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3. Espressione eterologa di proteine vegetali in Pichia pastoris

Author

Angelini, R., Balestrini, R., Barera, S., Bellincampi, D., Benedetti, M., Boccaccini, A., Carpaneto, A., Cona, A., Conti, S., Dall'Osto, L., De Caroli, M., Fiorilli, V., Fraudentali, I., Giuntoli, B., Licausi, F., Lionetti, V., Malacarne, G., Martignago, D., Morosinotto, T., Moser, C., Pilati, S., Piro, G., Ricci, A., Rigano, M. M., Rolli, E., Tavladoraki, P., Torelli, A., and Trifilò, P.

Published
2020

10. Effects of Light on Cultivation of Scenedesmus Obliquus in Batch and Continuous Flat Plate Photobioreactor

Author

Sforza, E., Gris, B., De Farias Silva, C. E., Morosinotto, T., and Bertucco, A.

Subjects
lcsh:Computer engineering. Computer hardware, lcsh:TP155-156, lcsh:TK7885-7895, lcsh:Chemical engineering
Abstract

In view of an efficient industrial autotrophic cultivation of microalgal biomass, the role of light into photobioreactor is still to be fully ascertained. The influence of different light intensities was investigated using both continuous illumination and alternation of light and dark cycles with different frequencies, which mimic illumination variations in a photobioreactor due to mixing. In order to minimize the effect of cell self- shading all experiments were carried out in thin layer flat plate reactor (which can be operated both in batch and continuous mode), where microalgae were cultivated under a non-limiting CO2 supply, with CO2- enriched air bubbled through the culture. In this apparatus, we measured growth rate, lipid content and photosynthetic performances of Scenedesmus obliquus. The species showed a maximum growth rate at about 150 µmol of photons m-2 s-1. Above this value the growth was inhibited, while the algae was found still able to exploit light, even if at lower efficiency, and showed a decreased pigment content. The resistance of the species to high irradiances was confirmed also in continuous experiments, where microalgae were found able to adapt to high illumination, leading to an increased steady state concentration and higher productivity at 350 µmol m-2 s-1 (about 3 g L-1 d-1). In addition, by changing the residence time, a maximum in productivity was found. The lipid content, constitutively high, was not affected by the variation light intensity, making this species suitable to industrial application. Furthermore, the alternation of light and dark cycles with different frequencies was studied both in batch and continuous experiments. Cultures exposed to pulsed light shows a drastically reduced growth compared to continuous light.

Published
2014

16. Pigment binding sites occupancy and functional architecture of the Photosystem II antenna complex Lhcb5

Author

Ballottari, M., Mozzo, M., Croce, R., Morosinotto, T., Bassi, R., Biophysics Photosynthesis/Energy, and LaserLaB - Energy

Subjects
food and beverages
Abstract

Lhcb5 is an antenna protein that is highly conserved in plants and green algae. It is part of the inner layer of photosystem II antenna system retained in high light acclimated plants. To study the structure-function relation and the role of individual pigments in this complex, we (i) "knocked out" each of the chromophores bound to multiple (nine total) chlorophyll sites and (ii) exchanged the xanthophylls bound to the three xanthophyll sites. The occupancy and associated energy of the pigment binding sites were determined. The role of the individual pigments in protein folding, stability, energy transfer, and dissipation was studied in vitro. The results indicate that lutein has a primary role in the folding and stability of the complex, whereas violaxanthin and zeaxanthin have a negative effect on folding yield and stability, respectively. The data showed a distinct function for the L1 and L2 carotenoid binding sites, the former preferentially involved in gathering the excitation energy to chlorophyll a (Chl a), whereas the latter modulates the concentration of chlorophyll singlet excited states dependent on the xanthophylls bound to it, likely via an interaction with Chl-603. Our results also underscored the role of zeaxanthin and lutein in quenching the excitation energy, whereas violaxanthin was shown to be very effective in energy transfer. The characteristics of the isolated proteins were consistent with the observed role of Lhcb5 in vivo in catalyzing fluorescence quenching upon zeaxanthin binding. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Published
2009

17. Singlet and triplet state transitions of carotenoids in the antenna complexes of higher-plant photosystem I

Author

Croce R, Mozzo M, Morosinotto T, Romeo A, Hienerwadel R, and Bassi R.

Subjects
food and beverages
Abstract

In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while beta-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The "red" lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima: one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The "blue"-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T-S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2-Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale.

Published
2007

20. Pigment-Pigment interactions in Lhca4 Antenna complex of Higher Plants

Author

Morosinotto T., Mozzo M., Bassi R., and Croce R.

Abstract

The red-most fluorescence emission of photosystem I (733 nm at 4 K) is associated with the Lhca4 subunit of the antenna complex. It has been proposed that this unique spectral feature originates from the low energy absorption band of an excitonic interaction involving chlorophyll A5 and a second chlorophyll a molecule, probably B5 (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Because of the short distances between chromophores in Lhc proteins, the possibility that other pigments are involved in the red-shifted spectral forms could not be ruled out. In this study, we have analyzed the pigment-pigment interactions between nearest neighboring chromophores in Lhca4. This was done by deleting individual chlorophyll binding sites by mutagenesis, and analyzing the changes in the spectroscopic properties of recombinant proteins refolded in vitro. The red-shifted (733 nm) fluorescence peak, the major target of this analysis, was lost upon mutations affecting sites A4, A5, and B5 and was modified by mutating site B6. In agreement with the shorter distance between chlorophylls A5 and B5 (7.9 A) versus A4 and A5 (12.2 A) in Lhca4 (Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635), we conclude that the low energy spectral form originates from an interaction involving pigments in sites A5 and B5. Mutation at site B6, although inducing a 15-nm blue-shift of the emission peak, maintains the red-shifted emission. This implies that chromophores responsible for the interaction are conserved and suggests a modification in the pigment organization. Besides the A5-B5 pair, evidence for additional pigment-pigment interactions between chlorophylls in sites B3-A3 and B6-A6 was obtained. However, these features do not affect the red-most spectral form responsible for the 733-nm fluorescence emission band.

Published
2005

22. Origin of the 701 nm fluorescence emission of the Lhca2 subunit of higher plant Photosystem I

Author

Croce R., Morosinotto T., Ihalainen J.A., Chojnicka A., Breton J., Dekker J.P., van Grondelle R., and Bassi R.

Abstract

Photosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes.

Published
2004

26. The nature of a chlorophyll ligand in Lhca proteins determines the far red fluorescence emission typical of Photosystem I

Author

Morosinotto T. 1, Breton J. 2, Bassi R. 1, and Croce R. 3

Subjects
polycyclic compounds, macromolecular substances
Abstract

Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at approximately 700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.

Published
2003

27. The recombinant Lhca2 and Lhca3 subunits of the photosystem I antenna system

Author

Castelletti S., Morosinotto T., Robert B., Caffarri S., Bassi R., and Croce R.

Subjects
food and beverages
Abstract

In this study, two gene products (Lhca2 and Lhca3), encoding higher plants (Arabidopsis thaliana) Photosystem I antenna complexes, were overexpressed in bacteria and reconstituted in vitro with purified chloroplast pigments. The chlorophyll-xanthophyll proteins thus obtained were characterized by biochemical and spectroscopic methods. Both complexes were shown to bind 10 chlorophyll (a and b) molecules per polypeptide, Lhca2 having higher chlorophyll b content as compared to Lhca3. The two proteins differed for the number of carotenoid binding sites: two and three for Lhca2 and Lhca3, respectively. beta-carotene was specifically bound to Lhca3 in addition to the xanthophylls violaxanthin and lutein, indicating a peculiar structure of carotenoid binding sites in this protein since it is the only one so far identified with the ability of binding beta-carotene. Analysis of the spectroscopic properties of the two pigment proteins showed the presence of low energy absorption forms (red forms) in both complexes, albeit with different energies and amplitudes. The fluorescence emission maximum at 77 K of Lhca2 was found at 701 nm, while in Lhca3 the major emission was at 725 nm. Reconstitution of Lhca3 without Chl b reveals that Chl b is not involved in originating the low energy absorption forms of this complex. The present data are discussed in comparison to the properties of the recombinant Lhca1 and Lhca4 complexes and of the native LHCI preparation, previously analyzed, thus showing a comprehensive description of the gene products composing the Photosystem I light harvesting system of A. thaliana.

Published
2003

29. Mutation analysis of Lhca1 complex: the low energy absorption forms originate from pigment-pigment interaction

Author

Morosinotto T. 1, Castelletti S. 1, 2, Breton J. 2, Bassi R. 1, and Croce R. 3

Abstract

The light harvesting complex Lhca1, one of the four gene products comprising the photosystem I antenna system, has been analyzed by site- directed mutagenesis with the aim of determining the chromophore(s) responsible for its long wavelength chlorophyll spectral form, a specific characteristic of the LHCI antenna complex. A family of mutant proteins, each carrying a mutation at a single chlorophyll-binding residue, was obtained and characterized by biochemical and spectroscopic methods. A map of the chromophores bound to each of the 10 chlorophyll-binding sites was drawn, and the energy levels of the Qy transition were determined in most cases. When compared with Lhcb proteins previously analyzed, Lhca1 is characterized by stronger interactions between individual chromophores as detected by both biochemical and spectroscopic methods; most mutations, although targeted to a single residue, lead to the loss of more than one chromophore and of conservative CD signals typical of chlorophyll- chlorophyll interactions. The lower energy absorption form (686 nm at 100K, 688 nm at room temperature), which is responsible for the red- shifted emission components at 690 and 701 nm, typical of Lhca1, is associated with a chlorophyll a/chlorophyll a excitonic interaction originating from a pigment cluster localized in the protein domain situated between helix C and the helix A/helix B cross. This cluster includes chlorophylls bound to sites A5-B5-B6 and a xanthophyll bound to site L2.

Published
2002

30. The Lhca antenna complexes of higher plants Photosystem I

Author

Croce R. 1, Morosinotto T. 2, Castelletti S. 2, Breton J. 3, and Bassi R. 2

Subjects
Recombinant Lhc protein, Chlorophyll-protein, Photosynthesis
Abstract

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI–LHCI complex. The red emission fluorescence is associated not only with Lhca1–4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1–4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/ b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while b-carotene is selectively bound to the Lhca1–4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.

Published
2002

31. The low-energy forms of photosystem I light-harvesting complexes: Spectroscopic properties and pigment-pigment interaction characteristics

Author

Croce, R., Chojnicka, A., Morosinotto, T., Ihalainen, J.A., van Mourik, F., Dekker, J.P., Bassi, R., van Grondelle, R., Croce, R., Chojnicka, A., Morosinotto, T., Ihalainen, J.A., van Mourik, F., Dekker, J.P., Bassi, R., and van Grondelle, R.

Abstract

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to "normal" chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm

Published
2007
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32. Excitation energy transfer pathways in Lhca4

Author

Gibasiewicz, K, Croce, R, Morosinotto, T., Ihalainen, J A, van Stokkum, I H M, Dekker, J P, Bassi, R., van Grondelle, R, Gibasiewicz, K, Croce, R, Morosinotto, T., Ihalainen, J A, van Stokkum, I H M, Dekker, J P, Bassi, R., and van Grondelle, R

Abstract

EET in reconstituted Lhca4, a peripheral light-harvesting complex from Photosystem I of Arabidopsis thaliana, containing 10 chlorophylls and 2 carotenoids, was studied at room temperature by femtosecond transient absorption spectroscopy. Two spectral forms of Lut were observed in the sites L1 and L2, characterized by significantly different interactions with nearby chlorophyll a molecules. A favorable interpretation of these differences is that the efficiency of EET to Chls is about two times lower from the "blue" Lut in the site L1 than from the "red" Lut in the site L2 due to fast IC in the former case. A major part of the energy absorbed by the "red" Lut, approximately 60%-70%, is transferred to Chls on a sub-100-fs timescale from the state S(2) but, in addition, minor EET from the hot S(1) state within 400-500 fs is also observed. EET from the S(1) state to chlorophylls occurs also within 2-3 ps and is ascribed to Vio and/or "blue" Lut. EET from Chl b to Chl a is biphasic and characterized by time constants of approximately 300 fs and 3.0 ps. These rates are ascribed to EET from Chl b spectral forms absorbing at approximately 644 nm and approximately 650 nm, respectively. About 25% of the excited Chls a decays very fast-within approximately 15 ps. This decay is proposed to be related to the presence of the interacting Chls A5 and B5 located next to the carotenoid in the site L2 and may imply some photoprotective role for Lhca4 in the photosystem I super-complex.

Published
2005
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35. Excitation Energy Transfer Pathways in Lhca4

Author

Gibasiewicz, K., primary, Croce, R., additional, Morosinotto, T., additional, Ihalainen, J.A., additional, van Stokkum, I.H.M., additional, Dekker, J.P., additional, Bassi, R., additional, and van Grondelle, R., additional

Published
2005
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37. Atmospheres in a Test Tube: State of the art at the Astronomical Observatory of Padova

Author

Erculiani, M. S., Claudi, R., Cocola, L., Giro, E., La Rocca, N., Morosinotto, T., Poletto, L., Barbisan, D., Billi, D., Bonato, M., D Alessandro, M., Galletta, G., Meneghini, M., Trivellin, N., Mariangela Cestelli Guidi, Pace, E., Schierano, D., and Micela, G.

Subjects
Climate simulation, Exoplanet, Atmosphere Characterization, Climate simulation, photosynthetic bacteria, photosynthetic bacteria, Atmosphere Characterization, Exoplanet

38. Atmosphere in a Test Tube

Author

Claudi, R., Pace, E., Ciaravella, A., Micela, G., Piccioni, G., DANIELA BILLI, Cestelli Guidi, M., Coccola, L., Erculiani, M. S., Fedel, M., Galletta, G., Giro, E., La Rocca, N., Morosinotto, T., Poletto, L., Schierano, D., and Stefani, S.

Subjects
Extrasolar Planets, Laboratory Analogs, Laboratory Analogs, Extrasolar Planets

40. Antenna complexes protect Photosystem I from Photoinhibition

Author

Hienerwadel Rainer, Ballottari Matteo, Alboresi Alessandro, Giacometti Giorgio M, and Morosinotto Tomas

Subjects
Botany, QK1-989
Abstract

Abstract Background Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II. Results In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex. Conclusion We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed.

Published
2009
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41. A study on PsbS and its role as a pH sensor

Author

Krishnan, M., Groot, H.J.M. de, Pandit, A., Overkleeft, H.S., Ubbink, M., Grip, W.J. de, Morosinotto, T., Wientjes, E., and Leiden

Subjects
Protonation sites, Photoprotection, Membrane proteins, food and beverages, Oligomerization
Abstract

Solar energy harnessed by plants and algae has great potential to be converted into biofuels for future generations. Understanding the regulatory mechanism of photosynthesis can increase biomass yield and drive the generation of biofuels to its maximum. PsbS, a membrane protein, is an essential component for NPQ and has been studied extensively in this thesis. PsbS is hypothesized to sense pH and to regulate photosynthesis under light stress conditions. The first objective in this thesis is to produce, purify and refold recombinant PsbS with sufficiently high yields for biochemical studies along with optimizing the detergent conditions for maintaining its stability. The second objective is the spectroscopic characterization of PsbS to understand its role as a pH sensor and its molecular mechanism during photoprotection.

Published
2019

42. Physcomitrium patens flavodiiron proteins form heterotetrametric complexes.

Author

Beraldo C, Traverso E, Boschin M, Cendron L, Morosinotto T, and Alboresi A

Subjects
Photosystem I Protein Complex metabolism, Photosystem I Protein Complex chemistry, Protein Multimerization, Iron metabolism, Iron chemistry, Escherichia coli metabolism, Escherichia coli genetics, Bryopsida metabolism, Plant Proteins metabolism, Plant Proteins chemistry
Abstract

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2024
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43. Mitochondrial respiration is essential for photosynthesis-dependent ATP supply of the plant cytosol.

Author

Vera-Vives AM, Novel P, Zheng K, Tan SL, Schwarzländer M, Alboresi A, and Morosinotto T

Subjects
Light, Adenosine Triphosphate metabolism, Photosynthesis, Cytosol metabolism, Mitochondria metabolism, Cell Respiration, Bryopsida metabolism, Bryopsida genetics, Bryopsida growth & development
Abstract

Plants rely on solar energy to synthesize ATP and NADPH for photosynthetic carbon fixation and all cellular need. Mitochondrial respiration is essential in plants, but this may be due to heterotrophic bottlenecks during plant development or because it is also necessary in photosynthetically active cells. In this study, we examined in vivo changes of cytosolic ATP concentration in response to light, employing a biosensing strategy in the moss Physcomitrium patens and revealing increased cytosolic ATP concentration caused by photosynthetic activity. Plants depleted of respiratory Complex I showed decreased cytosolic ATP accumulation, highlighting a critical role of mitochondrial respiration in light-dependent ATP supply of the cytosol. Consistently, targeting mitochondrial ATP production directly, through the construction of mutants deficient in mitochondrial ATPase (complex V), led to drastic growth reduction, despite only minor alterations in photosynthetic electron transport activity. Since P. patens is photoautotrophic throughout its development, we conclude that heterotrophic bottlenecks cannot account for the indispensable role of mitochondrial respiration in plants. Instead, our results support that mitochondrial respiration is essential for ATP provision to the cytosol in photosynthesizing cells. Mitochondrial respiration provides metabolic integration, ensuring supply of cytosolic ATP essential for supporting plant growth and development., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Published
2024
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44. Autotrophic poly-3-hydroxybutyrate accumulation in Cupriavidus necator for sustainable bioplastic production triggered by nutrient starvation.

Author

Santin A, Spatola Rossi T, Morlino MS, Gupte AP, Favaro L, Morosinotto T, Treu L, and Campanaro S

Subjects
Autotrophic Processes, Oxygen metabolism, Phosphorus metabolism, Carbon metabolism, Nutrients metabolism, Plastics metabolism, Hydrogen metabolism, Biodegradable Plastics metabolism, Magnesium metabolism, Polyhydroxybutyrates, Cupriavidus necator metabolism, Hydroxybutyrates metabolism, Polyesters metabolism, Nitrogen metabolism
Abstract

Cupriavidus necator is a facultative chemolithoautotrophic bacterium able to convert carbon dioxide into poly-3-hydroxybutyrate. This is highly promising as the conversion process allows the production of sustainable and biodegradable plastics. Poly-3-hydroxybutyrate accumulation is known to be induced by nutrient starvation, but information regarding the optimal stress conditions controlling the process is still heterogeneous and fragmentary. This study presents a comprehensive comparison of the effects of nutrient stress conditions, namely nitrogen, hydrogen, phosphorus, oxygen, and magnesium deprivation, on poly-3-hydroxybutyrate accumulation in C. necator DSM545. Nitrogen starvation exhibited the highest poly-3-hydroxybutyrate accumulation, achieving 54% of total cell dry weight after four days of nutrient stress, and a carbon conversion efficiency of 85%. The gas consumption patterns indicated flexible physiological mechanisms underlying polymer accumulation and depolymerization. These findings provide insights into strategies for efficient carbon conversion into bioplastics, and highlight the key role of C. necator for future industrial-scale applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Published
2024
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45. Deciphering the genetic landscape of enhanced poly-3-hydroxybutyrate production in Synechocystis sp. B12.

Author

Santin A, Collura F, Singh G, Morlino MS, Bizzotto E, Bellan A, Gupte AP, Favaro L, Campanaro S, Treu L, and Morosinotto T

Abstract

Background: Microbial biopolymers such as poly-3-hydroxybutyrate (PHB) are emerging as promising alternatives for sustainable production of biodegradable bioplastics. Their promise is heightened by the potential utilisation of photosynthetic organisms, thus exploiting sunlight and carbon dioxide as source of energy and carbon, respectively. The cyanobacterium Synechocystis sp. B12 is an attractive candidate for its superior ability to accumulate high amounts of PHB as well as for its high-light tolerance, which makes it extremely suitable for large-scale cultivation. Beyond its practical applications, B12 serves as an intriguing model for unravelling the molecular mechanisms behind PHB accumulation., Results: Through a multifaceted approach, integrating physiological, genomic and transcriptomic analyses, this work identified genes involved in the upregulation of chlorophyll biosynthesis and phycobilisome degradation as the possible candidates providing Synechocystis sp. B12 an advantage in growth under high-light conditions. Gene expression differences in pentose phosphate pathway and acetyl-CoA metabolism were instead recognised as mainly responsible for the increased Synechocystis sp. B12 PHB production during nitrogen starvation. In both response to strong illumination and PHB accumulation, Synechocystis sp. B12 showed a metabolic modulation similar but more pronounced than the reference strain, yielding in better performances., Conclusions: Our findings shed light on the molecular mechanisms of PHB biosynthesis, providing valuable insights for optimising the use of Synechocystis in economically viable and sustainable PHB production. In addition, this work supplies crucial knowledge about the metabolic processes involved in production and accumulation of these molecules, which can be seminal for the application to other microorganisms as well., (© 2024. The Author(s).)

Published
2024
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46. Net O 2 exchange rates under dark and light conditions across different stem compartments.

Author

Natale S, Peralta Ogorek LL, Caracciolo L, Morosinotto T, van Amerongen H, Casolo V, Pedersen O, and Nardini A

Subjects
Darkness, Fraxinus metabolism, Chloroplasts metabolism, Chloroplasts radiation effects, Plant Bark metabolism, Photosynthesis radiation effects, Photosystem II Protein Complex metabolism, Plant Stems metabolism, Plant Stems radiation effects, Oxygen metabolism, Light, Wood metabolism
Abstract

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O 2 production in whole stem, bark, and wood. We present the radial profile of O 2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O 2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O 2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Published
2024
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47. Biological carbon capture from biogas streams: Insights into Cupriavidus necator autotrophic growth and transcriptional profile.

Author

Serna-García R, Silvia Morlino M, Bucci L, Savio F, Favaro L, Morosinotto T, Seco A, Bouzas A, Campanaro S, and Treu L

Subjects
Carbon Dioxide, Biofuels, Rivers, Autotrophic Processes, Cupriavidus necator genetics, Polyhydroxyalkanoates metabolism
Abstract

Recycling carbon-rich wastes into high-value platform chemicals through biological processes provides a sustainable alternative to petrochemicals. Cupriavidus necator, known for converting carbon dioxide (CO 2 ) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO 2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H 2 and O 2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO 2 -adaptated cultures exhibited upregulation of hydrogenases and Calvin cycle enzymes, as well as genes related to electron transport, nutrient uptake, and glyoxylate cycle. Non-adapted samples displayed activation of stress response mechanisms, suggesting potential lags in large-scale processes. These findings showcase the setting of growth parameters for a pioneering biological biogas upgrading strategy, emphasizing the importance of inoculum adaptation for autotrophic growth and providing potential targets for genetic engineering to push PHA yields in future applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Published
2024
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48. Assessment of photosynthetic activity in dense microalgae cultures using oxygen production.

Author

Vera-Vives AM, Michelberger T, Morosinotto T, and Perin G

Subjects
Ecosystem, Oxygen metabolism, Photosynthesis, Photobioreactors, Biomass, Microalgae
Abstract

Microalgae are photosynthetic microorganisms playing a pivotal role in primary production in aquatic ecosystems, sustaining the entry of carbon in the biosphere. Microalgae have also been recognized as sustainable source of biomass to complement crops. For this objective they are cultivated in photobioreactors or ponds at high cell density to maximize biomass productivity and lower the cost of downstream processes. Photosynthesis depends on light availability, that is often not constant over time. In nature, sunlight fluctuates over diurnal cycles and weather conditions. In high-density microalgae cultures of photobioreactors outdoors, on top of natural variations, microalgae are subjected to further complexity in light exposure. Because of the high-density cells experience self-shading effects that heavily limit light availability in most of the mass culture volume. This limitation strongly affects biomass productivity of industrial microalgae cultivation plants with important implications on economic feasibility. Understanding how photosynthesis responds to cell density is informative to assess functionality in the inhomogeneous light environment of industrial photobioreactors. In this work we exploited a high-sensitivity Clark electrode to measure microalgae photosynthesis and compare cultures with different densities, using Nannochloropsis as model organism. We observed that cell density has a substantial impact on photosynthetic activity, and demonstrated the reduction of the cell's light-absorption capacity by genetic modification is a valuable strategy to increase photosynthetic functionality on a chlorophyll-basis of dense microalgae cultures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Published
2024
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49. Cupriavidus necator as a platform for polyhydroxyalkanoate production: An overview of strains, metabolism, and modeling approaches.

Author

Morlino MS, Serna García R, Savio F, Zampieri G, Morosinotto T, Treu L, and Campanaro S

Subjects
Fermentation, Biotechnology, Carbon metabolism, Polyhydroxyalkanoates genetics, Polyhydroxyalkanoates metabolism, Cupriavidus necator genetics, Cupriavidus necator metabolism
Abstract

Cupriavidus necator is a bacterium with a high phenotypic diversity and versatile metabolic capabilities. It has been extensively studied as a model hydrogen oxidizer, as well as a producer of polyhydroxyalkanoates (PHA), plastic-like biopolymers with a high potential to substitute petroleum-based materials. Thanks to its adaptability to diverse metabolic lifestyles and to the ability to accumulate large amounts of PHA, C. necator is employed in many biotechnological processes, with particular focus on PHA production from waste carbon sources. The large availability of genomic information has enabled a characterization of C. necator's metabolism, leading to the establishment of metabolic models which are used to devise and optimize culture conditions and genetic engineering approaches. In this work, the characteristics of available C. necator strains and genomes are reviewed, underlining how a thorough comprehension of the genetic variability of C. necator is lacking and it could be instrumental for wider application of this microorganism. The metabolic paradigms of C. necator and how they are connected to PHA production and accumulation are described, also recapitulating the variety of carbon substrates used for PHA accumulation, highlighting the most promising strategies to increase the yield. Finally, the review describes and critically analyzes currently available genome-scale metabolic models and reduced metabolic network applications commonly employed in the optimization of PHA production. Overall, it appears that the capacity of C. necator of performing CO 2 bioconversion to PHA is still underexplored, both in biotechnological applications and in metabolic modeling. However, the accurate characterization of this organism and the efforts in using it for gas fermentation can help tackle this challenging perspective in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2023
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50. Improving photosynthetic efficiency toward food security: Strategies, advances, and perspectives.

Author

Smith EN, van Aalst M, Tosens T, Niinemets Ü, Stich B, Morosinotto T, Alboresi A, Erb TJ, Gómez-Coronado PA, Tolleter D, Finazzi G, Curien G, Heinemann M, Ebenhöh O, Hibberd JM, Schlüter U, Sun T, and Weber APM

Subjects
Crops, Agricultural, Nutrients, Food Security, Plant Breeding, Photosynthesis
Abstract

Photosynthesis in crops and natural vegetation allows light energy to be converted into chemical energy and thus forms the foundation for almost all terrestrial trophic networks on Earth. The efficiency of photosynthetic energy conversion plays a crucial role in determining the portion of incident solar radiation that can be used to generate plant biomass throughout a growth season. Consequently, alongside the factors such as resource availability, crop management, crop selection, maintenance costs, and intrinsic yield potential, photosynthetic energy use efficiency significantly influences crop yield. Photosynthetic efficiency is relevant to sustainability and food security because it affects water use efficiency, nutrient use efficiency, and land use efficiency. This review focuses specifically on the potential for improvements in photosynthetic efficiency to drive a sustainable increase in crop yields. We discuss bypassing photorespiration, enhancing light use efficiency, harnessing natural variation in photosynthetic parameters for breeding purposes, and adopting new-to-nature approaches that show promise for achieving unprecedented gains in photosynthetic efficiency., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Published
2023
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