein LHCX1, while the LHC stress-related (LHCSR) and photosystem II subunit S proteins are essential for green algae and plants, respectively. Here, we report a biochemical and molecular characterization of LHCX1 to investigate its role in qE. We found that, when grown under intermittent light, Phaeodactylum tricornutum forms very large qE, due to LHCX1 constitutive upregulation. This "super qE" is abolished in LHCX1 knockout mutants. Biochemical and spectroscopic analyses of LHCX1 reveal that this protein might differ in the character of binding pigments relative to the major pool of light-harvesting antenna proteins. The possibility of transient pigment binding or not binding pigments at all is discussed. Targeted mutagenesis of putative protonatable residues (D95 and E205) in transgenic P. tricornutum lines does not alter qE capacity, showing that they are not involved in sensing lumen pH, differently from residues conserved in LHCSR3. Our results suggest functional divergence between LHCX1 and LHCSR3 in qE modulation. We propose that LHCX1 evolved independently to facilitate dynamic tracking of light fluctuations in turbulent waters. The evolution of LHCX(-like) proteins in organisms with secondary red plastids, such as diatoms, might have conferred a selective advantage in the control of dynamic photoprotection, ultimately resulting in their ecological success.

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes.

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679 nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.

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.

Direct impact of the sustained decline in the photosystem II efficiency upon plant productivity at different developmental stages.

The impact of chronic photoinhibition of photosystem II (PSII) on the productivity of plants remains unknown. The present study investigated the influences of persistent decline in the PSII yield on morphology and productivity of Arabidopsis plants that were exposed to lincomycin at two different developmental stages (seedling and rosette stage). The results indicated that, although retarded, the lincomycin treated plants were able to accomplish the entire growth period with only 50% of the maximum quantum yield of primary photochemistry (Fv/Fm) of the control plants. The decline in quantum yield limited the electron transport rate (ETR). The impact of lincomycin on NPQ was not significant in seedlings, but was pronounced in mature plants. The treated plants produced an above ground biomass of 50% compared to control plants. Moreover, a linear relationship was found between the above ground biomass and total rosette leaf area, and the slope was decreased due to photoinhibition. The starch accumulation was highly inhibited by lincomycin treatment. Lincomycin induced a significant decrease in seed yield with plants treated from the rosette state showing higher yield than those treated from the seedling stage. Our data suggest that the sustained decline of PSII efficiency decreases plant productivity by constraining the ETR, leaf development and starch production.

The xanthophyll cycle affects reversible interactions between PsbS and light-harvesting complex II to control non-photochemical quenching.

To maintain high photosynthetic rates, plants must adapt to their light environment on a timescale of seconds to minutes. Therefore, the light-harvesting antenna system of photosystem II in thylakoid membranes, light-harvesting complex II (LHCII), has a feedback mechanism, which determines the proportion of absorbed energy dissipated as heat: non-photochemical chlorophyll fluorescence quenching (NPQ). This is crucial to prevent photo-oxidative damage to photosystem II (PSII) and is controlled by the transmembrane pH differences (ΔpH). High ΔpH activates NPQ by protonation of the protein PsbS and the enzymatic de-epoxidation of LHCII-bound violaxanthin to zeaxanthin. But the precise role of PsbS and its interactions with different LHCII complexes remain uncertain. We have investigated PsbS-LHCII interactions in native thylakoid membranes using magnetic-bead-linked antibody pull-downs. The interaction of PsbS with the antenna system is affected by both ΔpH and the level of zeaxanthin. In the presence of ΔpH alone, PsbS is found to be mainly associated with the trimeric LHCII protein polypeptides, Lhcb1, Lhcb2 and Lhcb3. However, a combination of ΔpH and zeaxanthin increases the proportion of PsbS bound to the minor LHCII antenna complex proteins Lhcb4, Lhcb5 and Lhcb6. This pattern of interaction is not influenced by the presence of PSII reactions centres. Similar to LHCII particles in the photosynthetic membrane, PsbS protein forms clusters in the NPQ state. NPQ recovery in the dark requires uncoupling of PsbS. We suggest that PsbS acts as a 'seeding' centre for the LHCII antenna rearrangement that is involved in NPQ.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.

Light-harvesting superstructures of green plant chloroplasts lacking photosystems.

The light-harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures - stacks of dozens of thylakoids or supergrana. Freeze-fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light-harvesting membranes, organized into three-dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self-assembly of the three-dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.

Economic photoprotection in photosystem II that retains a complete light-harvesting system with slow energy traps.

The light-harvesting antenna of higher plant photosystem II has an intrinsic capability for self-defence against intense sunlight. The thermal dissipation of excess energy can be measured as the non-photochemical quenching of chlorophyll fluorescence. It has recently been proposed that the transition between the light-harvesting and self-defensive modes is associated with a reorganization of light-harvesting complexes. Here we show that despite structural changes, the photosystem II cross-section does not decrease. Our study reveals that the efficiency of energy trapping by the non-photochemical quencher(s) is lower than the efficiency of energy capture by the reaction centres. Consequently, the photoprotective mechanism works effectively for closed rather than open centres. This type of defence preserves the exceptional efficiency of electron transport in a broad range of light intensities, simultaneously ensuring high photosynthetic productivity and, under hazardous light conditions, sufficient photoprotection for both the reaction centre and the light-harvesting pigments of the antenna.

Development and staging of the water flea Daphnia magna (Straus, 1820; Cladocera, Daphniidae) based on morphological landmarks.

BACKGROUND: Crustaceans of the genus Daphnia are one of the oldest model organisms in ecotoxicology, ecology and evolutionary biology. The publication of the Daphnia pulex genome has facilitated the development of genetic tools to answer long-standing questions in these research fields (Science 331: 555-561, 2011). A particular focus is laid on understanding the genetic basis of the striking ability of daphnids to change their phenotype in response to environmental stressors. Furthermore, Daphnia have recently been developed into crustacean model organisms for EvoDevo research, contributing to the ongoing attempt to resolve arthropod phylogeny. These problems require the comparative analyses of gene expression and functional data, which in turn require a standardized developmental staging system for Daphnia. RESULTS: Here we provide a detailed staging system of the embryonic development of Daphnia magna based on morphological landmarks. The staging system does not rely on developmental hours and is therefore suitable for functional and ecological experiments, which often cause developmental delays in affected embryos and thus shifts in time reference points. We provide a detailed description of each stage and include schematic drawings of all stages showing relevant morphological landmarks in order to facilitate the application of this staging scheme. CONCLUSION: We present here a staging system for Daphnia magna, which is based on morphological landmarks. The staging system can be adopted for other daphnids with minor variations since the sequence of development is highly conserved during early stages and only minor heterochronic shifts occur in late embryonic stages.

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda).

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.

Unravelling the evolution of neural stem cells in arthropods: notch signalling in neural stem cell development in the crustacean Daphnia magna.

The genetic regulatory networks controlling major developmental processes seem to be conserved in bilaterians regardless of an independent or a common origin of the structures. This has been explained by the employment of a genetic toolkit that was repeatedly used during bilaterian evolution to build the various forms and body plans. However, it is not clear how genetic networks were incorporated into the formation of novel structures and how homologous genes can regulate the disparate morphological processes. Here we address this question by analysing the role of Notch signalling, which is part of the bilaterian toolkit, in neural stem cell evolution in arthropods. Within arthropods neural stem cells have evolved in the last common ancestor of insects and crustaceans (Tetraconata). We analyse here for the first time the role of Notch signalling in a crustacean, the branchiopod Daphnia magna, and show that it is required in neural stem cells for regulating the time of neural precursor production and for binary cell fate decisions in the ventral neuroectoderm. The function of Notch signalling has diverged in the ventral neuroectoderm of insects and crustaceans accompanied by changes in the morphogenetic processes. In the crustacean, Notch controlled mechanisms of neuroblast regulation have evolved that are surprisingly similar to vertebrates and thus present a remarkable case of parallel evolution. These new data on a representative of crustaceans complete the arthropod data set on Notch signalling in the nervous system and allow for reconstructing how the Notch signalling pathway has been co-opted from pre-existing structures to the development of the evolving neural stem cells in the Tetraconata ancestor.

Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans.

Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete-scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete-scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods - the absence of neuroblast segregation and proneural clusters - might be used to support or reject the possible groupings of paraphyletic crustaceans.

Axogenesis in the central and peripheral nervous system of the amphipod crustacean Orchestia cavimana.

We describe the formation of the major axon pathways in the embryonic central and peripheral nervous systems of the amphipod crustacean Orchestia cavimana Heller, 1865 by means of antibody staining against acetylated alpha-tubulin. The data add to a long list of previous studies of various other aspects of development in Orchestia and provide a basis for future studies of neurogenesis on a deeper cellular and molecular level. Orchestia exhibits a tripartite dorsal brain, which is a characteristic feature of euarthropods. Its anlagen are the first detectable structures in the developing nervous system and can be traced back to distinct neuronal cell clusters in the early embryo. The development of the ventral nervous system proceeds with an anteroposterior gradient of development. In each trunk segment, the longitudinal connectives and the anterior commissure form first, followed by the intersegmental nerve, the posterior commissure and segmental nerves, respectively. A single commissure of a vestigial seventh pleonal segment is found. In the peripheral nervous system we observe a spatial and temporal pattern of leg innervation, which is strikingly similar in both limb types, the uniramous pereopods and the biramous pleopods. A proximal leg nerve splitting distally into two separated nerves probably reflects a general feature of crustaceans.

The post-embryonic development of Remipedia (Crustacea)--additional results and new insights.

The post-embryonic development of a species of the enigmatic crustacean group Remipedia is described in detail for the first time under various aspects. Applying a molecular approach, we can clearly prove the species identity of the larvae as belonging to Pleomothra apletocheles. We document the cellular level of several larval stages and the differentiation of segments, limbs, and the general body morphology applying the techniques of confocal laser scanning microscopy and scanning electron microscopy. In addition, we document the swimming behavior and the peculiar movements of the naupliar appendages. A comparison of our results with published data on other Crustacea and their larval development tentatively supports ideas about phylogenetic affinities of the Remipedia to the Malacostraca.

The chelifores of sea spiders (Arthropoda, Pycnogonida) are the appendages of the deutocerebral segment.

Within the last decade, gene expression patterns and neuro-anatomical data have led to a new consensus concerning the long-debated association between anterior limbs and neuromeres in the arthropod head. According to this new view, the first appendage in all extant euarthropods is innervated by the second neuromere, the deutocerebrum, whereas the anterior-most head region bearing the protocerebrum lacks an appendage. This stands in contrast to the clearly protocerebrally targeted "antennae" of Onychophora and to some evidence for protocerebral limbs in fossil euarthropod representatives. Yet, the latter "frontal appendages" or "primary antennae" have most likely been reduced or lost in the lineage, leading to extant taxa. Surprisingly, a recent neuro-anatomical study on a pycnogonid challenged this evolutionary scenario, reporting a protocerebral innervation of the first appendages, the chelifores. However, this interpretation was soon after questioned by Hox gene expression data. To re-evaluate the unresolved controversy, we analyzed neuro-anatomy and neurogenesis in four pycnogonid species using immunohistochemical techniques. We clearly show the postprotocerebral innervation of the chelifores, which is resolved as the plesiomorphic condition in pycnogonids when evaluated against a recently published comprehensive phylogeny. By providing direct morphological support for the deutocerebral status of the cheliforal ganglia, we reconcile morphological and gene expression data and argue for a corresponding position between the anterior-most appendages in all extant euarthropods. Consequently, other structures have to be scrutinized to illuminate the fate of a presumptive protocerebral appendage in recent euarthropods. The labrum and the "frontal filaments" of some crustaceans are possible candidates for this approach.

Filling the gap between identified neuroblasts and neurons in crustaceans adds new support for Tetraconata.

The complex spatio-temporal patterns of development and anatomy of nervous systems play a key role in our understanding of arthropod evolution. However, the degree of resolution of neural processes is not always detailed enough to claim homology between arthropod groups. One example is neural precursors and their progeny in crustaceans and insects. Pioneer neurons of crustaceans and insects show some similarities that indicate homology. In contrast, the differentiation of insect and crustacean neuroblasts (NBs) shows profound differences and their homology is controversial. For Drosophila and grasshoppers, the complete lineage of several NBs up to formation of pioneer neurons is known. Apart from data on median NBs no comparable results exist for Crustacea. Accordingly, it is not clear where the crustacean pioneer neurons come from and whether there are NBs lateral to the midline homologous to those of insects. To fill this gap, individual NBs in the ventral neuroectoderm of the crustacean Orchestia cavimana were labelled in vivo with a fluorescent dye. A partial neuroblast map was established and for the first time lineages from individual NBs to identified pioneer neurons were established in a crustacean. Our data strongly suggest homology of NBs and their lineages, providing further evidence for a close insect-crustacean relationship.